Patent Publication Number: US-2019194670-A1

Title: Bacteria-Based Protein Delivery

Description:
THE FIELD OF THE INVENTION 
     The present invention relates to recombinant Gram-negative bacterial strains and the use thereof for delivery of repeated domains of a heterologous protein or two or more domains of different heterologous proteins into eukaryotic cells. 
     BACKGROUND OF THE INVENTION 
     Transient transfection techniques have been applied in cell biological research over many years to address protein functions. These methods generally result in a massive overrepresentation of the protein under study, which might lead to oversimplified models of signalling. For proteins controlling short-lived signalling processes, the protein of interest is present for far longer as the signalling event it controls. Even more, DNA transfection based transient over-expression leads to a heterogenous and unsynchronized cell population, which complicates functional studies and hampers -omics approaches. Besides this, the upscaling of such assays to a larger scale is very expensive. Some of the above mentioned points are covered by existing techniques as microinjection or proteo-fection of purified proteins, the inducible translocation strategy to rapidly target plasmid born small GTPases to the cell membrane or the addition of purified proteins fused to cell-permeable bacterial toxins. But these techniques are all time-consuming and cumbersome and to our knowledge none fulfils all mentioned criteria. 
     Bacteria have evolved different mechanisms to directly inject proteins into target cells [1]. The type III secretion system (T3SS) used by bacteria like  Yersinia, Shigella  and  Salmonella  [2] functions like a nano-syringe that injects so-called bacterial effector proteins into host cells. Bacterial proteins to be secreted via the T3SS, called effectors, harbour a short N-terminal secretion signal [3]. Inside bacteria, some effectors are bound by chaperones. Chaperones might mask toxic domains, they contribute to exposition of the secretion signal and keep the substrates in a secretion-competent conformation, therefore facilitating secretion. Upon induction of secretion, an ATPase adjacent to the T3SS removes the chaperones and the effectors travel unfolded or only partially folded through the needle, and refold once in the host cytoplasm. 
     T3S has been exploited to deliver hybrid peptides and proteins into target cells. Heterologous bacterial T3SS effectors have been delivered in case the bacterium under study is hardly accessible by genetics (like  Chlamydia trachomatis ). Often reporter proteins were fused to possible T3SS secretion signals as to study requirements for T3 SS dependent protein delivery, such as the  Bordetella pertussis  adenylate cyclase, murine DHFR or a phosphorylatable tag. Peptide delivery was mainly conducted with the aim of vaccination. This includes viral epitopes, bacterial epitopes (listeriolysin O) as well as peptides representing epitopes of human cancer cells. In few cases functional eukaryotic proteins have been delivered to modulate the host cell, as done with nanobodies [4], nuclear proteins (Cre-recombinase, MyoD) [5,6] or 110 and IL1ra [7]. None of the above-mentioned systems allows single-protein delivery as in each case one or multiple endogenous effector proteins are still encoded. Furthermore, the vectors used have not been designed in a way allowing simple cloning of other DNA fragments encoding proteins of choice, hindering broad application of the system. Surprisingly it has been found that delivery of repeated domains of heterologous proteins or combinations of domains of different heterologous proteins to eukaryotic cells enlarges the impact on a desired cellular pathway. 
     SUMMARY OF THE INVENTION 
     The present invention relates generally to recombinant Gram-negative bacterial strains and the use thereof for delivery of repeated domains of a heterologous protein or two or more domains of different heterologous proteins into eukaryotic cells. The present invention provides Gram-negative bacterial strains and the use thereof, which allows the translocation of repeated domains of a heterologous protein or two or more domains of different heterologous proteins such as various type III effectors, but also of type IV effectors, viral proteins and most importantly functional eukaryotic proteins. Means for fluorescent tracking of delivery, for relocalization to the nucleus and notably for removal of the bacterial appendage after delivery to the host cell are provided. The presented T3SS based system results in scalable, rapid, synchronized, homogenous and tunable delivery of repeated domains of a heterologous protein or two or more domains of different heterologous proteins of interest. The delivery system of the present invention is suitable to inject repeated domains of a eukaryotic protein or two or more domains of different eukaryotic proteins in living animals and can be used for therapeutic purposes. 
     In a first aspect the present invention relates to a recombinant Gram-negative bacterial strain transformed with a vector which comprises in the 5′ to 3′ direction: 
     a promoter; 
     a first DNA sequence encoding a delivery signal from a bacterial T3SS effector protein, operably linked to said promoter; and 
     a second DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins fused in frame to the 3′ end of said first DNA sequence, wherein the heterologous proteins are selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. 
     In a further aspect the present invention relates to a recombinant Gram-negative bacterial strain transformed with a vector which comprises in the 5′ to 3′ direction: a first DNA sequence encoding a delivery signal or a fragment thereof from a bacterial effector protein; and 
     a second DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins fused in frame to the 3′ end of said first DNA sequence, wherein the heterologous proteins are selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. 
     In a further aspect the present invention relates to a vector which comprises in the 5′ to 3′ direction: 
     a first DNA sequence encoding a delivery signal or a fragment thereof from a bacterial effector protein; and 
     a second DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins fused in frame to the 3′ end of said first DNA sequence, wherein the heterologous proteins are selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. 
     In a further aspect the present invention relates to a vector which comprises in the 5′ to 3′ direction: 
     a promoter; 
     a first DNA sequence encoding a delivery signal from a bacterial T3SS effector protein, operably linked to said promoter; 
     a second DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins fused in frame to the 3′ end of said first DNA sequence, 
     wherein the heterologous proteins are selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. 
     The present invention further relates to a method for delivering repeated domains of a heterologous protein or two or more domains of different heterologous proteins into a eukaryotic cell comprising the following steps: 
     i) culturing a Gram-negative bacterial strain; and 
     ii) contacting a eukaryotic cell with the Gram-negative bacterial strain of i) wherein a fusion protein which comprises a delivery signal from a bacterial T3SS effector protein and the repeated domains of a heterologous protein or two or more domains of different heterologous proteins is expressed by the Gram-negative bacterial strain and is translocated into the eukaryotic cell. 
     The present invention further relates to a method for delivering repeated domains of a heterologous protein or two or more domains of different heterologous proteins into a eukaryotic cell comprising the following steps: 
     i) culturing a Gram-negative bacterial strain; 
     ii) contacting a eukaryotic cell with the Gram-negative bacterial strain of i) wherein a fusion protein which comprises a delivery signal from a bacterial T3SS effector protein and the repeated domains of a heterologous protein or two or more domains of different heterologous proteins is expressed by the Gram-negative bacterial strain and is translocated into the eukaryotic cell; and 
     iii) cleaving the fusion protein so that the repeated domains of a heterologous protein or the two or more domains of different heterologous proteins are cleaved from the delivery signal from the bacterial T3SS effector protein. 
     In a further aspect the present invention relates to a library of Gram-negative bacterial strains, wherein the repeated domains of a heterologous protein or the two or more domains of different heterologous proteins encoded by the second DNA sequence of the expression vector of the Gram-negative bacterial strains are domains of a human or murine protein and, wherein each domain of a human or murine protein expressed by a Gram-negative bacterial strain is different in amino acid sequence. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 : Characterization of T3SS protein delivery. (A) Schematic representation of T3SS dependent protein secretion into the surrounding medium (in-vitro secretion)(left side) or into eukaryotic cells (right side). I: shows the type 3 secretion system. II indicates proteins secreted into the surrounding medium, III proteins translocated through the membrane into the cytosol of eukaryotic cells (VII). VI shows a stretch of the two bacterial membranes in which the T3SS is inserted and the bacterial cytosol underneath. IV is a fusion protein attached to the YopE 1-138  N-terminal fragment (V) (B) In-vitro secretion of I:  Y. enterocolitica  E40 wild type, II:  Y. enterocolitica  ΔHOPEMT asd or III:  Y. enterocolitica  ΔHOPEMT asd+pBadSi_2 as revealed by Western blotting on total bacterial lysates (IV) and precipitated culture supernatants (V) using an anti-YopE antibody. 
         FIG. 2 : Characterization of T3SS protein delivery into epithelial cells. (A) Anti-Myc immunofluorescence staining on HeLa cells infected at an MOI of 100 for 1 h with I:  Y. enterocolitica  ΔHOPEMT asd or II:  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2. (B) Quantification of anti-Myc immunofluorescence staining intensity from (A) within HeLa cells. Data were combined from n=20 sites, error bars indicated are standard error of the mean. I: uninfected, II:  Y. enterocolitica  ΔHOPEMT asd or III:  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2. Y-axis indicates anti-Myc staining intensity [arbitrary unit], x-axis indicates time of infection in minutes (C) Quantification of Anti-Myc immunofluorescence staining intensity within cells. HeLa cells were infected for 1 h with  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2 at an MOI indicated on the x-axis. Data were combined from n=20 sites, error bars indicated are standard error of the mean. Y-axis indicates anti-Myc staining intensity [a.u.]. 
         FIG. 3 : Modifications of the T3SS based protein delivery allow nuclear localization of a YopE 1-138  fusion protein (EGFP). EGFP signal in HeLa cells infected with I:  Y. enterocolitica  ΔHOPEMT asd or II:  Y. enterocolitica  ΔHOPEMT asd ΔyopB carrying the plasmids III: +YopE 1-138 -EGFP or IV: +YopE 1-138 -EGFP-NLS at an MOI of 100. EGFP signal is shown in “a”, for localization comparison nuclei were stained in “b”. 
         FIG. 4 : Modifications of the T3SS based protein delivery allow removal of the YopE 1-138  appendage. HeLa cells are infected with two different  Y. enterocolitica  strains at the same time, which is reached by simple mixing of the two bacterial suspensions. One strain is delivering the TEV protease fused to YopE 1-138 , while the other strain delivers a protein of interest fused to YopE 1-138  with a linker containing a double TEV protease cleavage site. After protein delivery into the eukaryotic cell, the TEV protease will cleave the YopE 1-138  appendage from the protein of interest (A) Digitonin lysed HeLa cells uninfected (II) or after infection (MOI of 100) for 2 h with I:  Y. enterocolitica  ΔHOPEMT asd and III: +pBadSi_2, IV: +YopE 1-138 -2×TEV cleavage site-Flag-INK4C, V: +YopE 1-138 -2×TEV cleavage site-Flag-INK4C and further overnight treatment with purified TEV protease and VI: +YopE 1-138 -2×TEV cleavage site-Flag-INK4C and a second strain+YopE 1-138 -TEV were analyzed by Western blotting anti-INK4C (shown in “a”) for the presence of YopE 1-138 -2×TEV cleavage site -Flag-INK4C or its cleaved form Flag-INK4C. As a loading control western blotting anti-Actin was performed (shown in “b”). In one case (V) the lysed cells were incubated overnight with purified TEV protease. (B) Actin normalized quantification of anti-INK4C staining intensity (shown as [a.u.] on the y-axis) from (A) at the size of full length YopE 1-138 -2×TEV cleavage site-Flag-INK4C, where sample IV is set to 100%. I:  Y. enterocolitica  ΔHOPEMT asd and IV: +YopE 1-138 -2×TEV cleavage site-Flag-INK4C, V: +YopE 1-138 -2×TEV cleavage site-Flag-INK4C and further overnight treatment with purified TEV protease and VI: +YopE 1-138 -2×TEV cleavage site-Flag-INK4C and a second strain+YopE 1-138 -TEV. Data were combined from n=2 independent experiments, error bars indicated are standard error of the mean (C) Digitonin lysed HeLa cells uninfected (II) or after infection (MOI of 100) for 2 h with I:  Y. enterocolitica  ΔHOPEMT asd and III: +pBadSi_2, IV: +YopE 1-138 -2×TEV cleavage site-ET1-Myc, V: +YopE 1-138 -2×TEV cleavage site-ET1-Myc and further overnight treatment with purified TEV protease and VI: +YopE 1-138 -2×TEV cleavage site-ET1-Myc and a second strain+YopE 1-138 -TEV were analyzed by Western blotting anti-Myc (shown in “a”) for the presence of YopE 1-138 -2×TEV cleavage site-ET1-Myc or its cleaved form ET1-Myc. As a loading control western blotting anti-Actin was performed (shown in “b”) In one case (V) the lysed cells were incubated overnight with purified TEV protease. 
         FIG. 5 : Delivery of bacterial effector proteins into eukaryotic cells (A) HeLa cells were infected with I:  Y. enterocolitica  ΔHOPEMT asd carrying II: pBad_Si2 or III: YopE 1-138 -SopE at an MOI of 100 for the time indicated above the images (2, 10 or 60 minutes). After fixation cells were stained for the actin cytoskeleton (B) HeLa cells were left uninfected (II) or infected with I:  Y. enterocolitica  ΔHOPEMT asd carrying III: YopE 1-138 -SopE-Myc and in some cases coinfected with IV: YopE 1-138 -SptP at the MOI indicated below the strain (MOI 50; MOI50:MOI50 or MOI50:MOI100) for 1 h. After fixation cells were stained for the actin cytoskeleton (shown in “a”) and the presence of the YopE 1-138 -SopE-Myc fusion protein was followed via staining anti-Myc (shown in “b”). 
         FIG. 6 : Delivery of bacterial effector proteins into eukaryotic cells (A) Phospho-p38 (“a”), total p38 (“b”) and actin (“c”) western blot analysis on HeLa cells left untreated (II) or infected for 75 min with I:  Y. enterocolitica  ΔHOPEMT asd carrying III: pBad_Si2 or IV: YopE 1-138 -OspF at an MOI of 100. Cells were stimulated with TNFα for the last 30 min of the infection as indicated (+ stands for addition of TNFα, − represent no treatment with TNFα) (B) Phospho-Akt T308 (“a”) and S473 (“b”) and actin (“c”) western blot analysis on HeLa cells left untreated (II) or infected for 22.5 or 45 min (indicated below the blots) with I:  Y. enterocolitica  ΔHOPEMT asd carrying III: pBad_Si2, IV: YopE 1-138 -SopE or V: YopE 1-138 -SopB at an MOI of 100 (C) cAMP levels (in fmol/well shown on y-axis) in HeLa cells left untreated (I) or infected for 2.5 h with V:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -BepA, VI:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -BepA E305-end , VII:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -BepG Bid  or VIII:  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2 at an MOI of 100. Cholera toxin (CT) was added for 1 h as positive control to samples II (1 μg/ml), III (25 μg/ml) or IV (50 μg/ml). Data were combined from n=3 independent experiments, error bars indicated are standard error of the mean. Statistical analysis was performed using an unpaired two-tailed t-test (ns indicates a non significant change, ** indicates a p value &lt;0.01, *** indicates a p value &lt;0.001). 
         FIG. 7 : Delivery of human tBid into eukaryotic cells induces massive apoptosis. (A) Cleaved Caspase 3 p17 (“a”) and actin (“b”) western blot analysis on HeLa cells left untreated (II) or infected for 60 min with I:  Y. enterocolitica  ΔHOPEMT asd carrying III: pBad_Si2, IV: YopE 1-138 -Bid or V: YopE 1-138 -t-Bid at an MOI of 100. In some cases, cells were treated with VI: 0.5 μM Staurosporine or VII: 1 μM Staurosporine (B) Digitonin lysed HeLa cells left untreated (II) or after infection for 1 h with I:  Y. enterocolitica  ΔHOPEMT asd carrying III: pBad_Si2, IV: YopE 1-138 -Bid or V: YopE 1-138 -t-Bid at an MOI of 100 were analyzed by Western blotting anti-Bid (“a”) allowing comparison of endogenous Bid levels (marked Z) to translocated YopE 1-138 -Bid (marked X) or YopE 1-138 -tBid (marked Y) levels. As a loading control western blotting anti-Actin was performed (shown in “b”). In some cases, cells were treated with VI: 0.5 μM Staurosporine or VII: 1 μM Staurosporine (C) HeLa cells were left untreated (I) or infected at an MOI of 100 for 1 h with II:  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2, III:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -Bid, IV:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -tBid. In some cases, cells were treated with V: 0.5 μM Staurosporine or VI: 1 μM Staurosporine. After fixation cells were stained for the actin cytoskeleton (gray). 
         FIG. 8 : T3SS dependent delivery of zebrafish BIM induces apoptosis in zebrafish embryos. (A) 2 dpf zebrafish embryos were infected with the EGFP expressing  Y. enterocolitica  ΔHOPEMT asd+pBad_Si1 control strain (I) or zBIM translocating strain (II:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -zBIM) by injection of about 400 bacteria into the hindbrain region. After 5.5 h the embryos were fixed, stained for activated Caspase 3 (cleaved Caspase 3, p17; shown in “c”) and analyzed for presence of bacteria (EGFP signal, shown in “b”). Maximum intensity z projections are shown for fluorescent images. Bright-field z projection are shown in “a” (B) Automated image analysis on maximum intensity z projections of recorded z-stack images of (A). Briefly, bacteria were detected via the GFP channel. Around each area of a bacterial spot a circle with a radius of 10 pixels was created. Overlapping regions were separated equally among the connecting members. In those areas closely surrounding bacteria, the Caspase 3 p17 staining intensity was measured and is plotted on the y-axis (as [a.u.]). Statistical analysis was performed using a Mann-Whitney test (*** indicates a p value &lt;0.001). Data were combined from n=14 for  Y. enterocolitica  ΔHOPEMT asd+pBad_Si1 control strain (I) or n=19 for II:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -zBIM infected animals, error bars indicated are standard error of the mean. 
         FIG. 9 : tBiD dependent phosphoproteome: HeLa cells were infected for 30 min with  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -t-Bid at an MOI of 100 and as a control with  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2. (A) Graphical representation of the tBID phosphoproteome. Proteins containing phosphopeptides that were significantly regulated in a tBid dependent manner (gray) (q-value &lt;0.01) as well as known apoptosis related proteins (dark gray) are represented in a STRING network of known and predicted protein-protein interactions (high-confidence, score 0.7). Only proteins with at least one connection in STRING are represented. (B) Confocal images of HeLa cells infected with either  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2 (I) or  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -t-Bid (II) reveal the induction of an apoptotic phenotype upon tBid delivery. Cells were stained for the nuclei with Hoechst (“a”), for F-actin with phalloidin (“b”), for tubulin with an anti-tubulin antibody (“c”) and for mitochondria with mitotracker (“d)”. Scale bar represents 40 μm. 
         FIG. 10 : Description of the type III secretion-based delivery toolbox. (A) Vector maps of the cloning plasmids pBad_Si1 and pBad_Si2 used to generate fusion constructs with YopE 1-138 . The chaperone SycE and the YopE 1-138 -fusion are under the native  Y. enterocolitica  promoter. The two plasmids only differ in presence of an arabinose inducible EGFP present on pBad_Si1 (B) Multiple cloning site directly following the yopE 1-138  fragment on pBad_Si1 and pBad_Si2 plasmids. 
         FIG. 11 : Characterization of T3SS protein delivery into various cell lines. Anti-Myc immunofluorescence staining on Swiss 3T3 fibroblasts (“a”), Jurkat cells (“b”) and HUVEC cells (“c”) left untreated (II) or infected with  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2 (I) at the MOI indicated above the images (MOI 25, 50, 100, 200 and 400 for HUVECs) for 1 h. 
         FIG. 12 : T3SS dependency of delivery of bacterial effector proteins into eukaryotic cell. Digitonin lysed HeLa cells after infection at an MOI of 100 for time indicated above the blots (0, 5, 15, 10, 60 and 120 minutes) with  Y. enterocolitica  ΔHOPEMT asd ΔyopB+YopE 1-138 -SopE-Myc (I) or  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -SopE-Myc (II) were analyzed by Western blotting anti-Myc. The size corresponding to YopE 1-138 -SopE-Myc is marked with “a”, while the size of the endogenous c-Myc protein is marked with “b”. 
         FIGS. 13 and 14 : T3SS dependent secretion of various other proteins into the culture supernatant. In-vitro secretion experiment of I:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138  fused to the protein as indicated. Protein content of total bacterial lysates (“A”) and precipitated culture supernatants (“B”) was analyzed by Western blotting using an anti-YopE antibody. Numbers written indicate molecular weight in kDa at the corresponding height. 
         FIGS. 15A  to N:  Y. enterocolitica  and  S. enterica  strains used in this study. 
       List of  Y. enterocolitica  and  S. enterica  strains used in this study providing information on background strains, plasmids and proteins for T3SS dependent delivery encoded on corresponding plasmids. Further, information on oligonucleotides used for construction of the corresponding plasmid, the backbone plasmid and antibiotic resistances is provided. 
         FIG. 16 : Delivery of murine tBid, murine Bid BH3 and murine Bax BH3 into B16F10 cells induces massive apoptosis. B16F10 cells uninfected (I) or after infection (MOI of 50) for 2.5 h with  Y. enterocolitica  ΔHOPEMT asd and II: +pBadSi_2, III: +YopE 1-138 - Y. enterocolitica  codon optimized murine tBid, IV: +YopE 1-138 - Y. enterocolitica  codon optimized murine Bid BH3 or V: +YopE 1-138 - Y enterocolitica  codon optimized murine Bax BH3. After fixation cells were stained for the actin cytoskeleton and nuclei (both in gray). 
         FIG. 17 : Delivery of murine tBid, murine Bid BH3 and murine Bax BH3 into D2A1 cells induces massive apoptosis. D2A1 cells uninfected (I) or after infection (MOI of 50) for 2.5 h with  Y. enterocolitica  ΔHOPEMT asd and II: +pBadSi_2, III: +YopE 1-138 - Y. enterocolitica  codon optimized murine tBid, IV: +YopE 1-138 - Y enterocolitica  codon optimized murine Bid BH3 or V: +YopE 1-138 - Y. enterocolitica  codon optimized murine Bax BH3. After fixation cells were stained for the actin cytoskeleton and nuclei (both in gray). 
         FIG. 18 : Delivery of murine tBid, murine Bid BH3 and murine Bax BH3 into HeLa cells induces massive apoptosis. HeLa cells uninfected (I) or after infection (MOI of 50) for 2.5 h with  Y. enterocolitica  ΔHOPEMT asd and II: +pBadSi_2, III: +YopE 1-138 - Y. enterocolitica  codon optimized murine tBid, IV: +YopE 1-138 - Y enterocolitica  codon optimized murine Bid BH3 or V: +YopE 1-138 - Y. enterocolitica  codon optimized murine Bax BH3. After fixation cells were stained for the actin cytoskeleton and nuclei (both in gray). 
         FIG. 19 : Delivery of murine tBid, murine Bid BH3 and murine Bax BH3 into 4T1 cells induces massive apoptosis. 4T1 cells uninfected (I) or after infection (MOI of 50) for 2.5 h with  Y. enterocolitica  ΔHOPEMT asd and II: +pBadSi_2, III: +YopE 1-138 - Y. enterocolitica  codon optimized murine tBid, IV: +YopE 1-138 - Y. enterocolitica  codon optimized murine Bid BH3 or V: +YopE 1-138 - Y. enterocolitica  codon optimized murine Bax BH3. After fixation cells were stained for the actin cytoskeleton and nuclei (both in gray). 
         FIG. 20 : Delivery of murine tBid by  S. enterica  grown under SPI-1 T3SS inducing conditions into eukaryotic cells induces apoptosis. Cleaved Caspase 3 p17 western blot analysis on HeLa cells left untreated (I) or infected for 4 h with III:  S. enterica  aroA carrying IV: SteA 1-20 -t-Bid, V: SteA FL -Bid, VI: SopE 1-81 -t-Bid or VII: SopE 1-105 -t-Bid at an MOI of 100. For this experiment, all  S. enterica  aroA strains were grown under SPI-1 T3SS inducing conditions. In some cases, cells were treated with II: 1 μM Staurosporine. Numbers written indicate molecular weight in kDa at the corresponding height. 
         FIG. 21 : Delivery of murine tBid by  S. enterica  grown under SPI-2 T3SS inducing conditions into eukaryotic cells induces apoptosis. Cleaved Caspase 3 p17 western blot analysis on HeLa cells left untreated (I) or infected for 4 h with III:  S. enterica  aroA carrying IV: SteA 1-20 -t-Bid, V: SteA FL -Bid, VI: SopE 1-81 -t-Bid or VII: SopE 1-105 -t-Bid at an MOI of 100. For this experiment, all  S. enterica  aroA strains were grown under SPI-2 T3SS inducing conditions. In some cases, cells were treated with II: 1 μM Staurosporine. Numbers written indicate molecular weight in kDa at the corresponding height. 
         FIG. 22 :  S. enterica  T3SS dependent secretion of various cell cycle proteins into the culture supernatant. In-vitro secretion experiment of  S. enterica  aroA+either SteA FL  (I, III, V, VII) or SopE 1-105  (II, IV, VI, VIII) fused to proteins as listed following. I and II: Ink4a-MycHis; III and IV: Ink4c-MycHis; V and VI: Mad2-MycHis; VII and VIII: Cdk1-MycHis. Protein content of precipitated culture supernatants (“A”) and total bacterial lysates (“B”) was analyzed by Western blotting using an anti-myc antibody. Numbers written indicate molecular weight in kDa at the corresponding height. 
         FIG. 23 : T3SS dependent secretion of various known cell cycle interfering peptides into the culture supernatant. In-vitro secretion experiment of I:  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2. II-VII:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138  fused to peptides as listed following: II: Ink4A 84-103 ; III: p107/RBL1 657-662 ; IV: p21 141-160D149A ; V: p21 145-160D149A ; VI: p2 117-33 ; VII: cyclin D2 139-147 . Protein content of precipitated culture supernatants (“A”) and total bacterial lysates (“B”) was analyzed by Western blotting using an anti-YopE antibody. Numbers written indicate molecular weight in kDa at the corresponding height. 
         FIG. 24 : Fusion of the T3SS delivered protein to Ubiquitin allows removal of the YopE 1-138  appendage. HeLa cells are infected with a strain delivering a protein of interest fused to YopE 1-138  with a directly fused Ubiquitin (YopE 1-138 -Ubi). After protein delivery into the eukaryotic cell, endogenous Ubiquitin specific proteases will cleave the YopE 1-138 -Ubi appendage from the protein of interest. Digitonin lysed HeLa cells uninfected (I) or after infection (MOI of 100) for 1 h with II:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -Flag-INK4C-MycHis or III: +YopE 1-138 -Flag-Ubiquitin-INK4C-MycHis were analyzed by Western blotting anti-INK4C for the presence of IV: YopE 1-138 -Flag-Ubiquitin-INK4C-MycHis or V: YopE 1-138 -Flag-INK4C-MycHis, the cleaved form VI: INK4C-MycHis and VII: the endogenous INK4C. 
         FIG. 25 : Schematic representation of heterologous proteins and domains thereof to be delivered via the bacteria T3SS. I: Human/murine full-length protein with domains colored in various grayscale, II: Truncated human/murine protein with domains colored in various grayscale, III: Motif/domain of human/murine full-length protein only, IV: Motif/domain only repeated (left) or combination of two different motifs/domains of human/murine full-length protein (right). 
         FIG. 26 : Delivery of BH3 domains of murine tBid and murine Bax into eukaryotic cells and fused repeats thereof induce apoptosis in cancerous cells. B16F10 murine melanoma cells were infected at a MOI of 5, 10, 25 and 50 (left to right in each condition) of corresponding bacteria as indicated for 4 h. Effect on cell viability was assessed by counting cell numbers via nuclear counting. I: nuclear count. II:  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2, III:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -Bid-BH3, IV:  Y enterocolitica  ΔHOPEMT asd+YopE 1-138 -(Bid-BH3) 2  and V:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -(Bid-BH3)-(Bax-BH3). Nuclei were stained with Hoechst. Images were acquired using an automated microscope and cell number was automatically determined using CellProfiler. 
         FIG. 27 : Delivery of BH3 domains of murine tBid and murine Bax into eukaryotic cells and fused repeats thereof induce apoptosis in cancerous cells. 4T1 murine breast cancer cells were infected at a MOI of 5, 10, 25 and 50 (left to right in each condition) of corresponding bacteria as indicated for 4 h. Effect on cell viability was assessed by counting cell numbers via nuclear counting. I: nuclear count. II:  Y. enterocolitica  ΔHOPEMT asd+pBad_Si2, III:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -Bid-BH3, IV:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -(Bid-BH3) 2  and V:  Y. enterocolitica  ΔHOPEMT asd+YopE 1-138 -(Bid-BH3)-(Bax-BH3). Nuclei were stained with Hoechst. Images were acquired using an automated microscope and cell number was automatically determined using CellProfiler. 
         FIG. 28 : Delivery of synthetic increased pro-apoptotic proteins. Delivery of single synthetic proteins consisting of single or tandem repeats of BH3 domains originating from pro-apoptotic proteins t-BID or BAX leads to enhanced apoptosis induction in 4T1 and B16F10 cancerous cells. 4T1 (I) or B16F10 (II) cells were infected with  Y. enterocolitica  ΔyopHOPEMT encoding on pBad-MycHisA IV: YopE 1-138 -tBID BH3 extended domain, V: YopE 1-138 -linker-tBID BH3, VI: YopE 1-138 -tBID BH3, VII: YopE 1-138 -(tBID BH3) 2 , VIII: YopE 1-138 -tBID BH3-BAX BH3 or IX: YopE 1-138 -BAX BH3-tBID BH3. A titration of the bacteria added to the cells (MOI) was performed for each strain, cell counts determined and IC50 calculated using non-linear regression. IC50 MOI is indicated (III). 
         FIG. 29 : Induction of apoptosis by pYV-encoded synthetic pro-apoptotic proteins. 
       Delivery of a single or a tandem repeat of BID BH3 domain encoded on the pYV leads to apoptosis induction in 4T1 and B16F10 cancerous cells. 4T1 (I) or B16F10 (II) cells were infected with  Y. enterocolitica  ΔHOPEMT+IV: pYV-YopE 1-138 -BH3-Bid, or V: +pYV-YopE 1-138 -(BH3-Bid) 2  or VI: with  Y. enterocolitica  ΔHOPEMT pBad-MycHisA-YopE 1-138 -(BH3-Bid) 2  for 3 hours. A titration of the bacteria added to the cells (MOI) was performed for each strain, cell counts determined and IC50 (III) calculated using non-linear regression. 
         FIG. 30 : Tumor colonization of i.v. injected  Y. enterocolitica  ΔyopH,O,P,E,M,T in the 4T1 breast cancer allograft model. Bacterial counts in tumors are indicated as colony forming units (CFU) per gram of tissue (III). Counts were assessed in tumors at day 8 (I) and 14 (II) post infection. Each dot represents an individual mouse. The horizontal dashed line indicates the detection limit. 
         FIG. 31 : Biodistribution of i.v. injected  Y. enterocolitica  ΔyopH,O,P,E,M,T in the 4T1 breast cancer allograft model. Bacterial counts in blood (I), spleen (II), liver (III), lung (IV) and tumor (V) are indicated as colony forming units (CFU) per gram of tissue or per ml of blood (VI). Counts were assessed at day 14 post infection. Each dot represents an individual mouse. The horizontal dashed line indicates the detection limit. * indicates a mouse with large metastases found on lung. 
         FIG. 32 : Delay of tumor progression in wildtype Balb/C mice allografted s.c. with 4T1 breast cancer cells. Wildtype Balb/C mice allografted s.c. with 4T1 breast cancer cells were i.v. injected with I: PBS or II: 1*10 7    Y. enterocolitica  dHOPEMT+pYV-YopE 1-138 (BH3-Bid) 2 , once the tumor had reached a size of 150-250 mm3. The day of the i.v. injection of bacteria was defined as day 0. Tumor volume was measured over the following days (III; day 0 to day 9 post i.v. injection of bacteria) with calipers. The relative tumor volume, normalized to the tumor volume at day 0, is indicated (IV) as mm 3 . The mean is indicated with symbols, error bars depicted show the standard error of the mean. Statistical significance is measured with a 2way ANOVA, * indicates p value &lt;0.05, ** a p value &lt;0.005. 
         FIG. 33 : Tumor progression in wildtype Balb/C mice allografted s.c. with 4T1 breast cancer cells. Wildtype Balb/C mice allografted s.c. with 4T1 breast cancer cells were i.v. injected with I: PBS or II: 1*10 7    Y. enterocolitica  dHOPEMT, once the tumor had reached a size of 150-250 mm3. The day of the i.v. injection of bacteria was defined as day 0. Tumor volume was measured over the following days (III; day 0 to day 9 post i.v. injection of bacteria) with calipers. The relative tumor volume, normalized to the tumor volume at day 0, is indicated (IV) as mm 3 . The mean is indicated with symbols, error bars depicted show the standard error of the mean. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention provides recombinant Gram-negative bacterial strains and the use thereof for delivery of repeated domains of a heterologous protein or two or more domains of different heterologous proteins into eukaryotic cells. 
     For the purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. 
     The term “Gram-negative bacterial strain” as used herein includes the following bacteria:  Aeromonas salmonicida, Aeromonas hydrophila, Aeromonas veronii, Anaeromyxobacter dehalogenans, Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei, Chlamydia muridarum, Chlamydia trachmoatis, Chlamydophila abortus, Chlamydophila pneumoniae, Chromobacterium violaceum, Citrobacter rodentium, Desulfovibrio vulgaris, Edwardsiella tarda, Endozoicomonas elysicola, Erwinia amylovora, Escherichia albertii, Escherichia coli, Lawsonia intracellularis, Mesorhizobium loti, Myxococcus xanthus, Pantoea agglomerans, Photobacterium damselae, Photorhabdus luminescens, Photorabdus temperate, Pseudoalteromonas spongiae, Pseudomonas aeruginosa, Pseudomonas plecoglossicida, Pseudomonas syringae, Ralstonia solanacearum, Rhizobium  sp,  Salmonella enterica  and other  Salmonella  sp,  Shigella flexneri  and other  Shigella  sp,  Sodalis glossinidius, Vibrio alginolyticus, Vibrio azureus, Vibrio campellii, Vibrio caribbenthicus, Vibrio harvey, Vibrio parahaemolyticus, Vibrio tasmaniensis, Vibrio tubiashii, Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas oryzae, Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis . Preferred Gram-negative bacterial strains of the invention are Gram-negative bacterial strains comprised by the family of Enterobacteriaceae and Pseudomonadaceae. The Gram-negative bacterial strain of the present invention is normally used for delivery of heterologous proteins by the bacterial T3SS into eukaryotic cells in vitro and/or in vivo, preferably in vivo. 
     The term “recombinant Gram-negative bacterial strain” used herein refers to a Gram-negative bacterial strain genetically transformed with a vector. A useful vector of the present invention is e.g an expression vector, a vector for chromosomal or virulence plasmid insertion or a DNA or RNA fragment for chromosomal or virulence plasmid insertion or modification. 
     The term “recombinant Gram-negative bacterial strains which are deficient in producing at least one T3SS functional effector protein” used herein refers to a recombinant Gram-negative bacterial strain in which at least one T3SS effector protein is mutated such that the resulting recombinant Gram-negative bacterial strain no longer produces a functional form of at least one T3SS effector protein i.e. that the expression of such effector gene is abolished so that the resulting recombinant Gram-negative bacterial strains does not produce any of the at least one T3SS effector protein or that the catalytic activity of the encoded effector protein is abolished so that the at least one T3SS effector protein produced does not have its catalytic activity. e.g. does not exercise its effector functions. For the purpose of delivering proteins, the secretion and translocation system of the recombinant Gram-negative bacterial strains which are deficient in producing at least one T3SS functional effector protein needs to be intact. The term “T3SS effector protein” or “bacterial T3SS effector protein” as used herein refers to proteins which are naturally injected by T3S systems into the cytosol of eukaryotic cells and to proteins which are naturally secreted by T3S systems that might e.g form the translocation pore into the eukaryotic membrane (including pore-forming tranlocators (as  Yersinia  YopB and YopD) and tip-proteins like  Yersinia  LcrV). Preferably proteins which are naturally injected by T3S systems into the cytosol of eukaryotic cells are used. These virulence factors will paralyze or reprogram the eukaryotic cell to the benefit of the pathogen. T3S effectors display a large repertoire of biochemical activities and modulate the function of crucial host regulatory molecules and include AvrA, AvrB, AvrBs2, AvrBS3, AvrBsT, AvrD, AvrD1, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, AvrRpm1, AvrRpt2, AvrXv3, CigR, EspF, EspG, EspH, EspZ, ExoS, ExoT, GogB, GtgA, GtgE, GALA family of proteins, HopAB2, HopAO1, HopI1, HopM1, HopN1, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, HopU1, HsvB, IcsB, IpaA, IpaB, IpaC, IpaH, IpaH7.8, IpaH9.8, IpgB1, IpgB2, IpgD, LcrV, Map, OspC1, OspE2, OspF, OspG, OspI, PipB, PipB2, PopB, PopP2, PthXo1, PthXo6, PthXo7, SifA, SifB, SipA/SspA, SipB, SipC/SspC, SipD/SspD, SlrP, SopA, SopB/SigD, SopD, SopE, SopE2, SpiC/SsaB, SptP, SpvB, SpvC, SrfH, SrfJ, Sse, SseB, SseC, SseD, SseF, SseG, SseI/SrfH, SseJ, SseK1, SseK2, SseK3, SseL, SspH1, SspH2, SteA, SteB, SteC, SteD, SteE, TccP2, Tir, VirA, VirPphA, VopF, XopD, YopB, YopD YopE, YopH, YopJ, YopM, YopO, YopP, YopT, YpkA. 
     The terms “Gram-negative bacterial strain deficient to produce an amino acid essential for growth” and “auxotroph mutant” are used herein interchangeably and refer to Gram-negative bacterial strains which can not grow in the absence of at least one exogenously provided essential amino acid or a precursor thereof. The amino acid the strain is deficient to produce is e.g. aspartate, meso-2,6-diaminopimelic acid, aromatic amino acids or leucine-arginine [8]. Such a strain can be generated by e.g. deletion of the aspartate-beta-semialdehyde dehydrogenase gene (Δasd). Such an auxotroph mutant cannot grow in absence of exogenous meso-2,6-diaminopimelic acid [9]. The mutation, e.g. deletion of the aspartate-beta-semialdehyde dehydrogenase gene is preferred herein for a Gram-negative bacterial strain deficient to produce an amino acid essential for growth of the present invention. 
     The term “Gram-negative bacterial strain deficient to produce adhesion proteins binding to the eukaryotic cell surface or extracellular matrix” refers to mutant Gram-negative bacterial strains which do not express at least one adhesion protein compared to the adhesion proteins expressed by the corresponding wild type strain. Adhesion proteins may include e.g. extended polymeric adhesion molecules like pili/fimbriae or non-fimbrial adhesins. Fimbrial adhesins include type-1 pili (such as  E. coli  Fim-pili with the FimH adhesin), P-pili (such as Pap-pili with the PapG adhesin from  E. coli ), type 4 pili (as pilin protein from e.g.  P. aeruginosa ) or curli (Csg proteins with the CsgA adhesin from  S. enterica ). Non-fimbrial adhesions include trimeric autotransporter adhesins such as YadA from  Y. enterocolitica , BpaA ( B. pseudomallei ), Hia ( H. influenzae ), BadA ( B. henselae ), NadA ( N. meningitidis ) or UspA1 ( M. catarrhalis ) as well as other autotransporter adhesins such as AIDA-1 ( E. coli ) as well as other adhesins/invasins such as InvA from  Y. enterocolitica  or Intimin ( E. coli ) or members of the Dr-family or Afa-family ( E. coli ). The terms YadA and InvA as used herein refer to proteins from  Y. enterocolitica . The autotransporter YadA [10,11] binds to different froms of collagen as well as fibronectin, while the invasin InvA [12-14] binds to β-integrins in the eukaryotic cell membrane. If the Gram-negative bacterial strain is a  Y. enterocolitica  strain the strain is preferably deficient in InvA and/or YadA. 
     As used herein, the term “family of Enterobacteriaceae” comprises a family of gram-negative, rod-shaped, facultatively anaerobic bacteria found in soil, water, plants, and animals, which frequently occur as pathogens in vertebrates. The bacteria of this family share a similar physiology and demonstrate a conservation within functional elements and genes of the respective genomes. As well as being oxidase negative, all members of this family are glucose fermenters and most are nitrate reducers. Enterobacteriaceae bacteria of the invention may be any bacteria from that family, and specifically includes, but is not limited to, bacteria of the following genera:  Escherichia, Shigella, Edwardsiella, Salmonella, Citrobacter, Klebsiella, Enterobacter, Serratia, Proteus, Erwinia, Morganella, Providencia , or  Yersinia . In more specific embodiments, the bacterium is of the  Escherichia coli, Escherichia blattae, Escherichia fergusonii, Escherichia hermanii, Escherichia vuneris, Salmonella enterica, Salmonella bongori, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Enterobacter aerogenes, Enterobacter gergoviae, Enterobacter sakazakii, Enterobacter cloacae, Enterobacter agglomerans, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Yersinia pseudotuberculosis, Yersinia pestis, Yersinia enterocolitica, Erwinia amylovora, Proteus mirabilis, Proteus vulgaris, Proteus penneri, Proteus hauseri, Providencia alcalifaciens , or  Morganella morganii  species. 
     Preferably the Gram-negative bacterial strain is selected from the group consisting of the genera  Yersinia, Escherichia, Salmonella, Shigella, Pseudomonas, Chlamydia, Erwinia, Pantoea, Vibrio, Burkholderia, Ralstonia, Xanthomonas, Chromobacterium, Sodalis, Citrobacter, Edwardsiella, Rhizobiae, Aeromonas, Photorhabdus, Bordetella  and  Desulfovibrio , more preferably from the group consisting of the genera  Yersinia, Escherichia, Salmonella , and  Pseudomonas , most preferably from the group consisting of the genera  Yersinia  and  Salmonella.    
     The term “ Yersinia ” as used herein includes all species of  Yersinia , including  Yersinia enterocolitica, Yersinia pseudotuberculosis  and  Yersinia pestis . Preferred is  Yersinia enterocolitica.    
     The term “ Salmonella ” as used herein includes all species of  Salmonella , including  Salmonella enterica  and  S. bongori . Preferred is  Salmonella enterica.    
     “Promoter” as used herein refers to a nucleic acid sequence that regulates expression of a transcriptional unit. A “promoter region” is a regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. Within the promoter region will be found a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase such as the putative −35 region and the Pribnow box. The term “operably linked” when describing the relationship between two DNA regions simply means that they are functionally related to each other and they are located on the same nucleic acid fragment. A promoter is operably linked to a structural gene if it controls the transcription of the gene and it is located on the same nucleic acid fragment as the gene. Usually the promoter is functional in said Gram-negative bacterial strain, i.e. the promoter is capable of expressing the fusion protein of the present invention, i.e. the promoter is capable of expressing the fusion protein of the present invention without further genetic engineering or expression of further proteins. Furthermore, a functional promoter must not be naturally counter-regulated to the bacterial T3SS. 
     The term “delivery” used herein refers to the transportation of a protein from a recombinant Gram-negative bacterial strain to a eukaryotic cell, including the steps of expressing the heterologous protein in the recombinant Gram-negative bacterial strain, secreting the expressed protein(s) from such Gram-negative bacterial strain and translocating the secreted protein(s) by such Gram-negative bacterial strain into the cytosol of the eukaryotic cell. Accordingly, the terms “delivery signal” or “secretion signal” which are used interchangeably herein refer to a polypeptide sequence which can be recognized by the secretion and translocation system of the Gram-negative bacterial strain and directs the delivery of a protein from the Gram-negative bacterial strain to eukaryotic cells. 
     The term “delivery signal from a bacterial effector protein” used herein refers to a delivery signal from a bacterial effector protein functional in the recombinant Gram-negative bacterial strain, i.e. which allows an expressed heterologous protein in the recombinant Gram-negative bacterial strain to be secreted from such recombinant Gram-negative bacterial strain by a secretion system such as e.g. the type III secretion system or to be translocated by such recombinant Gram-negative bacterial strain into the cytosol of a eukaryotic cell by a secretion system such as e.g. the type III secretion system. The term “delivery signal from a bacterial effector protein” used herein also comprises a fragment of a delivery signal from a bacterial effector protein i.e. shorter versions of a delivery signal e.g. a delivery signal comprising up to 10, preferably up to 20, more preferably up to 50, even more preferably up to 100, in particular up to 140 amino acids of a delivery signal e.g. of a naturally occurring delivery signal. Thus a nucleotide sequence such as e.g. a DNA sequence encoding a delivery signal from a bacterial effector protein may encode a full length delivery signal or a fragment thereof wherein the fragment usually comprises usually up to 30, preferably up to 60, more preferably up to 150, even more preferably up to 300, in particular up to 420 nucleic acids. 
     As used herein, the “secretion” of a protein refers to the transportation of a heterologous protein outward across the cell membrane of a recombinant Gram-negative bacterial strain. The “translocation” of a protein refers to the transportation of a heterologous protein from a recombinant Gram-negative bacterial strain across the plasma membrane of a eukaryotic cell into the cytosol of such eukaryotic cell. 
     The term “eukaryotic cells” as used herein includes e.g. the following eukaryotic cells: Hi-5, HeLa, Hek, HUVECs, 3T3, CHO, Jurkat, Sf-9, HepG2, Vero, MDCK, Mefs, THP-1, J774, RAW, Caco2, NCI60, DU145, Lncap, MCF-7, MDA-MB-438, PC3, T47D, A549, U87, SHSY5Y, Ea.Hy926, Saos-2, 4T1, D2A1, B16F10, and primary human hepatocytes. “Eukaryotic cells” as used herein, are also referred to as “target cells” or “target eukaryotic cells”. 
     The term “T3SS effector protein” as used herein refers to proteins which are naturally injected by T3S systems into the cytosol of eukaryotic cells and to proteins which are naturally secreted by T3S systems that might e.g form the translocation pore into the eukaryotic membrane (including pore-forming tranlocators (as  Yersinia  YopB and YopD) and tip-proteins like  Yersinia  LcrV). Preferably proteins which are naturally injected by T3S systems into the cytosol of eukaryotic cells are used. These virulence factors will paralyze or reprogram the eukaryotic cell to the benefit of the pathogen. T3S effectors display a large repertoire of biochemical activities and modulate the function of crucial host regulatory molecules [2,15] and include AvrA, AvrB, AvrBs2, AvrBS3, AvrBsT, AvrD, AvrD1, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, AvrRpm1, AvrRpt2, AvrXv3, CigR, EspF, EspG, EspH, EspZ, ExoS, ExoT, GogB, GtgA, GtgE, GALA family of proteins, HopAB2, HopAO1, HopI1, HopM1, HopN1, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, HopU1, HsvB, IcsB, IpaA, IpaB, IpaC, IpaH, IpaH7.8, IpaH9.8, IpgB1, IpgB2, IpgD, LcrV, Map, OspC1, OspE2, OspF, OspG, OspI, PipB, PipB2, PopB, PopP2, PthXo1, PthXo6, PthXo7, SifA, SifB, SipA/SspA, SipB, SipC/SspC, SipD/SspD, SlrP, SopA, SopB/SigD, SopD, SopE, SopE2, SpiC/SsaB, SptP, SpvB, SpvC, SrfH, SrfJ, Sse, SseB, SseC, SseD, SseF, SseG, SseI/SrfH, SseJ, SseK1, SseK2, SseK3, SseL, SspH1, SspH2, SteA, SteB, SteC, SteD, SteE, TccP2, Tir, VirA, VirPphA, VopF, XopD, YopB, YopD YopE, YopH, YopJ, YopM, YopO, YopP, YopT, YpkA. 
     T3SS effector genes of  Yersinia  have been cloned from e.g.  Y. enterocolitica  which are YopE, YopH, YopM, YopO, YopP/YopJ, and YopT [16]. The respective effector genes can be cloned from  Shigella flexneri  (e.g. OspF, IpgD, IpgB1),  Salmonella enterica  (e.g. SopE, SopB, SptP),  P. aeruginosa  (e.g ExoS, ExoT, ExoU, ExoY) or  E. coli  (e.g. Tir, Map, EspF, EspG, EspH, EspZ). The nucleic acid sequences of these genes are available to those skilled in the art, e.g., in the Genebank Database (yopH, yopO, yopE, yopP, yopM, yopT from NC_002120 GI:10955536;  S. flexneri  effector proteins from AF386526.1 GI: 18462515;  S. enterica  effectors from NC_016810.1 GI:378697983 or FQ312003.1 GI:301156631;  P. aeruginosa  effectors from AE004091.2 GI:110227054 or CP000438.1 GI:115583796 and  E. coli  effector proteins from NC_011601.1 GI:215485161). 
     For the purpose of the present invention, genes are denoted by letters of lower case and italicised to be distinguished from proteins. In case the genes (denoted by letters of lower case and italicised) are following a bacterial species name (like  E. coli ), they refer to a mutation of the corresponding gene in the corresponding bacterial species. For example, YopE refers to the effector protein encoded by the yopE gene.  Y. enterocolitica  yopE represents a  Y. enterocolitica  having a mutation in the yopE gene. 
     As used herein, the terms “polypeptide”, “peptide”, “protein”, “polypeptidic” and “peptidic” are used interchangeably to designate a series of amino acid residues connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. Preferred are proteins which have an amino acid sequence comprising at least 10 amino acids, more preferably at least 20 amino acids. 
     According to the present invention, “a domain of a heterologous protein” includes domains of naturally occurring proteins and also includes domains of artificially engineered proteins. As used herein, the term “domain of a heterologous protein” refers to a domain of a heterologous protein other than a domain of a T3SS effector protein or a domain other than a domain comprising the N-terminal fragment thereof to which it can be fused to achieve a fusion protein. In particular the domain of a heterologous protein as used herein refers to a domain of a heterologous protein, which do not belong to the proteome, i.e. the entire natural protein complement of the specific recombinant Gram-negative bacterial strain provided and used by the invention, e.g. which do not belong to the proteome, i.e. the entire natural protein complement of a specific bacterial strain of the genera  Yersinia, Escherichia, Salmonella  or  Pseudomonas . Usually the domain of the heterologous protein is of animal origin including human origin. Preferably the domain of the heterologous protein is a domain of a human protein. More preferably the domain of the heterologous protein is a domain of a protein selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. Particular preferably the domain of the heterologous protein is a domain of a protein selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, reporter proteins, small GTPases, GPCR related proteins, nanobody fusion constructs, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. Even more particular preferred are domains of heterologous proteins selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, and ankyrin repeat proteins. Most preferred are domains of proteins involved in apoptosis or apoptosis regulation, like animal proteins involved in apoptosis or apoptosis regulation, preferably domains of human heterologous proteins involved in apoptosis or apoptosis regulation. 
     The term “repeated domains of a heterologous protein” as used herein refers to a fusion protein consisting of several repetitions of a domain of a heterologous protein, where these domains might either be directly fused to each other or where a variable linker e.g. a linker between 1 and 30, preferably between 2 and 15, more preferably between 3 and 10 amino acids might be introduced in between the domains. Preferably repeated identical domains or repeated domains which have an amino acid sequence identity of more than 80%, usually more than 85%, preferably more than 90%, even more preferably more than 95%, in particular more than 96%, more particular more than 97%, even more particular more than 98%, most particular more than 99% are used. Also preferred are identical domains which have an amino acid identity of 100%. Preferably two repeated domains, more preferably two repeated identical domains or two repeated domains having an amino acid sequence identity of more than 90%, preferably more than 95% most preferably 100% are comprised by the fusion protein as referred herein. More than two, e.g. three, four, five or six repeated domains are also contemplated by the present invention. 
     The term “two or more domains of different heterologous proteins” as used herein refers to a fusion protein consisting of one or several repetitions of at least two domains of different heterologous proteins e.g. at least two domains of heterologous proteins having an amino acid sequence identity of 80% or less, where these different domains might either be directly fused to each other or where a variable linker e.g. a linker between 1 and 30, preferably between 2 and 15, more preferably between 3 and 10 amino acids might be introduced in between the domains. 
     Preferably two domains of different heterologous proteins are comprised by the fusion protein as referred herein. More than two, e.g. three, four, five or six domains of different heterologous proteins are also contemplated by the present invention. 
     The term “heterologous proteins which belong to the same functional class of proteins” as used herein refers to heterologous proteins which have the same function e.g. heterologous proteins having enzymatic activity, heterologous proteins which act in the same pathway such as e.g. cell cycle regulation, or share a common specific feature as e.g. belonging to the same class of bacterial effector proteins. Functional classes of proteins are e.g. proteins involved in apoptosis or apoptosis regulation, proteins which act as cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors or viral proteins which act jointly in the biological process of establishing virulence to eukaryotic cells. 
     The domain of a heterologous protein expressed by the recombinant Gram-negative bacterial strain has usually a molecular weight of between 1-50 kDa, preferably between 1-30 kDa, more preferably between 1-20 kDa, most preferably between 1-10 kDa. 
     According to the present invention “proteins involved in apoptosis or apoptosis regulation” or “human heterologous proteins involved in apoptosis or apoptosis regulation” include, but are not limited to, Bad, Bcl2, Bak, Bmt, Bax, Puma, Noxa, Bim, Bcl-xL, Apaf1, Caspase 9, Caspase 3, Caspase 6, Caspase 7, Caspase 10, DFFA, DFFB, ROCK1, APP, CAD, ICAD, CAD, EndoG, AIF, HtrA2, Smac/Diablo, Arts, ATM, ATR, Bok/Mtd, Bmf, Mcl-1(S), IAP family, LC8, PP2B, 14-3-3 proteins, PKA, PKC, PI3K, Erkl/2, p90RSK, TRAF2, TRADD, FADD, Daxx, Caspase8, Caspase2, RIP, RAIDD, MKK7, JNK, FLIPs, FKHR, GSK3, CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)), and the Cip1/Waf1/Kip1-2-family (p21 (Cip1/Waf1), p27(Kip1), p57(Kip2). Preferably Bad, Bmt, Bcl2, Bak, Bax, Puma, Noxa, Bim, Bcl-xL, Caspase9, Caspase3, Caspase6, Caspase7, Smac/Diablo, Bok/Mtd, Bmf, Mcl-1(S), LC8, PP2B, TRADD, Daxx, Caspase8, Caspase2, RIP, RAIDD, FKHR, CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)), most preferably BIM, Bid, truncated Bid, FADD, Caspase 3 (and subunits thereof), Bax, Bad, Akt, CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)) are used [17-19]. Additionally proteins involved in apoptosis or apoptosis regulation include DIVA, Bcl-Xs, Nbk/Bik, Hrk/Dp5, Bid and tBid, Egl-1, Bcl-Gs, Cytochrome C, Beclin, CED-13, BNIP1, BNIP3, Bcl-B, Bcl-W, Ced-9, A1, NR13, Bfl-1, Caspase 1, Caspase 2, Caspase 4, Caspase 5, Caspase 8. 
     Proteins involved in apoptosis or apoptosis regulation are selected from the group consisting of pro-apoptotic proteins, anti-apoptotic proteins, inhibitors of apoptosis-prevention pathways and inhibitors of pro-survival signalling or pathways. Pro-apoptotic proteins comprise proteins selected form the group consisting of Bax, Bak, Diva, Bcl-Xs, Nbk/Bik, Hrk/Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Apaf1, Smac/Diablo, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13, Cytochrome C, FADD, the Caspase family, and CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)) or selected from the group consisting of Bax, Bak, Diva, Bcl-Xs, Nbk/Bik, Hrk/Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Egl-1, Apaf1, Smac/Diablo, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13, Cytochrome C, FADD, and the Caspase family. Preferred are Bax, Bak, Diva, Bcl-Xs, Nbk/Bik, Hrk/Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Egl-1, Apaf1, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13, Smac/Diablo, FADD, the Caspase family, CDKs and their inhibitors like the INK4-family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)). Equally preferred are Bax, Bak, Diva, Bcl-Xs, Nbk/Bik, Hrk/Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Apaf1, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13, Smac/Diablo, FADD, the Caspase family. 
     Anti-apoptotic proteins comprise proteins selected form the group consisting of Bcl-2, Bcl-Xl, Bcl-B, Bcl-W, Mcl-1, Ced-9, A1, NR13, IAP family and Bfl-1. Preferred are Bcl-2, Bcl-Xl, Bcl-B, Bcl-W, Mcl-1, Ced-9, A1, NR13 and Bfl-1. 
     Inhibitors of apoptosis-prevention pathways comprise proteins selected form the group consisting of Bad, Noxa and Cdc25A. Preferred are Bad and Noxa. Inhibitors of pro-survival signalling or pathways comprise proteins selected form the group consisting of PTEN, ROCK, PP2A, PHLPP, JNK, p38. Preferred are PTEN, ROCK, PP2A and PHLPP. 
     In some embodiments the heterologous proteins involved in apoptosis or apoptosis regulation are selected from the group consisting of BH3-only proteins, caspases and intracellular signalling proteins of death receptor control of apoptosis. 
     BH3-only proteins comprise proteins selected form the group consisting of Bad, BIM, Bid and tBid, Puma, Bik/Nbk, Bod, Hrk/Dp5, BNIP1, BNIP3, Bmf, Noxa, Mcl-1, Bcl-Gs, Beclin 1, Egl-1 and CED-13. Preferred are Bad, BIM, Bid and tBid. Caspases comprise proteins selected form the group consisting of Caspase 1, Caspase 2, Caspase 3, Caspase 4, Caspase 5, Caspase 6, Caspase 7, Caspase 8, Caspase 9, Caspase 10. Preferred are Caspase 3, Caspase 8 and Caspase 9. 
     Intracellular signalling proteins of death receptor control of apoptosis comprise proteins selected form the group consisting of FADD, TRADD, ASC, BAP31, GULP1/CED-6, CIDEA, MFG-E8, CIDEC, RIPK1/RIP1, CRADD, RIPK3/RIP3, Crk, SHB, CrkL, DAXX, the 14-3-3 family, FLIP, DFF40 and 45, PEA-15, SODD. Preferred are FADD and TRADD. 
     In some embodiments two domains of a heterologous proteins involved in apoptosis or apoptosis regulation are comprised by the Gram-negative bacterial strain and/or the vector of the present invention, preferably two repeated, more preferably two identical repeated domains of a protein involved in apoptosis or apoptosis regulation or two domains of different proteins involved in apoptosis or apoptosis regulation. In some embodiments two domains of a heterologous proteins involved in apoptosis or apoptosis regulation are comprised by the Gram-negative bacterial strain and/or the vector of the present invention, wherein one is a domain of a pro-apoptotic protein and the other is a domain of a protein which is an inhibitor of apoptosis-prevention pathways or wherein one is a domain of a pro-apoptotic protein and the other domain is a domain of a protein which is an inhibitor of pro-survival signalling or pathways. 
     Pro-apoptotic proteins encompassed by the present invention have usually an alpha helical structure, preferably a hydrophobic helix surrounded by amphipathic helices and usually comprise at least one of BH1, BH2, BH3 or BH4 domains, preferably comprise at least one BH3 domain. Usually pro-apoptotic proteins encompassed by the present invention have no enzymatic activity. 
     Anti-apoptotic proteins encompassed by the present invention have usually an alpha helical structure, preferably a hydrophobic helix surrounded by amphipathic helices and comprises a combination of different BH1, BH2, BH3 and BH4 domains, preferably a combination of different BH1, BH2, BH3 and BH4 domains wherein a BH1 and a BH2 domain is present, more preferably BH4-BH3-BH1-BH2, BH1-BH2, BH4-BH1-BH2 or BH3-BH1-BH2 (from N- to the C-terminus). Additionally, proteins containing at least one BIR domain are also encompassed. 
     Inhibitors of apoptosis-prevention pathways encompassed by the present invention have usually an alpha helical structure, preferably a hydrophobic helix surrounded by amphipathic helices and usually comprise one BH3 domain. 
     BH1, BH2, BH3 or BH4 domains are each usually between about 5 to about 50 amino acids in length. Thus in some embodiments the domains of heterologous proteins are selected from the group consisting of domains of heterologous proteins which are about 5 to about 200, preferably about 5 to about 150, more preferably about 5 to about 100, most preferably about 5 to about 50, in particular about 5 to about 25 amino acids in length. 
     A particular preferred domain is the BH3 domain of apoptosis inducer tBID, more particular the BH3 domain comprising a sequence selected from the group consisting of SEQ ID NOs: 209, 210, 211 and 212, preferably SEQ ID NO: 211 or SEQ ID NO: 212. Equally preferred is the BH3 domain of apoptosis regulator BAX, more particular the BAX domain comprising a sequence selected from the group consisting of SEQ ID NOs: 213, 214, 215 and 216, preferably SEQ ID NO: 215 or SEQ ID NO: 216. The human and murine sequences are given in SEQ ID NOs 209-216, but tBID and BAX BH3 domains of all other species are equally included. 
     In some embodiments the repeated domains of the heterologous proteins are the BH3 domain, in particular repeated BH3 domains of apoptosis inducer tBID, more particular two repeated BH3 domains of apoptosis inducer tBID, most particular two repeated BH3 domains of apoptosis inducer tBID comprised by the sequence of SEQ ID NO: 202. Thus in a preferred embodiment the vector of the Gram-negative bacterial strain of the present invention comprises a second DNA sequence encoding two repeated domains of a BH3 domain, more preferably two repeated BH3 domains of apoptosis inducer tBID. The two repeated domains are preferably connected by a linker of 1-30 amino acid length, preferably 2-15 amino acids, more preferred 3-10 amino acids long. 
     In some embodiments the two or more domains of different heterologous proteins are domains of heterologous proteins which belong to the same functional class of proteins, preferably the different heterologous proteins of the two or more domains are different heterologous proteins from the class of proteins involved in apoptosis or apoptosis regulation. In a preferred embodiment the two or more domains of different heterologous proteins are the BH3 domain of apoptosis inducer tBID and the BH3 domain of apoptosis regulator BAX, in particular the fused BH3 domains comprised by the sequence of SEQ ID NO: 203. The two domains of different heterologous proteins are preferably connected by a linker of 1-30 amino acid length, preferably 2-15 amino acids, more preferred 3-10 amino acids long. 
     In some embodiments the heterologous proteins is a pro-drug converting enzyme. In these embodiments the recombinant virulence attenuated Gram-negative bacterial strain expresses, preferably expresses and secretes a pro-drug converting enzyme. A prodrug converting enzyme as referred herein comprises enzymes converting non-toxic prodrugs into a toxic drug, preferably enzymes selected from the group consisting of cytosine deaminase, purine nucleoside phosphorylase, thymidine kinase, beta-galactosidase, carboxylesterases, nitroreductase, carboxypeptidases and beta-glucuronidases, more preferably enzymes selected from the group consisting of cytosine deaminase, purine nucleoside phosphorylase, thymidine kinase, and beta-galactosidase. 
     The term “protease cleavage site” as used herein refers to a specific amino acid motif within an amino acid sequence e.g. within an amino acid sequence of a protein or a fusion protein, which is cleaved by a specific protease, which recognizes the amino acid motif. For review see [20]. Examples of protease cleavage sites are amino acid motifs, which are cleaved by a protease selected from the group consisting of enterokinase (light chain), enteropeptidase, prescission protease, human rhinovirus protease (HRV 3C), TEV protease, TVMV protease, FactorXa protease and thrombin. 
     The following amino acid motif is recognized by the respective protease:
         Asp-Asp-Asp-Asp-Lys: Enterokinase (light chain)/Enteropeptidase   Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro: PreScission Protease/human Rhinovirus protease (HRV 3C)   Glu-Asn-Leu-Tyr-Phe-Gln-Ser and modified motifs based on the Glu-X-X-Tyr-X-Gln-Gly/Ser (where X is any amino acid) recognized by TEV protease (tobacco etch virus)   Glu-Thr-Val-Arg-Phe-Gln-Ser: TVMV protease   Ile-(Glu or Asp)-Gly-Arg: FactorXa protease   Leu-Val-Pro-Arg/Gly-Ser: Thrombin.       

     Encompassed by the protease cleavage sites as used herein is ubiquitin. Thus in some preferred embodiments ubiquitin is used as protease cleavage site, i.e. the third DNA sequence encodes ubiquitin as protease cleavage site, which can be cleaved by a specific ubiquitin processing proteases at the N-terminal site, e.g. which can be cleaved by a specific ubiquitin processing proteases called Deubiquitinating enzymes at the N-terminal site endogenously in the cell where the fusion protein has been delivered to. Ubiquitin is processed at its C-terminus by a group of endogenous Ubiquitin-specific C-terminal proteases (Deubiquitinating enzymes, DUBs). The cleavage of Ubiquitin by DUBs is supposed to happen at the very C-terminus of Ubiquitin (after G76). 
     An “individual,” “subject” or “patient” is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including human and non-human primates) and rodents (e.g., mice and rats). In certain embodiments, a mammal is a human. 
     The term “mutation” is used herein as a general term and includes changes of both single base pair and multiple base pairs. Such mutations may include substitutions, frame-shift mutations, deletions, insertions and truncations. 
     The term “labelling molecule or an acceptor site for a labelling molecule” as used herein refers to a small chemical compound binding to a specific amino acid sequence resulting in fluorescence of the bound chemical compound, preferably coumarine ligase/coumarine acceptor site (and derivates thereof), resorufin ligase/resorufin acceptor site (and derivates thereof) and the tetra-Cysteine motif (as Cys-Cys-Pro-Gly-Cys-Cys and derivates thereof) in use with F1AsH/ReAsH dye (life technologies) or a fluorescent protein as Enhanced Green Fluorescent Protein (EGFP). 
     The term “nuclear localization signal” as used herein refers to an amino acid sequence that marks a protein for import into the nucleus of a eukaryotic cell and includes preferably a viral nuclear localization signal such as the SV40 large T-antigen derived NLS (PPKKKRKV). 
     The term “multiple cloning site” as used herein refers to a short DNA sequence containing several restriction sites for cleavage by restriction endonucleases such as AclI, HindIII, SspI, MluCI, Tsp509I, PciI, AgeI, BspMI, BfuAI, SexAI, MluI, BceAI, HpyCH4IV, HpyCH4III, BaeI, BsaXI, AflIII, SpeI, BsrI, BmrI, BglII, AfeI, AluI, StuI, ScaI, ClaI, BspDI, PI-SceI, NsiI, AseI, SwaI, CspCI, MfeI, BssSI, BmgBI, PmlI, DraIII, AleI, EcoP 151, PvuII, AlwNI, BtsIMutI, TspRI, NdeI, NlaIII, CviAII, FatI, MslI, FspEI, XcmI, BstXI, PflMI, BccI, NcoI, BseYI, FauI, SmaI, XmaI, TspMI, Nt.CviPII, LpnPI, AciI, SacII, BsrBI, MspI, HpaII, ScrFI, BssKI, StyD4I, BsaJI, BslI, BtgI, NciI, AvrII, MnlI, BbvCI, Nb.BbvCI, Nt.BbvCI, SbfI, Bpu10I, Bsu36I, EcoNI, HpyAV, BstNI, PspGI, StyI, BcgI, PvuI, BstUI, EagI, RsrII, BsiEI, BsiWI, BsmBI, Hpy99I, MspAlI, MspJI, SgrAI, BfaI, BspCNI, XhoI, EarI, AcuI, PstI, BpmI, DdeI, SfcI, AflII, BpuEI, SmlI, AvaI, BsoBI, MboII, BbsI, XmnI, BsmI, Nb.BsmI, EcoRI, HgaI, AatII, ZraI, Tth111I PflFI, PshAI, AhdI, DrdI, Eco53kI, SacI, BseRI, PleI, Nt.BstNBI, MlyI, HinfI, EcoRV, MboI, Sau3AI, DpnII BfuCI, DpnI, BsaBI, TfiI, BsrDI, Nb.BsrDI, BbvI, BtsI, Nb.BtsI, BstAPI, SfaNI, SphI, NmeAIII, NaeI, NgoMIV, BglI, AsiSI, BtgZI, HinPI, HhaI, BssHII, NotI, Fnu4HI, Cac8I, MwoI, NheI, BmtI, SapI, BspQI, Nt.BspQI, BlpI, TseI, ApeKI, Bsp1286I, AlwI, Nt.AlwI, BamHI, FokI, BtsCI, HaeIII, PhoI, FseI, SfiI, NarI, KasI, SfoI, PluTI, AscI, EciI, BsmFI, ApaI, PspOMI, Sau961, NlaIV, KpnI, Acc65I, BsaI, HphI, BstEII, AvaII, BanI, BaeGI, BsaHI, BanII, RsaI, CviQI, BstZ17I, BciVI, SalI, Nt.BsmAI, BsmAI, BcoDI, ApaLI, BsgI, AccI, Hpyl66II, Tsp45I, HpaI, PmeI, HincII, BsiHKAI, ApoI, NspI, BsrFI, BstYI, HaeII, CviKI-1, EcoO109I, PpuMI, I-CeuI, SnaBI, I-SceI, BspHI, BspEI, MmeI, TaqαI, NruI, Hpy188I, Hpyl88III, XbaI, BclI, HpyCH4V, FspI, PI-PspI, MscI, BsrGI, MseI, PacI, PsiI, BstBI, DraI, PspXI, BsaWI, BsaAI, EaeI, preferably XhoI, XbaI, HindIII, NcoI, NotI, EcoRI, EcoRV, BamHI, NheI, SacI, SalI, BstBI. The term “multiple cloning site” as used herein further refers to a short DNA sequence used for recombination events as e.g in Gateway cloning strategy or for methods such as Gibbson assembly or topo cloning. 
     The term “ Yersinia  wild type strain” as used herein refers to a naturally occurring variant (as  Y. enterocolitica  E40) or a naturally occurring variant containing genetic modifications allowing the use of vectors, such as deletion mutations in restriction endonucleases or antibiotic resistance genes (as  Y. enterocolitica  MRS40, the Ampicillin sensitive derivate of  Y. enterocolitica  E40) These strains contain chromosomal DNA as well as an unmodified virulence plasmid (called pYV). 
     The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. 
     The term “about” refers to a range of values+10% of a specified value. For example, the phrase “about 200” includes ±10% of 200, or from 180 to 220. 
     In one embodiment of the present invention the recombinant Gram-negative bacterial strain is transformed with a vector which comprises in the 5′ to 3′ direction: a first DNA sequence encoding a delivery signal or a fragment thereof from a bacterial effector protein; 
     a second DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins fused in frame to the 3′ end of said first DNA sequence, wherein the heterologous proteins are selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. Preferably the DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins is flanked on its 3′ end by a DNA sequence homologous to the DNA sequence of the chromosome or of the endogenous virulence plasmid at the 3′ end of the delivery signal from a bacterial effector protein or to a fragment thereof. More preferably, this DNA sequence flanking the homologous protein on its 3′ end is homologous to the DNA sequence and is lying within 10 kbp on the chromosome or on an endogenous virulence plasmid at the 3′ end of the delivery signal from a bacterial effector protein or to a fragment thereof. In particular, this nucleotide sequence flanking the homologous protein on its 3′ end is homologous to the DNA sequence and is within the same operon on the chromosome or on an endogenous virulence plasmid as the delivery signal from a bacterial effector protein or a fragment thereof. In this embodiment, transformation is usually performed so that the fused first and the second DNA sequence are inserted by homologous recombination on an endogenous virulence plasmid or a chromosome, preferably on an endogenous virulence plasmid, of the recombinant virulence attenuated Gram-negative bacterial strain, and the fused first and the second DNA sequence is operably linked to a promoter of an endogenous virulence plasmid or of a chromosome e.g. of a chromosomal pathogenicity island. Preferably the fused first and the second DNA sequence is operably linked to a promoter of an endogenous virulence plasmid. In this embodiment the first DNA sequence comprises a delivery signal or fragment thereof from a bacterial effector protein, preferably a fragment thereof, which provides for homologous recombination at the homologous site at the chromosome or at an endogenous virulence plasmid to result in the second DNA sequence be placed in frame to the 3′ end of the chromosomal or endogenous virulence plasmid delivery signal which is operatively linked to the endogenous promoter. 
     In a further embodiment of the present invention the recombinant Gram-negative bacterial strain, is transformed with a nucleotide molecule, preferably a DNA nucleotide molecule, comprising a nucleotide sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins and a nucleotide sequence which is homologous or identical to a nucleotide sequence encoding a delivery signal from a bacterial effector protein or which is homologous or identical to a nucleotide sequence encoding a fragment of a delivery signal from a bacterial effector protein, wherein the delivery signal from a bacterial effector protein is encoded on the chromosome or on an endogenous virulence plasmid of the recombinant virulence attenuated Gram-negative bacterial strain. Preferably the nucleotide sequence which is homologous or identical to a nucleotide sequence of a delivery signal from a bacterial effector protein or to a fragment thereof is located on the 5′ end of the nucleotide sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins. More preferably the nucleotide sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins is flanked on its 3′ end by a nucleotide sequence homologous to the DNA sequence of the chromosome or of the endogenous virulence plasmid at the 3′ end of the delivery signal from a bacterial effector protein or to a fragment thereof. Even more preferably, this nucleotide sequence flanking the homologous protein on its 3′ end is homologous to the DNA sequence lying within 10 kbp on the chromosome or on an endogenous virulence plasmid at the 3′ end of the delivery signal from a bacterial effector protein or to a fragment thereof. In particular, this nucleotide sequence flanking the homologous protein on its 3′ end is homologous to the DNA sequence is within the same operon on the chromosome or on an endogenous virulence plasmid as the delivery signal from a bacterial effector protein or a fragment thereof. In this embodiment, transformation is usually performed so that the nucleotide sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins is inserted on an endogenous virulence plasmid or a chromosome of the recombinant virulence attenuated Gram-negative bacterial strain at the 3′ end of a delivery signal from a bacterial effector protein encoded by the chromosome or the endogenous virulence plasmid, wherein the repeated domains of a heterologous protein or two or more domains of different heterologous proteins fused to the delivery signal are expressed and secreted. 
     In case the recombinant virulence attenuated Gram-negative bacterial strain is a  Yersinia  strain the endogenous virulence plasmid for insertion is pYV (plasmid of  Yersinia  Virulence). In case the recombinant virulence attenuated Gram-negative bacterial strain is a  Salmonella  strain, the endogenous location for insertion is one of the gene clusters called SpiI or SpiII (for  Salmonella  pathogenicity island), a position where an effector protein is elsewhere encoded or alternatively one of the  Salmonella  virulence plasmids (SVPs). 
     Preferably the first and the second DNA sequence or the nucleotide molecule are inserted on an endogenous virulence plasmid at the native site of a bacterial effector protein e.g. at the native site of a virulence factor, preferably in case the recombinant virulence attenuated Gram-negative bacterial strain is a  Yersinia  strain, at the native site of YopE or another Yop (YopH, YopO, YopP, YopM, YopT), preferably at the native site of YopE or in case the recombinant virulence attenuated Gram-negative bacterial strain is a  Salmonella  strain at the native site of an effector protein encoded within SpiI, SpiII or encoded elsewhere, preferably at the native site of an effector protein encoded within SpiI or SpiII, more preferably at the native site of SopE or SteA. Preferably the first and the second DNA sequence or the nucleotide molecule are operably linked to a native promoter of a bacterial effector protein present on an endogenous virulence plasmid e.g. in case the recombinant virulence attenuated Gram-negative bacterial strain is a  Yersinia  strain to a native promoter from a  Yersinia  virulon gene as outlined below, more preferably to the native YopE promoter or another Yop (YopH, YopO, YopP, YopM, YopT) promoter, preferably to the native YopE promoter or in case the recombinant virulence attenuated Gram-negative bacterial strain is a  Salmonella  strain to a native promoter from SpiI or SpiII pathogenicity island or from an effector protein elsewhere encoded as outlined below, more preferably to the native SopE, InvB or SteA promoter. 
     In one embodiment the present invention provides a recombinant Gram-negative bacterial strain, wherein the Gram-negative bacterial strain is selected from the group consisting of the genera  Yersinia, Escherichia, Salmonella  and  Pseudomonas . In one embodiment the present invention provides a recombinant Gram-negative bacterial strain, wherein the Gram-negative bacterial strain is selected from the group consisting of the genera  Yersinia  and  Salmonella . Preferably the Gram-negative bacterial strain is a  Yersinia  strain, more preferably a  Yersinia enterocolitica  strain. Most preferred is  Yersinia enterocolitica  E40 (0:9, biotype 2) [21] or Ampicilline sensitive derivates thereof as  Y. enterocolitica  MRS40 (also named  Y. enterocolitica  subsp.  palearctica  MRS40) as described in [22].  Y. enterocolitica  E40 and its derivate  Y. enterocolitica  MRS40 as described in [22] is identical to  Y. enterocolitica  subsp.  palearctica  E40 and its derivate  Y. enterocolitica  subsp.  palearctica  MRS40 as described in [23-25]. Also preferably the Gram-negative bacterial strain is a  Salmonella  strain, more preferably a  Salmonella enterica  strain. Most preferred is  Salmonella enterica  Serovar  Typhimurium  SL1344 as described by the Public health England culture collection (NCTC 13347). 
     In one embodiment of the present invention the delivery signal from a bacterial T3SS effector protein comprises a bacterial T3SS effector protein or a N-terminal fragment thereof wherein the T3SS effector protein or a N-terminal fragment thereof may comprise a chaperone binding site. A T3SS effector protein or a N-terminal fragment thereof which comprises a chaperone binding site is particular useful as delivery signal in the present invention. Preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of SopE, SopE2, SptP, YopE, ExoS, SipA, SipB, SipD, SopA, SopB, SopD, IpgB1, IpgD, SipC, SifA, SseJ, Sse, SrfH, YopJ, AvrA, AvrBsT, YopT, YopH, YpkA, Tir, EspF, TccP2, IpgB2, OspF, Map, OspG, OspI, IpaH, SspH1, VopF, ExoS, ExoT, HopAB2, XopD, AvrRpt2, HopAO1, HopPtoD2, HopU1, GALA family of proteins, AvrBs2, AvrD1, AvrBS3, YopO, YopP, YopE, YopM, YopT, EspG, EspH, EspZ, IpaA, IpaB, IpaC, VirA, IcsB, OspC1, OspE2, IpaH9.8, IpaH7.8, AvrB, AvrD, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, VirPphA, AvrRpm1, HopPtoE, HopPtoF, HopPtoN, PopB, PopP2, AvrBs3, XopD, and AvrXv3. More preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of SopE, SptP, YopE, ExoS, SopB, IpgB1, IpgD, YopJ, YopH, EspF, OspF, ExoS, YopO, YopP, YopE, YopM, YopT, whereof most preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of IpgB1, SopE, SopB, SptP, OspF, IpgD, YopH, YopO, YopP, YopE, YopM, YopT, in particular YopE or an N-terminal fragment thereof. Equally preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of SopE, SopE2, SptP, SteA, SipA, SipB, SipD, SopA, SopB, SopD, IpgB1, IpgD, SipC, SifA, SifB, SseJ, Sse, SrfH, YopJ, AvrA, AvrBsT, YopH, YpkA, Tir, EspF, TccP2, IpgB2, OspF, Map, OspG, OspI, IpaH, VopF, ExoS, ExoT, HopAB2, AvrRpt2, HopAO, HopU1, GALA family of proteins, AvrBs2, AvrD1, YopO, YopP, YopE, YopT, EspG, EspH, EspZ, IpaA, IpaB, IpaC, VirA, IcsB, OspC1, OspE2, IpaH9.8, IpaH7.8, AvrB, AvrD, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, VirPphA, AvrRpm1, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, PopB, PopP2, AvrBs3, XopD, and AvrXv3. Equally more preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of SopE, SptP, SteA, SifB, SopB, IpgB1, IpgD, YopJ, YopH, EspF, OspF, ExoS, YopO, YopP, YopE, YopT, whereof equally most preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of IpgB1, SopE, SopB, SptP, SteA, SifB, OspF, IpgD, YopH, YopO, YopP, YopE, and YopT, in particular SopE, SteA, or YopE or an N-terminal fragment thereof, more particular SteA or YopE or an N-terminal fragment thereof, most particular YopE or an N-terminal fragment thereof. 
     In some embodiments the delivery signal from the bacterial T3SS effector protein encoded by the first DNA sequence comprises the bacterial T3SS effector protein or an N-terminal fragment thereof, wherein the N-terminal fragment thereof includes at least the first 10, preferably at least the first 20, more preferably at least the first 100 amino acids of the bacterial T3SS effector protein. 
     In some embodiments the delivery signal from the bacterial T3SS effector protein encoded by the first DNA sequence comprises the bacterial T3SS effector protein or an N-terminal fragment thereof, wherein the bacterial T3SS effector protein or the N-terminal fragment thereof comprises a chaperone binding site. 
     Preferred T3SS effector proteins or a N-terminal fragment thereof, which comprise a chaperone binding site comprise the following combinations of chaperone binding site and T3SS effector protein or N-terminal fragment thereof: SycE-YopE, InvB-SopE, SicP-SptP, SycT-YopT, SycO-YopO, SycN/YscB-YopN, SycH-YopH, SpcS-ExoS, CesF-EspF, SycD-YopB, SycD-YopD. More preferred are SycE-YopE, InvB-SopE, SycT-YopT, SycO-YopO, SycN/YscB-YopN, SycH-YopH, SpcS-ExoS, CesF-EspF. Most preferred is a YopE or an N-terminal fragment thereof comprising the SycE chaperone binding site such as an N-terminal fragment of a YopE effector protein containing the N-terminal 138 amino acids of the YopE effector protein designated herein as YopE 1-138  and as shown in SEQ ID NO. 2 or a SopE or an N-terminal fragment thereof comprising the InvB chaperone binding site such as an N-terminal fragment of a SopE effector protein containing the N-terminal 81 or 105 amino acids of the SopE effector protein designated herein as SopE 1-81  or SopE 1-105  respectively, and as shown in SEQ ID NO.: 142 or 143. 
     In one embodiment of the present invention the recombinant Gram-negative bacterial strain is a  Yersinia  strain and the delivery signal from the bacterial T3SS effector protein encoded by the first DNA sequence comprises a YopE effector protein or an N-terminal part, preferably the  Y. enterocolitica  YopE effector protein or an N-terminal part thereof. Preferably the SycE binding site is comprised within the N-terminal part of the YopE effector protein. In this connection an N-terminal fragment of a YopE effector protein may comprise the N-terminal 12, 16, 18, 52, 53, 80 or 138 amino acids [26-28]. Most preferred is an N-terminal fragment of a YopE effector protein containing the N-terminal 138 amino acids of the YopE effector protein e.g. as described in Forsberg and Wolf-Watz [29] designated herein as YopE 1-138  and as shown in SEQ ID NO.: 2. 
     In one embodiment of the present invention the recombinant Gram-negative bacterial strain is a  Salmonella  strain and the delivery signal from the bacterial T3SS effector protein encoded by the first DNA sequence comprises a SopE or SteA effector protein or an N-terminal part thereof, preferably the  Salmonella enterica  SopE or SteA effector protein or an N-terminal part thereof. Preferably the chaperon binding site is comprised within the N-terminal part of the SopE effector protein. In this connection an N-terminal fragment of a SopE effector protein protein may comprise the N-terminal 81 or 105 amino acids. Most preferred is the full length SteA and an N-terminal fragment of a SopE effector protein containing the N-terminal 105 amino acids of the effector protein e.g. as described in SEQ ID NO. 142 or 143. 
     One skilled in the art is familiar with methods for identifying the polypeptide sequences of an effector protein that are capable of delivering a protein. For example, one such method is described by Sory et al. [21]. Briefly, polypeptide sequences from e.g. various portions of the Yop proteins can be fused in-frame to a reporter enzyme such as the calmodulin-activated adenylate cyclase domain (or Cya) of the  Bordetella pertussis  cyclolysin. Delivery of a Yop-Cya hybrid protein into the cytosol of eukaryotic cells is indicated by the appearance of cyclase activity in the infected eukaryotic cells that leads to the accumulation of cAMP. By employing such an approach, one skilled in the art can determine, if desired, the minimal sequence requirement, i.e., a contiguous amino acid sequence of the shortest length, that is capable of delivering a protein, see, e.g. [21]. Accordingly, preferred delivery signals of the present invention consists of at least the minimal sequence of amino acids of a T3SS effector protein that is capable of delivering a protein. 
     In one embodiment the present invention provides mutant recombinant Gram-negative bacterial strains in particular recombinant Gram-negative bacterial strains which are deficient in producing at least one T3SS functional effector protein. According to the present invention, such a mutant Gram-negative bacterial strain e.g. such a mutant  Yersinia  strain can be generated by introducing at least one mutation into at least one effector-encoding gene. Preferably, such effector-encoding genes include YopE, YopH, YopO/YpkA, YopM, YopP/YopJ and YopT as far as a  Yersinia  strain is concerned. Preferably, such effector-encoding genes include AvrA, CigR, GogB, GtgA, GtgE, PipB, SifB, SipA/SspA, SipB, SipC/SspC, SipD/SspD, SlrP, SopB/SigD, SopA, SpiC/SsaB, SseB, SseC, SseD, SseF, SseG, SseI/SrfH, SopD, SopE, SopE2, SspH1, SspH2, PipB2, SifA, SopD2, SseJ, SseK1, SseK2, SseK3, SseL, SteC, SteA, SteB, SteD, SteE, SpvB, SpvC, SpvD, SrfJ, SptP, as far as a  Salmonella  strain is concerned. Most preferably, all effector-encoding genes are deleted. The skilled artisan may employ any number of standard techniques to generate mutations in these T3SS effector genes. Sambrook et al. describe in general such techniques. See Sambrook et al. [30]. 
     In accordance with the present invention, the mutation can be generated in the promoter region of an effector-encoding gene so that the expression of such effector gene is abolished. 
     The mutation can also be generated in the coding region of an effector-encoding gene such that the catalytic activity of the encoded effector protein is abolished. The “catalytic activity” of an effector protein refers normally to the anti-target cell function of an effector protein, i.e., toxicity. Such activity is governed by the catalytic motifs in the catalytic domain of an effector protein. The approaches for identifying the catalytic domain and/or the catalytic motifs of an effector protein are well known by those skilled in the art. See, for example, [31,32]. 
     Accordingly, one preferred mutation of the present invention is a deletion of the entire catalytic domain. Another preferred mutation is a frameshift mutation in an effector-encoding gene such that the catalytic domain is not present in the protein product expressed from such “frameshifted” gene. A most preferred mutation is a mutation with the deletion of the entire coding region of the effector protein. Other mutations are also contemplated by the present invention, such as small deletions or base pair substitutions, which are generated in the catalytic motifs of an effector protein leading to destruction of the catalytic activity of a given effector protein. 
     The mutations that are generated in the genes of the T3SS functional effector proteins may be introduced into the particular strain by a number of methods. One such method involves cloning a mutated gene into a “suicide” vector which is capable of introducing the mutated sequence into the strain via allelic exchange. An example of such a “suicide” vector is described by [33]. 
     In this manner, mutations generated in multiple genes may be introduced successively into a Gram-negative bacterial strain giving rise to polymutant, e.g a sixtuple mutant recombinant strain. The order in which these mutated sequences are introduced is not important. Under some circumstances, it may be desired to mutate only some but not all of the effector genes. Accordingly, the present invention further contemplates polymutant  Yersinia  other than sixtuple-mutant  Yersinia , e.g., double-mutant, triple-mutant, quadruple-mutant and quintuple-mutant strains. For the purpose of delivering proteins, the secretion and translocation system of the instant mutant strain needs to be intact. 
     A preferred recombinant Gram-negative bacterial strain of the present invention is a recombinant Gram-negative bacterial strain which is deficient in producing at least one, preferably at least two, more preferably at least three, even more preferably at least four, in particular at least five, more particular at least six, most particular all T3SS effector proteins e.g. a sixtuple-mutant Gram-negative bacterial strain in which all the effector-encoding genes are mutated such that the resulting Gram-negative bacterial strain no longer produce any functional effector proteins. 
     A more preferred recombinant Gram-negative bacterial strain of the present invention is a sixtuple-mutant  Yersinia  strain in which all the effector-encoding genes are mutated such that the resulting  Yersinia  no longer produce any functional effector proteins. Such sixtuple-mutant  Yersinia  strain is designated as ΔyopH,O,P,E,M,T for  Y. enterocolitica . As an example such a sixtuple-mutant can be produced from the  Y. enterocolitica  MRS40 strain giving rise to  Y. enterocolitica  MRS40 ΔyopH,O,P,E,M,T, which is preferred. 
     A further aspect of the present invention is directed to a vector for use in combination with the recombinant Gram-negative bacterial strains to deliver a desired protein into eukaryotic cells, wherein the vector comprises in the 5′ to 3′ direction: 
     a promoter; 
     a first DNA sequence encoding a delivery signal from a bacterial T3SS effector protein, operably linked to said promoter; 
     a second DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins fused in frame to the 3′ end of said first DNA sequence, 
     wherein the heterologous proteins are selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. 
     Promoter, heterologous protein and protease cleavage site as described supra can be used for the vector of the Gram-negative bacterial strain. 
     A further aspect of the present invention is directed to a vector for use in combination with the recombinant Gram-negative bacterial strains to deliver a desired protein into eukaryotic cells, wherein the vector comprises in the 5′ to 3′ direction: 
     a first DNA sequence encoding a delivery signal or a fragment thereof from a bacterial effector protein; 
     a second DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins fused in frame to the 3′ end of said first DNA sequence, 
     wherein the heterologous proteins are selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. 
     Preferably the DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins of the vector is flanked on its 3′ end by a DNA sequence homologous to the DNA sequence of the chromosome or of the endogenous virulence plasmid at the 3′ end of the delivery signal from a bacterial effector protein or to a fragment thereof. More preferably, this DNA sequence flanking the homologous protein on its 3′ end is homologous to the DNA sequence and is lying within 10 kbp on the chromosome or on an endogenous virulence plasmid at the 3′ end of the delivery signal from a bacterial effector protein or to a fragment thereof. In particular, this nucleotide sequence flanking the homologous protein on its 3′ end is homologous to the DNA sequence and is within the same operon on the chromosome or on an endogenous virulence plasmid as the delivery signal from a bacterial effector protein or a fragment thereof. Heterologous protein and protease cleavage site as described supra can be used for the vector of the Gram-negative bacterial strain. 
     Vectors which can be used according to the invention depend on the Gram-negative bacterial strains used as known to the skilled person. Vectors which can be used according to the invention include expression vectors (including synthetic or otherwise generated modified versions of endogenous virulence plasmids), vectors for chromosomal or virulence plasmid insertion and DNA fragments for chromosomal or virulence plasmid insertion. Expression vectors which are useful in e.g.  Yersinia, Escherichia, Salmonella  or  Pseudomonas  strain are e.g pUC, pBad, pACYC, pUCP20 and pET plasmids. Vectors for chromosomal or virulence plasmid insertion which are useful in e.g.  Yersinia, Escherichia, Salmonella  or  Pseudomonas  strain are e.g pKNG101. DNA fragments for chromosomal or virulence plasmid insertion refer to methods used in e.g.  Yersinia, Escherichia, Salmonella  or  Pseudomonas  strain as e.g. lambda-red genetic engineering. Vectors for chromosomal or virulence plasmid insertion or DNA fragments for chromosomal or virulence plasmid insertion may insert the first, second and/or third DNA sequence of the present invention so that the first, second and/or third DNA sequence is operably linked to an endogenous promoter of the recombinant Gram-negative bacterial strain. Thus if a vector for chromosomal or virulence plasmid insertion or a DNA fragment for chromosomal or virulence plasmid insertion is used, an endogenous promoter can be encoded on the endogenous bacterial DNA (chromosomal or plasmid DNA) and only the first and second DNA sequence will be provided by the engineered vector for chromosomal or virulence plasmid insertion or DNA fragment for chromosomal or virulence plasmid insertion. Alternatively, if a vector for chromosomal or virulence plasmid insertion or a nucleotide molecule such as e.g. a DNA sequence for chromosomal or virulence plasmid insertion is used, an endogenous promoter and the delivery signal from a bacterial effector protein can be encoded on the endogenous bacterial DNA (chromosomal or plasmid DNA) and only the nucleotide molecule such as e.g. a DNA sequence encoding the heterologous protein will be provided by a vector for chromosomal or virulence plasmid insertion or by a nucleotide molecule such as e.g. a DNA sequence for chromosomal or virulence plasmid insertion. Thus a promoter is not necessarily needed to be comprised by the vector used for transformation of the recombinant Gram-negative bacterial strains i.e. the recombinant Gram-negative bacterial strains of the present invention may be transformed with a vector which dose not comprise a promoter. The vector of the present invention is normally used for delivery of the heterologous proteins by the bacterial T3SS into eukaryotic cells in vitro and in vivo. 
     A preferred vector e.g. a preferred expression vector for  Yersinia  is selected from the group consisting of pBad_Si_1 and pBad_Si_2. pBad_Si2 was constructed by cloning of the SycE-YopE 1-138  fragment containing endogenous promoters for YopE and SycE from purified pYV40 into KpnI/HindIII site of pBad-MycHisA (Invitrogen). Additional modifications include removal of the NcoI/BglII fragment of pBad-MycHisA by digest, Klenow fragment treatment and religation. Further at the 3′ end of YopE 1-138  the following cleavage sites were added: XbaI-XhoI-BstBI-(HindIII). pBad_Si1 is equal to pBad_Si2 but encodes EGFP amplified from pEGFP-C1 (Clontech) in the NcoI/BglII site under the Arabinose inducible promoter. Equally preferred is the use of modified versions of the endogenous  Yersinia  virulence plasmid pYV encoding heterologous proteins as fusions to a T3SS signal sequence. A preferred vector e.g. a preferred expression vector for  Salmonella  is selected from the group consisting of pSi_266, pSi_267, pSi_268 and pSi_269. Plasmids pSi_266, pSi_267, pSi_268 and pSi_269 containing the corresponding endogenous promoter and the SteA 1-20  fragment (pSi_266), the full length SteA sequence (pSi_267), the SopE 1-81  fragment (pSi_268) or the SopE 1-105  fragment (pSi_269) were amplified from  S. enterica  SL1344 genomic DNA and cloned into NcoI/KpnI site of pBad-MycHisA (Invitrogen). 
     The vectors of the instant invention may include other sequence elements such as a 3′ termination sequence (including a stop codon and a poly A sequence), or a gene conferring a drug resistance which allows the selection of transformants having received the instant vector. 
     The vectors of the present invention may be transformed by a number of known methods into the recombinant Gram-negative bacterial strains. For the purpose of the present invention, the methods of transformation for introducing a vector include, but are not limited to, electroporation, calcium phosphate mediated transformation, conjugation, or combinations thereof. For example, a vector can be transformed into a first bacteria strain by a standard electroporation procedure. Subsequently, such a vector can be transferred from the first bacteria strain into the desired strain by conjugation, a process also called “mobilization”. Transformant (i.e., Gram-negative bacterial strains having taken up the vector) may be selected, e.g., with antibiotics. These techniques are well known in the art. See, for example, [21]. 
     In accordance with the present invention, the promoter of the expression vector of the recombinant Gram-negative bacterial strain of the invention can be a native promoter of a T3SS effector protein of the respective strain or a compatible bacterial strain or a promoter used in expression vectors which are useful in e.g.  Yersinia, Escherichia, Salmonella  or  Pseudomonas  strain e.g pUC and pBad. Such promoters are the T7 promoter, Plac promoter or Ara-bad promoter. 
     If the recombinant Gram-negative bacterial strain is a  Yersinia  strain the promoter can be from a  Yersinia  virulon gene. A “ Yersinia  virulon gene” refers to genes on the  Yersinia  pYV plasmid, the expression of which is controlled both by temperature and by contact with a target cell. Such genes include genes coding for elements of the secretion machinery (the Ysc genes), genes coding for translocators (YopB, YopD, and LcrV), genes coding for the control elements (YopN, TyeA and LcrG), genes coding for T3SS effector chaperones (SycD, SycE, SycH, SycN, SycO and SycT), and genes coding for effectors (YopE, YopH, YopO/YpkA, YopM, YopT and YopP/YopJ) as well as other pYV encoded proteins as VirF and YadA. 
     In a preferred embodiment of the present invention, the promoter is the native promoter of a T3SS functional effector encoding gene. If the recombinant Gram-negative bacterial strain is a  Yersinia  strain the promoter is selected from any one of YopE, YopH, YopO/YpkA, YopM and YopP/YopJ. More preferably, the promoter is from YopE or SycE. 
     If the recombinant Gram-negative bacterial strain is a  Salmonella  strain the promoter can be from SpiI or SpiII pathogenicity island or from an effector protein elsewhere encoded. Such genes include genes coding for elements of the secretion machinery, genes coding for translocators, genes coding for the control elements, genes coding for T3SS effector chaperones, and genes coding for effectors as well as other proteins encoded by SPI-1 or SPI-2. In a preferred embodiment of the present invention, the promoter is the native promoter of a T3SS functional effector encoding gene. If the recombinant Gram-negative bacterial strain is a  Salmonella  strain the promoter is selected from any one of the effector proteins. More preferably, the promoter is from SopE, InvB or SteA. 
     In a preferred embodiment the vector e.g. the expression vector comprises a DNA sequence encoding a protease cleavage site. Generation of a functional and generally applicable cleavage site allows cleaving off the delivery signal after translocation. As the delivery signal can interfere with correct localization and/or function of the translocated protein within the target cells the introduction of a protease cleavage site between the delivery signal and the protein of interest provides for the first time delivery of almost native proteins into eukaryotic cells. Preferably the protease cleavage site is an amino acid motif which is cleaved by a protease or the catalytic domains thereof selected from the group consisting of enterokinase (light chain), enteropeptidase, prescission protease, human rhinovirus protease 3C, TEV protease, TVMV protease, FactorXa protease and thrombin, more preferably an amino acid motif which is cleaved by TEV protease. Equally preferable the protease cleavage site is an amino acid motif which is cleaved by a protease or the catalytic domains thereof selected from the group consisting of enterokinase (light chain), enteropeptidase, prescission protease, human rhinovirus protease 3C, TEV protease, TVMV protease, FactorXa protease, ubiquitin processing protease, called Deubiquitinating enzymes, and thrombin. Most preferred is an amino acid motif which is cleaved by TEV protease or by an ubiquitin processing protease. 
     Thus in a further embodiment of the present invention, the heterologous protein is cleaved from the delivery signal from a bacterial T3SS effector protein by a protease. 
     Preferred methods of cleavage are methods wherein: 
     a) the protease is translocated into the eukaryotic cell by a recombinant Gram-negative bacterial strain as described herein which expresses a fusion protein which comprises the delivery signal from the bacterial T3SS effector protein and the protease as heterologous protein; or 
     b) the protease is expressed constitutively or transiently in the eukaryotic cell. 
     Usually the recombinant Gram-negative bacterial strain used to deliver a desired protein into a eukaryotic cell and the recombinant Gram-negative bacterial strain translocating the protease into the eukaryotic cell are different. 
     In one embodiment of the present invention the vector comprises a further DNA sequence encoding a labelling molecule or an acceptor site for a labelling molecule. The further DNA sequence encoding a labelling molecule or an acceptor site for a labelling molecule is usually fused to the 5′ end or to the 3′ end of the second DNA sequence. A preferred labelling molecule or an acceptor site for a labelling molecule is selected from the group consisting of enhanced green fluorescent protein (EGFP), coumarin, coumarin ligase acceptor site, resorufin, resurofin ligase acceptor site, the tetra-Cysteine motif in use with FlAsH/ReAsH dye (life technologies). Most preferred is resorufin and a resurofin ligase acceptor site or EGFP. The use of a labelling molecule or an acceptor site for a labelling molecule will lead to the attachment of a labelling molecule to the heterologous protein of interest, which will then be delivered as such into the eukaryotic cell and enables tracking of the protein by e.g. live cell microscopy. 
     In one embodiment of the present invention the vector comprises a further DNA sequence encoding a peptide tag. The further DNA sequence encoding a peptide tag is usually fused to the 5′ end or to the 3′ end of the second DNA sequence. A preferred peptide tag is selected from the group consisting of Myc-tag, His-tag, Flag-tag, HA tag, Strep tag or V5 tag or a combination of two or more tags out of these groups. Most preferred is Myc-tag, Flag-tag, His-tag and combined Myc- and His-tags. The use of a peptide tag will lead to traceability of the tagged protein e.g by immunofluorescence or Western blotting using anti-tag antibodies. Further, the use of a peptide tag allows affinity purification of the desired protein either after secretion into the culture supernatant or after translocation into eukaryotic cells, in both cases using a purification method suiting the corresponding tag (e.g. metal-chelate affinity purification in use with a His-tag or anti-Flag antibody based purification in use with the Flag-tag). 
     In one embodiment of the present invention the vector comprises a further DNA sequence encoding a nuclear localization signal (NLS). The further DNA sequence encoding a nuclear localization signal (NLS) is usually fused to the 5′ end or to the 3′ end of the second DNA sequence wherein said further DNA sequence encodes a nuclear localization signal (NLS). A preferred NLS is selected from the group consisting of SV40 large T-antigen NLS and derivates thereof [34] as well as other viral NLS. Most preferred is SV40 large T-antigen NLS and derivates thereof. 
     In one embodiment of the present invention the vector comprises a multiple cloning site. The multiple cloning site is usually located at the 3′ end of the first DNA sequence and/or at the 5′ end or 3′ end of the second DNA sequence. One or more than one multiple cloning sites can be comprised by the vector. A preferred multiple cloning site is selected from the group of restriction enzymes consisting of XhoI, XbaI, HindIII, NcoI, NotI, EcoRI, EcoRV, BamHI, NheI, SacI, SalI, BstBI. Most preferred is XbaI, XhoI, BstBI and HindIII. 
     The fused protein expressed from the first and second and optional third DNA sequences of the vector is also termed as a “fusion protein” or a “hybrid protein”, i.e., a fused protein or hybrid of delivery signal and repeated domains of a heterologous protein or two or more domains of different heterologous proteins. 
     The present invention contemplates a method for delivering repeated domains of a heterologous protein or two or more domains of different heterologous proteins as hereinabove described into eukaryotic cells in cell culture as well as in-vivo. 
     Thus in one embodiment the method for delivering repeated domains of a heterologous protein or two or more domains of different heterologous proteins comprises 
     i) culturing the Gram-negative bacterial strain as described herein; 
     ii) contacting a eukaryotic cell with the Gram-negative bacterial strain of i) wherein a fusion protein which comprises a delivery signal from a bacterial T3SS effector protein and the repeated domains of a heterologous protein or the two or more domains of different heterologous proteins is expressed by the Gram-negative bacterial strain and is translocated into the eukaryotic cell; and optionally 
     iii) cleaving the fusion protein so that the repeated domains of a heterologous protein or the two or more domains of different heterologous proteins are cleaved from the delivery signal from the bacterial T3SS effector protein. 
     In some embodiments at least two fusion proteins which comprises each a delivery signal from a bacterial effector protein and repeated domains of a heterologous protein or the two or more domains of different heterologous proteins are expressed by the recombinant virulence attenuated Gram-negative bacterial strain and are translocated into the eukaryotic cell by the methods of the present inventions. 
     The recombinant Gram-negative bacterial strain can be cultured so that a fusion protein is expressed which comprises the delivery signal from the bacterial T3SS effector protein and the repeated domains of a heterologous protein or the two or more domains of different heterologous proteins according to methods known in the art (e.g. FDA, Bacteriological Analytical Manual (BAM), chapter 8:  Yersinia enterocolitica ). Preferably the recombinant Gram-negative bacterial strain can be cultured in Brain Heart infusion broth e.g. at 28° C. For induction of expression of T3SS and e.g. YopE/SycE promoter dependent genes, bacteria can be grown at 37° C. 
     In a preferred embodiment, the eukaryotic cell is contacted with two Gram-negative bacterial strains of i), wherein the first Gram-negative bacterial strain expresses a first fusion protein which comprises the delivery signal from the bacterial T3SS effector protein and repeated domains of a heterologous protein or two or more domains of different heterologous proteins and the second Gram-negative bacterial strain expresses a second fusion protein which comprises the delivery signal from the bacterial T3SS effector protein and repeated domains of a second heterologous protein or two or more domains of a different second heterologous proteins of a second heterologous protein, so that the first and the second fusion protein are translocated into the eukaryotic cell. This embodiment provided for co-infection of e.g eukaryotic cells with two bacterial strains as a valid method to deliver e.g. two different hybrid proteins into single cells to address their functional interaction. 
     Those skilled in the art can also use a number of assays to determine whether the delivery of a fusion protein is successful. For example, the fusion protein may be detected via immunofluorescence using antibodies recognizing a fused tag (like Myc-tag). The determination can also be based on the enzymatic activity of the protein being delivered. 
     The present invention contemplates a wide range of eukaryotic cells that may be targeted by the instant recombinant Gram-negative bacterial strain e.g. Hi-5 (BTI-TN-5B1-4; life technologies B855-02), HeLa cells, e.g. HeLa Ccl2 (as ATCC No. CCL-2), fibroblast cells, e.g. 3T3 fibroblast cells (as ATCC No. CCL-92) or Mef (as ATCC No. SCRC-1040), Hek (as ATCC No. CRL-1573), HUVECs (as ATCC No. PCS-100-013), CHO (as ATCC No. CCL-61), Jurkat (as ATCC No. TIB-152), Sf-9 (as ATCC No. CRL-1711), HepG2 (as ATCC No. HB-8065), Vero (as ATCC No. CCL-81), MDCK (as ATCC No. CCL-34), THP-1 (as ATCC No. TIB-202), J774 (as ATCC No. TIB-67), RAW (as ATCC No. TIB-71), Caco2 (as ATCC No. HTB-37), NCI cell lines (as ATCC No. HTB-182), DU145 (as ATCC No. HTB-81), Lncap (as ATCC No. CRL-1740), MCF-7 (as ATCC No. HTB-22), MDA-MB cell lines (as ATCC No. HTB-128), PC3 (as ATCC No. CRL-1435), T47D (as ATCC No. CRL-2865), A549 (as ATCC No. CCL-185), U87 (as ATCC No. HTB-14), SHSY5Y (as ATCC No. CRL-2266s), Ea.Hy926 (as ATCC No. CRL-2922), Saos-2 (as ATCC No. HTBH-85), 4T1 (as ATCC No. CRL-2539), B16F10 (as ATCC No. CRL-6475), or primary human hepatocytes (as life technologies HMCPIS), preferably HeLa, Hek, HUVECs, 3T3, CHO, Jurkat, Sf-9, HepG2 Vero, THP-1, Caco2, Mef, A549, 4T1, B16F10 and primary human hepatocytes and most preferably HeLa, Hek, HUVECs, 3T3, CHO, Jurkat, THP-1, A549 and Mef. By “target”, is meant the extracellular adhesion of the recombinant Gram-negative bacterial strain to a eukaryotic cell. 
     In accordance with the present invention, the delivery of a protein can be achieved by contacting a eukaryotic cell with a recombinant Gram-negative bacterial strain under appropriate conditions. Various references and techniques are conventionally available for those skilled in the art regarding the conditions for inducing the expression and translocation of virulon genes, including the desired temperature, Ca ++  concentration, addition of inducers as Congo Red, manners in which the recombinant Gram-negative bacterial strain and target cells are mixed, and the like. See, for example, [35]. The conditions may vary depending on the type of eukaryotic cells to be targeted and the recombinant bacterial strain to be used. Such variations can be addressed by those skilled in the art using conventional techniques. 
     Those skilled in the art can also use a number of assays to determine whether the delivery of a fusion protein is successful. For example, the fusion protein may be detected via immunofluorescence using antibodies recognizing a fused tag (like Myc-tag). The determination can also be based on the enzymatic activity of the protein being delivered, e.g., the assay described by [21]. 
     In one embodiment the present invention provides the recombinant Gram-negative bacterial strain as described herein for use in medicine. 
     In one embodiment the present invention provides the recombinant Gram-negative bacterial strain as described herein for use in the delivery of repeated domains of a heterologous protein or two or more domains of different heterologous proteins as a medicament or as a vaccine to a subject. The repeated domains of a heterologous protein or the two or more domains of different heterologous proteins can be delivered to a subject as a vaccine by contacting the Gram-negative bacterial strain with eukaryotic cells, e.g. with a living animal in vivo so that the repeated domains of a heterologous protein or the two or more domains of different heterologous proteins are translocated into the living animal which then produces antibodies against the repeated domains of a heterologous protein or the two or more domains of different heterologous proteins. The antibodies produced can be directly used or be isolated and purified and used in diagnosis, in research use as well as in therapy. The B-cells producing the antibodies or the therein contained DNA sequence can be used for further production of specific antibodies for use in diagnosis, in research use as well as in therapy. 
     In one embodiment the present invention provides a method for delivering repeated domains of a heterologous protein or the two or more domains of different heterologous proteins, wherein the repeated domains of a heterologous protein or two or more domains of different heterologous proteins are delivered in vitro into a eukaryotic cell. 
     In a further embodiment the present invention provides a method for delivering repeated domains of a heterologous protein or two or more domains of different heterologous proteins, wherein the eukaryotic cell is a living animal wherein the living animal is contacted with the Gram-negative bacterial strain in vivo so that a fusion protein is translocated into the living animal. The preferred animal is a mammal, more preferably a human being. 
     In a further embodiment the present invention provides the use of the recombinant Gram-negative bacterial strain as described supre for High Throughput Screenings of inhibitors for a cellular pathway or event triggered by the translocated heterologous protein(s). 
     In a further aspect the present invention provides a library of Gram-negative bacterial strains, wherein the repeated domains of a heterologous protein or the two or more domains of different heterologous proteins encoded by the second DNA sequence of the expression vector of the Gram-negative bacterial strains are domains of a human or murine protein, preferably a domain of a human protein and, wherein each domain of a human or murine protein expressed by a Gram-negative bacterial strain is different in amino acid sequence. As cloning vector for expression the above described expression vectors can be used. 
     In a further aspect the present invention provides a kit comprising a vector as described herein and a bacterial strain expressing and secreting a protease capable of cleaving the protease cleavage site comprised by the vector. A particular useful vector is a vector for use in combination with the bacterial strain to deliver a desired protein into eukaryotic cells as described above, wherein the vector comprises in the 5′ to 3′ direction: 
     a promoter; 
     a first DNA sequence encoding a delivery signal from a bacterial T3SS effector protein, operably linked to said promoter; 
     a second DNA sequence encoding repeated domains of a heterologous protein or two or more domains of different heterologous proteins fused in frame to the 3′ end of said first DNA sequence, 
     wherein the heterologous proteins are selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, small GTPases, GPCR related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins. 
     EXAMPLES 
     Example 1 
     A) Materials and Methods 
     Bacterial strains and growth conditions. The strains used in this study are listed in  FIGS. 15A  to N.  E. coli  Top 10, used for plasmid purification and cloning, and  E. coli  Sm10λ pir, used for conjugation, as well as  E. coli  BW19610 [36], used to propagate pKNG101, were routinely grown on LB agar plates and in LB broth at 37° C. Ampicillin was used at a concentration of 200 μg/ml ( Yersinia ) or 100 μg/ml ( E. coli ) to select for expression vectors. Streptomycin was used at a concentration of 100 μg/ml to select for suicide vectors.  Y. enterocolitica  MRS40 [22] a non Ampicillin resistant E40-derivate [21] and strains derived thereof were routinely grown on Brain Heart Infusion (BHI; Difco) at RT. To all  Y. enterocolitica  strains Nalidixic acid was added (35 μg/ml) and all  Y. enterocolitica  asd strains were additionally supplemented with 100 μg/ml meso-2,6-Diaminopimelic acid (mDAP, Sigma Aldrich).  S. enterica  SL1344 were routinely grown on LB agar plates and in LB broth at 37° C. Ampicillin was used at a concentration of 100 μg/ml to select for expression vectors in  S. enterica.    
     Genetic Manipulations of  Y. enterocolitica.    
     Genetic manipulations of  Y. enterocolitica  has been described [37,38]. Briefly, mutators for modification or deletion of genes in the pYV plasmids or on the chromosome were constructed by 2-fragment overlapping PCR using purified pYV40 plasmid or genomic DNA as template, leading to 200-250 bp of flanking sequences on both sides of the deleted or modified part of the respective gene. Resulting fragments were cloned in pKNG101 [33] in  E. coli  BW19610 [36]. Sequence verified plasmids were transformed into  E. coli  Sm10λ pir, from where plasmids were mobilized into the corresponding  Y. enterocolitica  strain. Mutants carrying the integrated vector were propagated for several generations without selection pressure. Then sucrose was used to select for clones that have lost the vector. Finally mutants were identified by colony PCR. Specific mutators (pSi_408, pSi_419) are listed in Table III. 
     Construction of Plasmids. 
     Plasmid pBad_Si2 or pBad_Si1 ( FIG. 10 ) were used for cloning of fusion proteins with the N-terminal 138 amino acids of YopE (SEQ ID No. 2). pBad_Si2 was constructed by cloning of the SycE-YopE 1-138  fragment containing endogenous promoters for YopE and SycE from purified pYV40 into KpnI/HindIII site of pBad-MycHisA (Invitrogen). Additional modifications include removal of the NcoI/BglII fragment of pBad-MycHisA by digestion, Klenow fragment treatment and religation. A bidirectional transcriptional terminator (BBa_B1006; iGEM foundation) was cloned into KpnI cut and Klenow treated (pBad_Si2) or BglII cut site (pBad_Si1). Further at the 3′ end of YopE 1-138  the following cleavage sites were added: XbaI-XhoI-BstBI-(HindIII) ( FIG. 10  B). pBad_Si1 is equal to pBad_Si2 but encodes EGFP amplified from pEGFP-C1 (Clontech) in the NcoI/BglII site under the Arabinose inducible promoter. Plasmids pSi_266, pSi_267, pSi_268 and pSi_269 containing the corresponding endogenous promoter and the SteA 1-20  fragment (pSi_266), the full length SteA sequence (pSi_267), the SopE 1-81  fragment (pSi_268) or the SopE 1-105  fragment (pSi_269) were amplified from  S. enterica  SL1344 genomic DNA and cloned into NcoI/KpnI site of pBad-MycHisA (Invitrogen). 
     Full length genes or fragments thereof were amplified with the specific primers listed in Table I below and cloned as fusions to YopE 1-138  into plasmid pBad_Si2 or in case of z-BIM (SEQ ID No. 21) into pBad_Si1 (see Table II below). For fusion to SteA or SopE, synthetic DNA constructs were cleaved by KpnI/HindII and cloned into pSi_266, pSi_267, pSi_268 or pSi_269 respectively. In case of genes of bacterial species, purified genomic DNA was used as template ( S. flexneri  M90T,  Salmonella enterica  subsp.  enterica  serovar  Typhimurium  SL1344,  Bartonella henselae  ATCC 49882). For human genes a universal cDNA library (Clontech) was used if not otherwise stated ( FIGS. 15A  to N), zebrafish genes were amplified from a cDNA library (a kind gift of M. Affolter). Ligated plasmids were cloned in  E. coli  Top 10. Sequenced plasmids were electroporated into the desired  Y. enterocolitica  or  S. enterica  strain using settings as for standard  E. coli  electroporation. 
     
       
         
           
               
             
               
                 TABLE I 
               
               
                   
               
               
                 (Primer Nr. Si_: Sequence) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
            
               
                 285: CATACCATGGGAGTGAGCAAGGGCGAG 
               
               
                   
               
               
                 286: GGAAGATCTttACTTGTACAGCTCGTCCAT 
               
               
                   
               
               
                 287: CGGGGTACCTCAACTAAATGACCGTGGTG 
               
               
                   
               
               
                 288: GTTAAAGCTTttcgaatctagactcgagCGTGGCGAACTGGTC 
               
               
                   
               
               
                 292: CAGTctcgagCAAATTCTAAACAAAATACTTCCAC 
               
               
                   
               
               
                 293: cagtTTCGAATTAATTTGTATTGCTTTGACGG 
               
               
                   
               
               
                 296: CAGTctcgagACTAACATAACACTATCCACCCAG 
               
               
                   
               
               
                 297: GTTAAAGCTTTCAGGAGGCATTCTGAAG 
               
               
                   
               
               
                 299: CAGTctcgagCAGGCCATCAAGTGTGTG 
               
               
                   
               
               
                 300: cagtTTCGAATCATTTTCTCTTCCTCTTCTTCA 
               
               
                   
               
               
                 301: CAGTctcgagGCTGCCATCCGGAA 
               
               
                   
               
               
                 302: cagtTTCGAATCACAAGACAAGGCACCC 
               
               
                   
               
               
                 306: GTTAAAGCTTGGAGGCATTCTGAAGatacttatt 
               
               
                   
               
               
                 307: CAGTctcgagCAAATACAGAGCTTCTATCACTCAG 
               
               
                   
               
               
                 308: GTTAAAGCTTTCAAGATGTGATTAATGAAGAAATG 
               
               
                   
               
               
                 317: cagtTTCGAACCCATAAAAAAGCCCTGTC 
               
               
                   
               
               
                 318: GTTAAAGCTTCTACTCTATCATCAAACGATAAAATGg 
               
               
                   
               
               
                 324: CAGTctcgagTTCACTCAAGAAACGCAAA 
               
               
                   
               
               
                 339: cagtTTCGAATTTTCTCTTCCTCTTCTTCAcg 
               
               
                   
               
               
                 341: cgtaTCTAGAAAAATGATGAAAATGGAGACTG 
               
               
                   
               
               
                 342: GTTAAAGCTTttaGCTGGAGACGGTGAC 
               
               
                   
               
               
                 346: CAGTctcgagTTCCAGATCCCAGAGTTTG 
               
               
                   
               
               
                 347: GTTAAAGCTTTCACTGGGAGGGGG 
               
               
                   
               
               
                 351: CAGTctcgagctcgagTTATCTACTCATAGAAACTACTTTTGCAG 
               
               
                   
               
               
                 352: cgcGGATCCtcagtgtctctgcggcatta 
               
               
                   
               
               
                 353: CATTTATTCCTCCTAGTTAGTCAcagcaactgctgctcctttc 
               
               
                   
               
               
                 354: gaaaggagcagcagttgctgTGACTAACTAGGAGGAATAAATG 
               
               
                   
               
               
                 355: cgattcacggattgctttctCATTATTCCCTCCAGGTACTA 
               
               
                   
               
               
                 356: TAGTACCTGGAGGGAATAATGagaaagcaatccgtgaatcg 
               
               
                   
               
               
                 357: cgtaTCTAGAcggetttaagtgcgacattc 
               
               
                   
               
               
                 364: cgtaTCTAGACTAAAGTATGAGGAGAGAAAATTGAA 
               
               
                   
               
               
                 365: GTTAAAGCTTTCAGCTTGCCGTCGT 
               
               
                   
               
               
                 367: CGTAtctagaGACCCGTTCCTGGTGC 
               
               
                   
               
               
                 369: cgtaTCTAGAccccccaagaagaagc 
               
               
                   
               
               
                 373: GTTAAAGCTTGCTGGAGACGGTGACC 
               
               
                   
               
               
                 386: CGTAtctagaTCAGGACGCTTCGGAGGTAG 
               
               
                   
               
               
                 387: CGTAtctagaATGGACTGTGAGGTCAACAA 
               
               
                   
               
               
                 389: CGTAtctagaGGCAACCGCAGCA 
               
               
                   
               
               
                 391: GTTAAAGCTTTCAGTCCATCCCATTTCTg 
               
               
                   
               
               
                 403: CGTAtctagatctggaatatccctggaca 
               
               
                   
               
               
                 406: GTTAAAGCTTgtctgtctcaatgccacagt 
               
               
                   
               
               
                 410: CAGTctcgagATGTCCGGGGTGGTg 
               
               
                   
               
               
                 413: cagtTTCGAATCACTGCAGCATGATGTC 
               
               
                   
               
               
                 417: CAGTctcgagAGTGGTGTTGATGATGACATG 
               
               
                   
               
               
                 420: cagtTTCGAATTAGTGATAAAAATAGAGTTCTTTTGTGAG 
               
               
                   
               
               
                 423: CAGTctcgagATGCACATAACTAATTTGGGATT 
               
               
                   
               
               
                 424: cagtTTCGAATTATACAAATGACGAATACCCTTT 
               
               
                   
               
               
                 425: GTTAAAGCTTttacaccttgcgcttcttcttgggcggGCTGGAG 
               
               
                 ACGGTGAC 
               
               
                   
               
               
                 428: CGTAtctagaATGGACTTCAACAGGAACTTT 
               
               
                   
               
               
                 429: CGTAtctagaGGACATAGTCCACCAGCG 
               
               
                   
               
               
                 430: GTTAAAGCTTTCAGTTGGATCCGAAAAAC 
               
               
                   
               
               
                 433: CGTAtctagaGAATTAAAAAAAACACTCATCCCA 
               
               
                   
               
               
                 434: CGTAtctagaCCAAAGGCAAAAGCAAAAA 
               
               
                   
               
               
                 435: GTTAAAGCTTTTAGCTAGCCATGGCAAGC 
               
               
                   
               
               
                 436: CGTAtctagaATGCCCCGCCCC 
               
               
                   
               
               
                 437: GTTAAAGCTTCTACCCACCGTACTCGTCAAT 
               
               
                   
               
               
                 438: CGTAtctagaATGTCTGACACGTCCAGAGAG 
               
               
                   
               
               
                 439: GTTAAAGCTTTCATCTTCTTCGCAGGAAAAAG 
               
               
                   
               
               
                 445: cgcGGATCCttatgggttctcacagcaaaa 
               
               
                   
               
               
                 446: CATTTATTCCTCCTAGTTAGTCAaggcaacagccaatcaagag 
               
               
                   
               
               
                 447: ctettgattggctgttgcctTGACTAACTAGGAGGAATAAATG 
               
               
                   
               
               
                 448: ttgattgcagtgacatggtgCATTATTCCCTCCAGGTACTA 
               
               
                   
               
               
                 449: TAGTACCTGGAGGGAATAATGcaccatgtcactgcaatcaa 
               
               
                   
               
               
                 450: cgtaTCTAGAtagccgcagatgttggtatg 
               
               
                   
               
               
                 451: CGTAtctagaGATCAAGTCCAACTGGTGG 
               
               
                   
               
               
                 463: CAGTctcgaggaaagettgtttaaggggc 
               
               
                   
               
               
                 464: cagtTTCGAAttagcgacggcgacg 
               
               
                   
               
               
                 476: GTTAAAGCTTttACTTGTACAGCTCGTCCAT 
               
               
                   
               
               
                 477: CGTAtctagaGTGAGCAAGGGCGAG 
               
               
                   
               
               
                 478: CAGTctcgagATGGAAGATTATACCAAAATAGAGAAA 
               
               
                   
               
               
                 479: GTTAAAGCTTCTACATCTTCTTAATCTGATTGTCCa 
               
               
                   
               
               
                 482: CGTAtctagaATGGCGCTGCAGCt 
               
               
                   
               
               
                 483: GTTAAAGCTTTCAGTCATTGACAGGAATTTTg 
               
               
                   
               
               
                 486: CGTAtctagaATGGAGCCGGCGGCG 
               
               
                   
               
               
                 487: GTTAAAGCTTTCAATCGGGGATGTCTg 
               
               
                   
               
               
                 492: CGTAtctagaATGCGCGAGGAGAACAAGGG 
               
               
                   
               
               
                 493: GTTAAAGCTTTCAGTCCCCTGTGGCTGTGc 
               
               
                   
               
               
                 494: CGTAtctagaATGGCCGAGCCTTG 
               
               
                   
               
               
                 495: GTTAAAGCTTttaTTGAAGATTTGTGGCTCC 
               
               
                   
               
               
                 504: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATT 
               
               
                 TTCAAAGTATGCCCCGCCCC 
               
               
                   
               
               
                 505: GTTAAAGCTTCCCACCGTACTCGTCAATtc 
               
               
                   
               
               
                 508: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATT 
               
               
                 TTCAAAGTATGGCCGAGCCTTG 
               
               
                   
               
               
                 509: GTTAAAGCTTTTGAAGATTTGTGGCTCCc 
               
               
                   
               
               
                 511: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATT 
               
               
                 TTCAAAGTGTGAGCAAGGGCGAG 
               
               
                   
               
               
                 512: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATT 
               
               
                 TTCAAAGTCCGCCGAAAAAAAAACGTAAAGTTGTGAGCAAGGGCGAG 
               
               
                   
               
               
                 513: GTTAAAGCTTttAAACTTTACGTTTTTTTTTCGGCGGCTTGTAC 
               
               
                 AGCTCGTCCAT 
               
               
                   
               
               
                 515: CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATT 
               
               
                 TTCAAAGTGATTATAAAGATGATGATGATAAAATGGCCGAGCCTTG 
               
               
                   
               
               
                 558: CGTATCTAGAATGACCAGTTTTGAAGATGC 
               
               
                   
               
               
                 559: GTTAAAGCTTTCATGACTCATTTTCATCCAT 
               
               
                   
               
               
                 561: CGTATCTAGAATGAGTCTCTTAAACTGTGAGAACAG 
               
               
                   
               
               
                 562: GTTAAAGCTTCTACACCCCCGCATCA 
               
               
                   
               
               
                 580: catgccatggATTTATGGTCATAGATATGACCTC 
               
               
                   
               
               
                 585: CAGTctcgagATGCAGATCTTCGTCAAGAC 
               
               
                   
               
               
                 586: GTTAAAGCTTgctagcttcgaaACCACCACGTAGACGTAAGAC 
               
               
                   
               
               
                 588: cagtTTCGAAGATTATAAAGATGATGATGATAAAATGGCCGAGCC 
               
               
                 TTG 
               
               
                   
               
               
                 612: CGGGGTACCatgaggtagcttatttcctgataaag 
               
               
                   
               
               
                 613: CGGGGTACCataattgtccaaatagttatggtagc 
               
               
                   
               
               
                 614: catgccatggCGGCAAGGCTCCTC 
               
               
                   
               
               
                 615: cggggtaccTTTATTTGTCAACACTGCCC 
               
               
                   
               
               
                 616: cggggtaccTGCGGGGTCTTTACTCG 
               
               
                   
               
               
                 677: TTACTATTCGAAGAAATTATTCATAATATTGCCCGCCATCTGGCC 
               
               
                 CAAATTGGTGATGAAATGGATCATTAAGCTTGGAGTA 
               
               
                   
               
               
                 678: TACTCCAAGCTTAATGATCCATTTCATCACCAATTTGGGCCAGAT 
               
               
                 GGCGGGCAATATTATGAATAATTTCTTCGAATAGTAA 
               
               
                   
               
               
                 682: TTACTACTCGAGAAAAAACTGAGCGAATGTCTGCGCCGCATTGGT 
               
               
                 GATGAACTGGATAGCTAAGCTTGGAGTA 
               
               
                   
               
               
                 683: TACTCCAAGCTTAGCTATCCAGTTCATCACCAATGCGGCGCAGAC 
               
               
                 ATTCGCTCAGTTTTTTCTCGAGTAGTAA 
               
               
                   
               
               
                 725: TTACTATTCGAAGAAATTATTCATAATATTGCC 
               
               
                   
               
               
                 726: TACTCCAAGCTTACGGTTGAATATTATGATCCATTTCATCACCAA 
               
               
                 TTTGG 
               
               
                   
               
               
                 727: TTACTATTCGAAGCCGGTGGTGCCGAAGAAATTATTCATAATATT 
               
               
                 GCCC 
               
               
                   
               
               
                 728: TACTCCAAGCTTAATGATCCATTTCATCA 
               
               
                   
               
               
                 733: TTACTACTCGAGGGTGCCATCGATGCCGAAGAAATTATTCATAAT 
               
               
                 ATTGCCCG 
               
               
                   
               
               
                 734: TACTCCTTCGAAGGCACCATGATCCATTTCATCACCAATTTGG 
               
               
                   
               
               
                 735: TACTCCTTCGAATTAATGATCCATTTCATCACCAATTTG 
               
               
                   
               
               
                 736: TTACTACTCGAGGGTGCCATCGATGCCAAAAAACTGAGCGAATGT 
               
               
                 CTGCG 
               
               
                   
               
               
                 737: TACTCCTTCGAAGGCACCGCTATCCAGTTCATCACCAATG 
               
               
                   
               
               
                 738: TACTCCTTCGAATTAGCTATCCAGTTCATCACCAATG 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE II 
               
             
            
               
                   
               
               
                 Cloned fusion proteins 
               
            
           
           
               
               
               
               
               
               
            
               
                 Protein to be 
                 Protein 
                   
                 Resulting 
                   
                 Primer 
               
               
                 delivred by 
                 Seq. ID. 
                 Backbone 
                 plasmid 
                 Primers. 
                 Seq. ID 
               
               
                 T3SS 
                 No. 
                 plasmid 
                 name 
                 Si_Nr.: 
                 No. 
               
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                 YopE1-138- 
                 3 
                 pBad- 
                 pBad_Si_1 
                 285/286 
                 44/45 and 
               
               
                 MycHis 
                   
                 MycHisA 
                   
                 (EGFP), 
                 46/47 
               
               
                   
                   
                 (Invitrogen) 
                   
                 287/288 
                   
               
               
                   
                   
                   
                   
                 (sycE- 
                   
               
               
                   
                   
                   
                   
                 YopE1-138) 
                   
               
               
                 YopE1-138- 
                 3 
                 pBad- 
                 pBad_Si_2 
                 287/288 
                 46/47 
               
               
                 MycHis 
                   
                 MycHisA 
                   
                 (sycE- 
                   
               
               
                   
                   
                 (Invitrogen) 
                   
                 YopE1-138) 
                   
               
               
                 YopE1-138- 
                 4 
                 pBad_Si_2 
                 pSi_16 
                 292/293 
                 48/49 
               
               
                 IpgB1 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 5 
                 pBad_Si_2 
                 pSi_20 
                 296/297 
                 50/51 
               
               
                 SopE 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 26 
                 pBad_Si_2 
                 pSi_22 
                 299/300 
                 52/53 
               
               
                 Rac1 Q61L 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 27 
                 pBad_Si_2 
                 pSi_24 
                 301/302 
                 54/55 
               
               
                 RhoA Q61E 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 135 
                 pBad_Si_2 
                 pSi_28 
                 296/306 
                 50/56 
               
               
                 SopE-MycHis 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 6 
                 pBad_Si_2 
                 pSi_30 
                 307/308 
                 57/58 
               
               
                 SopB 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 28 
                 pBad_Si_2 
                 pSi_37 
                 367/386 
                 76/79 
               
               
                 FADD 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 7 
                 pBad_Si_2 
                 pSi_38 
                 317/318 
                 59/60 
               
               
                 OspF 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 136 
                 pBad_Si_2 
                 pSi_43 
                 324/351 
                 61/67 
               
               
                 BepG 715-end 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 137 
                 pBad_Si_2 
                 pSi_51 
                 299/339 
                 52/62 
               
               
                 Rac1 Q61L- 
                   
                   
                   
                   
                   
               
               
                 MycHis 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 32 
                 pBad_Si_2 
                 pSi_53 
                 341/342 
                 63/64 
               
               
                 Slmb1-VhH4 
                   
                   
                   
                   
                   
               
               
                 YopE1-138-Bad 
                 29 
                 pBad_Si_2 
                 pSi_57 
                 346/347 
                 65/66 
               
               
                 YopE1-138- 
                 8 
                 pBad_Si_2 
                 pSi_64 
                 364/365 
                 74/75 
               
               
                 SptP 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 33 
                 pBad_Si_2 
                 pSi_70 
                 369/342 
                 77/64 
               
               
                 NLS-Slmb1- 
                   
                   
                   
                   
                   
               
               
                 VhH4 
                   
                   
                   
                   
                   
               
               
                 YopE1-138-Bid 
                 24 
                 pBad_Si_2 
                 pSi_85 
                 387/391 
                 80/82 
               
               
                 YopE1-138-t- 
                 25 
                 pBad_Si_2 
                 pSi_87 
                 389/391 
                 81/82 
               
               
                 Bid 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 22 
                 pBad_Si_2 
                 pSi_97 
                 403/406 
                 83/84 
               
               
                 Caspase3 p17 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 30 
                 pBad_Si_2 
                 pSi_103 
                 410/413 
                 85/86 
               
               
                 GPCR GNA12 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 23 
                 pBad_Si_2 
                 pSi_106 
                 417/420 
                 87/88 
               
               
                 Caspase3 
                   
                   
                   
                   
                   
               
               
                 p10/12 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 9 
                 pBad_Si_2 
                 pSi_111 
                 423/424 
                 89/90 
               
               
                 IpgD 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 34 
                 pBad_Si_2 
                 pSi_112 
                 341/425 
                 63/91 
               
               
                 Slmb1-VhH4- 
                   
                   
                   
                   
                   
               
               
                 NLS 
                   
                   
                   
                   
                   
               
               
                 YopE1-138-z-Bid 
                 19 
                 pBad_Si_2 
                 pSi_116 
                 428/430 
                 92/94 
               
               
                 YopE1-138-z-t-Bid 
                 20 
                 pBad_Si_2 
                 pSi_117 
                 429/430 
                 93/94 
               
               
                 YopE1-138- 
                 11 
                 pBad_Si_2 
                 pSi_118 
                 433/435 
                 95/97 
               
               
                 BepA E305-end 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 10 
                 pBad_Si_2 
                 pSi_119 
                 434/435 
                 96/97 
               
               
                 BepA 
                   
                   
                   
                   
                   
               
               
                 YopE1-138-ET1 
                 36 
                 pBad_Si_2 
                 pSi_120 
                 436/437 
                 98/99 
               
               
                 YopE1-138-z- 
                 21 
                 pbad_Si_1 
                 pSi_121 
                 438/439 
                 100/101 
               
               
                 BIM 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 31 
                 pBad_Si_2 
                 pSi_124 
                 451/373 
                 108/78 
               
               
                 VhH4 nanobody 
                   
                   
                   
                   
                   
               
               
                 recognizing 
                   
                   
                   
                   
                   
               
               
                 EGFP 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 42 
                 pBad_Si_2 
                 pSi_132 
                 463/464 
                 109/110 
               
               
                 TEV protease 
                   
                   
                   
                   
                   
               
               
                 S219V 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 37 
                 pBad_Si_2 
                 pSi_140 
                 477/476 
                 112/111 
               
               
                 EGFP 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 14 
                 pBad_Si_2 
                 pSi_143 
                 478/479 
                 113/114 
               
               
                 Cdkl 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 15 
                 pBad_Si_2 
                 pSi_145 
                 482/483 
                 115/116 
               
               
                 Mad2 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 16 
                 pBad_Si_2 
                 pSi_147 
                 486/487 
                 117/118 
               
               
                 Ink4A 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 17 
                 pBad_Si_2 
                 pSi_150 
                 492/493 
                 119/120 
               
               
                 Ink4B 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 18 
                 pBad_Si_2 
                 pSi_151 
                 494/495 
                 121/122 
               
               
                 Ink4C 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 13 
                 pBad_Si_2 
                 pSi_153 
                 558/559 
                 131/132 
               
               
                 TIFA 
                   
                   
                   
                   
                   
               
               
                 YopE1-138-2x 
                 41 
                 pBad_Si_2 
                 pSi_156 
                 504/505 
                 123/124 
               
               
                 TEVsite - ET1 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 39 
                 pBad_Si_2 
                 pSi_159 
                 511/513 
                 127/129 
               
               
                 2xTEVsite 
                   
                   
                   
                   
                   
               
               
                 EGFP-NLS 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 38 
                 pBad_Si_2 
                 pSi_160 
                 512/476 
                 128/111 
               
               
                 2xTEVsite 
                   
                   
                   
                   
                   
               
               
                 NLS-EGFP 
                   
                   
                   
                   
                   
               
               
                 YopE1-138-2x 
                 40 
                 pBad_Si_2 
                 pSi_161 
                 508/509 
                 125/126 
               
               
                 TEVsite 
                   
                   
                   
                   
                   
               
               
                 INK4C 
                   
                   
                   
                   
                   
               
               
                 YopE1-138-2x 
                 43 
                 pBad_Si_2 
                 pSi_164 
                 515/509 
                 130/126 
               
               
                 TEVsite - Flag - 
                   
                   
                   
                   
                   
               
               
                 INK4C 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 12 
                 pBad_Si_2 
                 pSi_166 
                 561/562 
                 133/134 
               
               
                 murine Traf6 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- Y . 
                 138 
                 pBad_Si_2 
                 pSi_318 
                 677/678 
                 148/149 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 BH3 part 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- Y . 
                 139 
                 pBad_Si_2 
                 pSi_322 
                 682/683 
                 150/151 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine Bax 
                   
                   
                   
                   
                   
               
               
                 BH3 part 
                   
                   
                   
                   
                   
               
               
                 pBad-MycHisA 
                   
                   
                   
                   
                   
               
               
                 SteA1-20 
                 140 
                 pBad-MycHisA 
                 pSi_266 
                 580/612 
                 152/153 
               
               
                   
                   
                 (Invitrogen) 
                   
                   
                   
               
               
                 SteA 
                 141 
                 pBad-MycHisA 
                 pSi_267 
                 580/613 
                 152/154 
               
               
                   
                   
                 (Invitrogen) 
                   
                   
                   
               
               
                 SopE1-81 
                 142 
                 pBad-MycHisA 
                 pSi_268 
                 614/615 
                 155/156 
               
               
                   
                   
                 (Invitrogen) 
                   
                   
                   
               
               
                 SopE1-105 
                 143 
                 pBad-MycHisA 
                 pSi_269 
                 614/616 
                 155/157 
               
               
                   
                   
                 (Invitrogen) 
                   
                   
                   
               
               
                 SteA1-20- S . 
                 144 
                 pSi_266 
                 pSi_270 
                 synthetic 
                 / 
               
               
                   enterica  codon 
                   
                   
                   
                 construct 
                   
               
               
                 optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 SteA- S. enterica   
                 145 
                 pSi_267 
                 pSi_271 
                 synthetic 
                 / 
               
               
                 codon optimized 
                   
                   
                   
                 construct 
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 SopE1-81- S . 
                 146 
                 pSi_268 
                 pSi_272 
                 synthetic 
                 / 
               
               
                   enterica  codon 
                   
                   
                   
                 construct 
                   
               
               
                 optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 SopE1-105- S . 
                 147 
                 pSi_269 
                 pSi_273 
                 synthetic 
                 / 
               
               
                   enterica  codon 
                   
                   
                   
                 construct 
                   
               
               
                 optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- Y . 
                 158 
                 pBad_Si_2 
                 pSi_362 
                 745/746 
                 172/173 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 Ink4A 84-103 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- Y . 
                 159 
                 pBad_Si_2 
                 pSi_363 
                 747/748 
                 174/175 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 p107/RBL1 
                   
                   
                   
                   
                   
               
               
                 657-662 
                   
                   
                   
                   
                   
               
               
                 (AAA02489.1) 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- Y . 
                 160 
                 pBad_Si_2 
                 pSi_364 
                 749/750 
                 176/177 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 p21 141-160 
                   
                   
                   
                   
                   
               
               
                 (AAH13967.1) 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- Y . 
                 161 
                 pBad_Si_2 
                 pSi_366 
                 753/754 
                 178/179 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 p21 145-160 
                   
                   
                   
                   
                   
               
               
                 (AAH13967.1) 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- Y . 
                 162 
                 pBad_Si_2 
                 pSi_367 
                 755/756 
                 180/181 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 p21 17-33 
                   
                   
                   
                   
                   
               
               
                 (AAH13967.1) 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- Y . 
                 163 
                 pBad_Si_2 
                 pSi_368 
                 757/758 
                 182/183 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 cyclin D2 139- 
                   
                   
                   
                   
                   
               
               
                 147 (CAA48493.1) 
                   
                   
                   
                   
                   
               
               
                 SteA-Ink4a- 
                 164 
                 pSi_267 
                 pSi_333 
                 703/704 
                 184/185 
               
               
                 MycHis 
                   
                   
                   
                   
                   
               
               
                 SopE1-105- 
                 165 
                 pSi_269 
                 pSi_334 
                 703/704 
                 184/185 
               
               
                 Ink4a-MycHis 
                   
                   
                   
                   
                   
               
               
                 SteA-Ink4c- 
                 166 
                 pSi_267 
                 pSi_335 
                 PCR1: 
                 186/187, 
               
               
                 MycHis 
                   
                   
                   
                 pSi_267 
                 188/189 
               
               
                   
                   
                   
                   
                 705/706; 
                   
               
               
                   
                   
                   
                   
                 PCR2: 
                   
               
               
                   
                   
                   
                   
                 707/708; 
                   
               
               
                   
                   
                   
                   
                 overlapping 
                   
               
               
                   
                   
                   
                   
                 PCR: 
                   
               
               
                   
                   
                   
                   
                 705/708 
                   
               
               
                 SopE1-105- 
                 167 
                 pSi_269 
                 pSi_336 
                 PCR1: 
                 186/187, 
               
               
                 Ink4c-MycHis 
                   
                   
                   
                 705/706; 
                 188/189 
               
               
                   
                   
                   
                   
                 PCR2: 
                   
               
               
                   
                   
                   
                   
                 707/708; 
                   
               
               
                   
                   
                   
                   
                 overlapping 
                   
               
               
                   
                   
                   
                   
                 PCR: 
                   
               
               
                   
                   
                   
                   
                 705/708 
                   
               
               
                 SteA-Mad2- 
                 168 
                 pSi_267 
                 pSi_337 
                 709/710 
                 190/191 
               
               
                 MycHis 
                   
                   
                   
                   
                   
               
               
                 SopE1-105- 
                 169 
                 pSi_269 
                 pSi_338 
                 709/710 
                 190/191 
               
               
                 Mad2-MycHis 
                   
                   
                   
                   
                   
               
               
                 SteA-Cdk1- 
                 170 
                 pSi_267 
                 pSi_339 
                 711/712 
                 192/193 
               
               
                 MycHis 
                   
                   
                   
                   
                   
               
               
                 SopE1-105- 
                 171 
                 pSi_269 
                 pSi_340 
                 711/712 
                 192/193 
               
               
                 Cdkl-MycHis 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- Y . 
                 194 
                 pBad_Si_2 
                 pSi_315 
                 synthetic 
                 / 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                 construct 
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 195 
                 pBad_Si_2 
                 pSi_236 
                 585/586 
                 197/198 
               
               
                 Ubiquitin 
                   
                   
                   
                   
                   
               
               
                 YopE1-138- 
                 196 
                 pSi_236 
                 pSi_237_II 
                 588/509 
                 199/126 
               
               
                 Ubiquitin-Flag- 
                   
                   
                   
                   
                   
               
               
                 INK4C-MycHis 
                 200 
                 pBad_Si_2 
                 pSi_357 
                 733/735 
                 204/205 
               
               
                 YopE1-138-( Y . 
                   
                   
                   
                   
                   
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 BH3 part) ready 
                   
                   
                   
                   
                   
               
               
                 for insertion of 
                   
                   
                   
                   
                   
               
               
                 further domains 
                   
                   
                   
                   
                   
               
               
                 YopE1-138-( Y . 
                 201 
                 pBad_Si_2 
                 pSi_358 
                 736/738 
                 206/207 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine BAX 
                   
                   
                   
                   
                   
               
               
                 BH3 part) ready 
                   
                   
                   
                   
                   
               
               
                 for insertion of 
                   
                   
                   
                   
                   
               
               
                 further domains 
                   
                   
                   
                   
                   
               
               
                 YopE1-138-( Y . 
                 202 
                 pSi_357 
                 pSi_371 
                 733/734 
                 204/208 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 BH3 part) 2   
                   
                   
                   
                   
                   
               
               
                 YopE1-(138- Y . 
                 203 
                 pSi_358 
                 pSi_373 
                 733/734 
                 204/208 
               
               
                 
                   enterocolitica 
                 
                 209 
                 pBad_Si_2 
                 pSi_353 
                 725/726 
                 212/213 
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 BH3 part- Y . 
                   
                   
                   
                   
                   
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine BAX 
                   
                   
                   
                   
                   
               
               
                 BH3 part 
                   
                   
                   
                   
                   
               
               
                 YopE 1 _138- 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 BH3 extended 
                   
                   
                   
                   
                   
               
               
                 part 
                   
                   
                   
                   
                   
               
               
                 YopE 1-138 -10 Aa 
                 210 
                 pBad_Si_2 
                 pSi_354 
                 727/728 
                 214/215 
               
               
                 linker -  Y . 
                   
                   
                   
                   
                   
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 BH3 part 
                   
                   
                   
                   
                   
               
               
                 YopE1-(138- Y . 
                 211 
                 pSI_357 
                 pSi_374 
                 736/737 
                 206/216 
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine Bax 
                   
                   
                   
                   
                   
               
               
                 BH3 part-  Y . 
                   
                   
                   
                   
                   
               
               
                 
                   enterocolitica 
                 
                   
                   
                   
                   
                   
               
               
                 codon optimized 
                   
                   
                   
                   
                   
               
               
                 murine tBid 
                   
                   
                   
                   
                   
               
               
                 BH3 part 
               
               
                   
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE III 
               
             
            
               
                   
               
               
                 Mutators for genetic modification 
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Mutator/ 
                 To be 
                 Backbone 
                 Resulting 
                 Primers 
                 Primers 
                 used with 
               
               
                 Construct 
                 inserted onto: 
                 plasmid 
                 plasmid name 
                 Si_Nr.: 
                 Seq. Id No. 
                 parent strain 
               
               
                   
               
               
                 YopE 1-138 - 
                 pYV 
                 pKNG101 
                 pSi_408 
                 Synthetic 
                 / 
                 / 
               
               
                 tBID BH3 
                   
                   
                   
                 gene 
               
               
                 YopE 1-138 - 
                 pYV 
                 pKNG101 
                 pSi_419 
                 Synthetic 
                 / 
                 Strain 
               
               
                 (tBID BH3) 2   
                   
                   
                   
                 gene 
                   
                 mutated 
               
               
                   
                   
                   
                   
                   
                   
                 with 
               
               
                   
                   
                   
                   
                   
                   
                 pSi_408 
               
               
                   
               
            
           
         
       
     
     Yop Secretion. 
     Induction of the yop regulon was performed by shifting the culture to 37° C. in BHI-Ox (secretion-permissive conditions) [39]. As carbon source glucose was added (4 mg/ml). 
     Total cell and supernatant fractions were separated by centrifugation at 20 800 g for 10 min at 4° C. The cell pellet was taken as total cell fraction. Proteins in the supernatant were precipitated with trichloroacetic acid 10% (w/v) final for 1 h at 4° C. After centrifugation (20 800 g for 15 min) and removal of the supernatant, the resulting pellet was washed in ice-cold Acetone over-night. The samples were centrifuged again, the supernatant was discarded and the pellet was air-dried and resuspened in 1×SDS loading dye. 
     Secreted proteins were analysed by SDS-PAGE; in each case, proteins secreted by 3×10 8  bacteria were loaded per lane. Detection of specific secreted proteins by immunoblotting was performed using 12.5% SDS-PAGE gels. For detection of proteins in total cells, 2×10 8  bacteria were loaded per lane, if not stated otherwise, and proteins were separated on 12.5% SDS-PAGE gels before detection by immunoblotting. 
     Immunoblotting was carried out using rat monoclonal antibodies against YopE (MIPA193-13A9; 1:1000, [40]). The antiserum was preabsorbed twice overnight against  Y. enterocolitica  ΔHOPEMT asd to reduce background staining. Detection was performed with secondary antibodies directed against rat antibodies and conjugated to horseradish peroxidase (1:5000; Southern biotech), before development with ECL chemiluminescent substrate (LumiGlo, KPM). 
     Cell Culture and Infections. 
     HeLa Ccl2, swiss 3T3 fibroblast cells, 4T1, B16F10 and D2A1 were cultured in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% FCS and 2 mM L-Glutamine (cDMEM). HUVECs were isolated and cultivated as described [41]. Jurkat and 4T1 cells were cultured in RPMI 1640 supplemented with 10% FCS and 2 mM L-Glutamine.  Y. enterocolitica  were grown in BHI with additives overnight at RT, diluted in fresh BHI to an OD 600  of 0.2 and grown for 2 h at RT before a temperature shift to a 37° C. waterbath shaker for further 30 min or for 1 h in case of delivery of EGFP. Finally, the bacteria were collected by centrifugation (6000 rcf, 30 sec) and washed once with DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine.  S. enterica  were grown in LB with additives overnight at 37° C. and either diluted 1:40 in fresh LB and grown for 2.5 h at 37° C. (SpiI T3SS inducting conditions) or the overnight culture was further incubated at 37° C. (SpiII T3SS inducing conditions). Finally, the bacteria were collected by centrifugation (6000 rcf, 30 sec) and washed once with DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine. Cells seeded in 96-well (for Immunofluorescence) or 6-well (for Western blotting) plates were infected at indicated MOIs in DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine. After adding bacteria, plates were centrifuged for 1 min at 1750 rpm and placed at 37° C. for indicated time periods. Extracellular bacteria were killed by gentamicin (100 mg/ml) if indicated. In case of immunofluorescence analysis, infection assays were stopped by 4% PFA fixation. For Western blot analysis cells were washed twice with ice-cold PBS and Phospho-safe lysis buffer (Novagen) was added to lyse the cells. After incubation on ice, the cells were centrifuged (16 000 rcf, 25 min, 4° C.). Supernatants were collected and analyzed for total protein content by Bradford BCA assay (Pierce) before SDS PAGE and Western blotting using anti-Phospho-Akt (Ser473 and T308, both Cell Signaling), anti-Actin (Millipore), Anti-Bid (Cell Signaling), anti-Myc (Santa Cruz), anti-p38 (Cell Signaling), anti-phospho-p-38 (Thr180/Tyr182; Cell Signaling), anti-Caspase-3 p17 (Cell Signaling) and anti-Ink4C (Cell Signaling) antibody. 
     Secretion Analysis with  S. enterica.    
     For induction of protein secretion by  S. enterica, S. enterica  were cultivated overnight in LB containing 0.3 M NaCl on an orbital shaker (set to 150 rpm).  S. enterica  were then diluted 1:50 in fresh LB containing 0.3 M NaCl and grown for 4 h at 37° C. without shaking. 
     Total cell and supernatant fractions were separated by centrifugation at 20 800 g for 20 min at 4° C. The cell pellet was taken as total cell fraction. Proteins in the supernatant were precipitated with trichloroacetic acid 10% (w/v) final for 1 h at 4° C. After centrifugation (20 800 g for 15 min) and removal of the supernatant, the resulting pellet was washed in ice-cold Acetone over-night. The samples were centrifuged again, the supernatant was discarded and the pellet was air-dried and resuspended in 1×SDS loading dye. 
     Secreted proteins were analysed by SDS-PAGE; in each case, proteins secreted by 3×108 bacteria were loaded per lane. Detection of specific secreted proteins by immunoblotting was performed using 12.5% SDS-PAGE gels. For detection of proteins in total cells, 2×10 8  bacteria were loaded per lane, if not stated otherwise, and proteins were separated on 12.5% SDS-PAGE gels before detection by immunoblotting. Immunoblotting was carried out using anti-Myc (Santa Cruz) antibody. 
     Western Blotting of T3SS Translocated Proteins from Infected Cells. 
     HeLa cells in 6-well plates were infected at an MOI of 100 as described above. In case of coinfection with the TEV protease translocating  Y. enterocolitica  strain, the OD 600  of the strains was set and the two bacterial suspensions were mixed in a tube at a ratio of 1:1 (if not otherwise indicated) before addition to the cells. At the end of the infection, the cells were washed twice with ice-cold PBS and collected by scraping in a small volume of ice-cold PBS. After centrifugation (16 000 rcf, 5 min, 4° C.) the pellet was dissolved in 0.002% digitonin supplemented with a protease inhibitor cocktail (Roche complete, Roche). The dissolved pellets were incubated for 5 minutes on ice and then centrifuged (16 000 rcf, 25 min, 4° C.). Supernatants were collected and analyzed for total protein content by Bradford BCA assay (Pierce) before SDS PAGE and Western blotting using an anti-Myc (Santa Cruz, 9E11) or anti-Ink4C (Cell Signaling) antibody. 
     Immunofluorescence. 
     Cell seeded in 96-well plates (Corning) were infected as described above and after fixation with 4% PFA the cells were washed three times with PBS. The wells were then blocked using 5% goat serum in PBS 0.3% Triton X-100 for 1 h at RT. The primary antibody (anti-Myc, Santa Cruz, 1:100) was diluted in PBS with 1% BSA and 0.3% Triton X-100 and cells were incubated overnight at 4° C. 
     Cells were washed 4 times with PBS before the secondary antibody (AF 488 anti-mouse, life technologies, 1:250) diluted in PBS with 1% BSA and 0.3% Triton X-100 was added. If needed Hoechst DNA staining (life technologies, 1:2500) and/or actin staining (Dy647-Phalloidin, DyeOmics) were included. In some cases only the DNA and/or actin stain was applied directly after washing the PFA off. Cells were incubated for 1 h at RT, washed three times with PBS and analyzed by automated image analysis as described below. 
     Automated Microscopy and Image Analysis. 
     Images were automatically acquired with an ImageXpress Micro (Molecular devices, Sunnyvale, USA). Quantification of anti-Myc staining intensities was performed using MetaXpress (Molecular devices, Sunnyvale, USA). Regions within cells excluding nuclear regions and regions containing bacteria were manually chosen (circles with an area of 40 pixels) and average intensity was recorded. 
     TNFα Stimulation and Western Blotting of Phospho-p38. 
     HeLa cells seeded in 6-well plates were infected with an MOI of 100 as described above. 30 min p.i Gentamicin was added and 45 min p.i. TNFα was added (10 ng/ml). 1 h 15 min p.i. cells were washed twice with ice-cold PBS and Phospho-safe lysis buffer (Novagen) was added to lyse the cells. After incubation on ice, the cells were centrifuged (16 000 rcf, 25 min, 4° C.). Supernatants were collected and analyzed for total protein content by Bradford BCA assay (Pierce) before SDS PAGE and Western blotting using an anti-Phospho-p38, total p38 antibodies (Cell Signaling) and anti-Actin antibody (Millipore). 
     cAMP Level Determination of Infected HeLa Cells. 
     HeLa cells seeded in 96-well plates were infected as described above. 30 min before the infection cDMEM was changed to DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine and 100 uM 3-Isobutyl-1-methylxanthin (IBMX, Sigma Aldrich). 60 min p.i. Gentamicin was added and cells were further incubated at 37° C. for another 90 min. Determination of cAMP was performed using a competitive ELISA according to the manufacturers instructions (Amersham, cAMP Biotrak, RPN225). As a positive control indicated amount of cholera toxin (C8052, Sigma Aldrich) was added for 1 h to cells in DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine and 100 uM IBMX. 
     Zebrafish Embryo Infections, Imaging and Automated Image Quantification. 
     All animal experiments were performed according to approved guidelines. Zebrafish were maintained at standard conditions [42]. Embryos were staged by hours postfertilization (hpf) at 28.5° C. [43]. The following zebrafish lines were used in this study: wild type fish (AB/EK and EK/TL). Infection protocol followed guidelines given in [44]. 12 hpf embryos were maintained in E3 medium containing 0.2 mM N-phenylthiourea (PTU) to prevent pigment formation. 2 days postfertilization (dpf) embryos were anesthetized by 0.2 mg/ml Tricaine and aligned on 1% agar plates in E3 using a hair loop tool [44].  Y. enterocolitica  were grown in BHI supplemented with 0.4% Arabinose and antibiotics and mDap overnight at RT, diluted in fresh BHI with 0.5% Arabinose and other additives to an OD 600  of 0.2 and grown for 2 h at RT before a temperature shift to a 37° C. waterbath shaker for further 45 min. Finally, the bacteria were collected by centrifugation (6000 rcf, 30 sec) and washed once with PBS. The OD 600  was set to 2 in PBS containing mDAP. 1-2 nL of this suspension were injected into the hindbrain of aligned zebrafish embryos using an Femtojet Microinjector (Eppendorf) using Femtotips II (Eppendorf), where the tip of the needle had been broken off with fine tweezers. The injection time was set to 0.2 s and the compensation pressure to 15 hPa (Eppendorf, Femtojet) and the injection pressure was adjusted between 600 and 800 hPa. Drop size and thus the inoculum was checked by microscopy and by control plating. Following microinjection the fish were collected in E3 containing Tricaine and PTU and incubated for 30 min at 37° C. and incubated for further 5 h at 28° C. A fluorescence binocular (Leica) was used to observe bacterial EGFP fluorescence 1 h post infection in zebrafish hindbrains, and embryos that are not properly injected were discarded. At the end of the infection, fish were fixed with 2% ice-cold PFA for 1 h on ice and further with fresh ice-cold PFA overnight at 4° C. Antibody staining was performed as described previously [45,46]. Briefly, embryos were washed 4 times with PBS 0.1% Tween for 5 min each wash and permeabilized with PBS-T+0.5% Triton X-100 for 30 min at RT. Embryos were blocked in blocking solution (PBS 0.1% Tween 0.1% TritonX-100 5% goat serum and 1% BSA) at 4° C. overnight. Antibody (Cleaved Caspase-3 (Asp175), Cell Signaling) was diluted 1:100 in blocking solution and incubated under shaking at 4° C. in the dark. Fish were washed 7 times with PBS 0.1% Tween for 30 min before the secondary antibody (goat anti-rabbit AF647, Invitrogen, 1:500) diluted in blocking solution was added and incubated at 4° C. overnight. Larvae were washed with PBS 0.1% Tween four times 30 min at 4° C. and once overnight and further washed 3-4 times. Images were taken with Leica TCS SP5 confocal microscope using a 40× water immersion objective. Images were analyzed using Imaris (Bitplane) and Image J software (http://imagej.nih.gov/ij/). 
     Image analysis (on n=14 for pBad_Si2 or n=19 for z-BIM) was performed via CellProfiler [47] on maximum intensity z projections of recorded z-stack images. 
     Briefly, bacteria were detected via the GFP channel. Around each area of a bacterial spot a circle with a radius of 10 pixels was created. Overlapping regions were separated equally among the connecting members. In those areas closely surrounding bacteria, the Caspase 3 p17 staining intensity was measured. 
     Sample Preparation for Phosphoproteomics. 
     For each condition, two 6-well plates of HeLa CCL-2 cells were grown to confluency. Cells were infected for 30 min as described above. At the indicated time-points, the plates were put on ice and washed twice with ice-cold PBS. Samples were then collected in urea solution [8 M Urea (AppliChem), 0.1 M Ammoniumbicarbonate (Sigma), 0.1% RapiGest (Waters), 1× PhosSTOP (Roche)]. The samples were briefly vortexed, sonicated at 4° C. (Hielscher), shaked for 5 min on a thermomixer (Eppendorf) and centrifuged for 20 min at 4° C. and 16′000 g. Supernatants were collected and stored at −80° C. for further processing. BCA Protein Assay (Pierce) was used to measure protein concentration. 
     Phosphopeptide Enrichment. 
     Disulfide bonds were reduced with tris(2-carboxyethyl)phosphine at a final concentration of 10 mM at 37° C. for 1 h. Free thiols were alkylated with 20 mM iodoacetamide (Sigma) at room temperature for 30 min in the dark. The excess of iodoacetamide was quenched with N-acetyl cysteine at a final concentration of 25 mM for 10 min at room temperature. Lys-C endopeptidase (Wako) was added to a final enzyme/protein ratio of 1:200 (w/w) and incubated for 4 h at 37° C. The solution was subsequently diluted with 0.1 M ammoniumbicarbonate (Sigma) to a final concentration below 2 M urea and digested overnight at 37° C. with sequencing-grade modified trypsin (Promega) at a protein-to-enzyme ratio of 50:1. Peptides were desalted on a C18 Sep-Pak cartridge (Waters) and dried under vacuum. Phosphopeptides were isolated from 2 mg of total peptide mass with TiO 2  as described previously [48]. Briefly, dried peptides were dissolved in an 80% acetonitrile (ACN)-2.5% trifluoroacetic acid (TFA) solution saturated with phthalic acid. Peptides were added to the same amount of equilibrated TiO 2  (5-μm bead size, GL Sciences) in a blocked Mobicol spin column (MoBiTec) that was incubated for 30 min with end-over-end rotation. The column was washed twice with the saturated phthalic acid solution, twice with 80% ACN and 0.1% TFA, and finally twice with 0.1% TFA. The peptides were eluted with a 0.3 M NH 4 OH solution. The pH of the eluates was adjusted to be below 2.5 with 5% TFA solution and 2 M HCl. Phosphopeptides were again desalted with microspin C18 cartridges (Harvard Apparatus). 
     LC-MS/MS Analysis. 
     Chromatographic separation of peptides was carried out using an EASY nano-LC system (Thermo Fisher Scientific), equipped with a heated RP-HPLC column (75 μm×45 cm) packed in-house with 1.9 μm C18 resin (Reprosil-AQ Pur, Dr. Maisch). Aliquots of 1 μg total phosphopeptide sample were analyzed per LC-MS/MS run using a linear gradient ranging from 98% solvent A (0.15% formic acid) and 2% solvent B (98% acetonitrile, 2% water, 0.15% formic acid) to 30% solvent B over 120 minutes at a flow rate of 200 nl/min. Mass spectrometry analysis was performed on a dual pressure LTQ-Orbitrap mass spectrometer equipped with a nanoelectrospray ion source (both Thermo Fisher Scientific). Each MS1 scan (acquired in the Orbitrap) was followed by collision-induced dissociation (CID, acquired in the LTQ) of the 20 most abundant precursor ions with dynamic exclusion for 30 seconds. For phosphopeptide analysis the 10 most abundant precursor ions were subjected to CID with enabled multistage activation. Total cycle time was approximately 2 s. For MS1, 10 6  ions were accumulated in the Orbitrap cell over a maximum time of 300 ms and scanned at a resolution of 60,000 FWHM (at 400 m/z). MS2 scans were acquired using the normal scan mode, a target setting of 10 4  ions, and accumulation time of 25 ms. Singly charged ions and ions with unassigned charge state were excluded from triggering MS2 events. The normalized collision energy was set to 32%, and one microscan was acquired for each spectrum. 
     Label-Free Quantification and Database Searching. 
     The acquired raw-files were imported into the Progenesis software tool (Nonlinear Dynamics, Version 4.0) for label-free quantification using the default parameters. MS2 spectra were exported directly from Progenesis in mgf format and searched using the MASCOT algorithm (Matrix Science, Version 2.4) against a decoy database [49] containing normal and reverse sequences of the predicted SwissProt entries of  Homo sapiens  (www.ebi.ac.uk, release date May 16, 2012) and commonly observed contaminants (in total 41,250 sequences) generated using the SequenceReverser tool from the MaxQuant software (Version 1.0.13.13). To identify proteins originating from  Y. enterocolitica , non phosphopeptide enriched samples were searched against the same database above including predicted SwissProt entries of  Y. enterocolitica  (www.ebi.ac.uk, release date Aug. 15, 2013) The precursor ion tolerance was set to 10 ppm and fragment ion tolerance was set to 0.6 Da. The search criteria were set as follows: full tryptic specificity was required (cleavage after lysine or arginine residues unless followed by proline), 2 missed cleavages were allowed, carbamidomethylation (C) was set as fixed modification and phosphorylation (S,T,Y) or oxidation (M) as a variable modification for TiO2 enriched or not enriched samples, respectively. Finally, the database search results were exported as an xml-file and imported back to the Progenesis software for MS1 feature assignment. For phosphopeptide quantification, a csv-file containing the MS1 peak abundances of all detected features was exported and for not enriched samples, a csv-file containing all protein measurements based on the summed feature intensities of all identified peptides per protein was created. Importantly, the Progenesis software was set that proteins identified by similar sets of peptides are grouped together and that only non-conflicting peptides with specific sequences for single proteins in the database were employed for protein quantification. Both files were further processed using the in-house developed SafeQuant v1.0 R script (unpublished data, available at https://github.com/eahrne/SafeQuant/). In brief, the software sets the identification level False Discovery Rate to 1% (based on the number of decoy protein sequence database hits) and normalizes the identified MS1 peak abundances (Extracted Ion Chromatogram, XIC) across all samples, i.e. the summed XIC of all confidently identified peptide features is scaled to be equal for all LC-MS runs. Next, all quantified phosphopeptides/proteins are assigned an abundance ratio for each time point, based on the median XIC per time point. The statistical significance of each ratio is given by its q-value (False Discovery Rate adjusted p-values), obtained by calculating modified t-statistic p-values [50] and adjusting for multiple testing [51]. The location of the phosphorylated residues was automatically assigned by MASCOT (score &gt;10). All annotated spectra together with the MS raw files and search parameters employed, will be deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository [52]. Sequence alignment was performed using EMBL-EBI web based ClustalW2 multiple sequence alignment tool at htp://www.ebi.ac.ukTools/msa/clustalw2/. 
     Biodistribution in 4T1 Tumor Allograft Mouse Models 
     All animal experiments were approved (license 1908; Kantonales Veteriniramt Basel-Stadt) and performed according to local guidelines (Tierschutz-Verordnung, Basel-Stadt) and the Swiss animal protection law (Tierschutz-Gesetz). 6 week old BALB/c mice were ordered from Janvier Labs. After at least one week of accommodation, mice were anesthetized using isoflurane and 100 ul 4T1 cells (1×10 5 -1×10 6  cells) were subcutaneously injected into the flank of BALB/c mice. Throughout the experiment, mice were scored for behavior and physical appearance, and surface temperature, as well as body weight was measured. 
     Once tumors had developed, mice were administered an 8 mg/ml desferal solution (10 ml/kg) through i.p. injection. On the following day, mice were infected with  Y. enterocolitica  MRS40 or  Y. enterocolitica  MRS40 ΔHOPEMT (2×10 5 , 1×10 6  or 1×10 7  bacteria) by injection into the tail vein. The inoculum i.v. administered to the mice was validated by dilution plating. In some experiments, tumor progression was followed by daily measurements of tumor length and width with digital calipers. Tumor volume was determined as 0.523×length×width 2 . On respective days postinfection, mice were sacrificed by CO 2  inhalation. A blood sample was immediately isolated through aspiration from the heart. Liver, spleen, lung and the tumor were isolated and their weight determined. The organs and the tumor were homogenized. CFU in each sample was determined by spotting of serial dilutions onto LB agar plates containing nalidixic acid (35 ug/ml). 
     B) Results 
     A Protein Delivery System Based on Type 3 Secretion of YopE Fusion Proteins 
     While the very N-terminus of the  Y. enterocolitica  T3SS effector YopE (SEQ ID No. 1) contains the secretion signal sufficient to translocate heterologous proteins [26], the chaperone-binding site (CBS) for its chaperone (SycE) is not included [53]. We selected the N-terminal 138 amino acids of YopE (SEQ ID No. 2) to be fused to proteins to be delivered, as this had been shown to give best results for translocation of other heterologous T3S substrates [28]. As these N-terminal 138 amino acids of YopE contain the CBS, we further decided to coexpress SycE. The SycE-YopE 1-138  fragment cloned from purified  Y. enterocolitica  pYV40 virulence plasmid contains the endogenous promoters of YopE and of its chaperone SycE ( FIG. 10 ). Therefore, SycE and any YopE 1-138  fusion protein are induced by a rapid temperature shift from growth at RT to 37° C. Culture time at 37° C. will affect fusion protein amount present in bacteria. A multiple cloning site (MCS) was added at the 3′ end of YopE 1-138  ( FIG. 10  B) followed by a Myc and a 6×His tag and a Stop codon. 
     The background strain was carefully selected. First, to limit the translocation of endogenous effectors, we used a  Y. enterocolitica  strain that was deleted for all known effectors, Yop H, O, P, E, M and T (named ΔHOPEMT) [54]. In addition, we occasionally used an auxotroph mutant that cannot grow in absence of exogenous meso-2,6-diaminopimelic acid [55]. This strain was deleted for the aspartate-beta-semialdehyde dehydrogenase gene (Δasd), and classified as biosafety level 1 by the Swiss safety agency (amendment to A010088/2). In addition, we deleted the adhesion proteins YadA and/or InvA to offer a larger choice of background strains. While the use of the yadA or yadA/invA strains reduce the background signalling induced [56], the delivered protein amount is affected as well [57]. 
     Characterization of YopE Fusion Protein Delivery into Eukaryotic Cells 
     In an in-vitro secretion assay (see  FIG. 1  A), protein secretion into the surrounding liquid is artificially induced. After TCA based protein precipitation, Western blot analysis with anti-YopE antibody was used to determine protein amounts secreted ( FIG. 1  B). While a wt strain secreted full length YopE, the ΔHOPEMT asd strains did not. Upon presence of YopE 1-138 -Myc-His (further termed YopE 1-138 -Myc; SEQ_ID No._3) a smaller YopE band became visible ( FIG. 1  B). Hence, the YopE 1-138  fragment is well secreted in the set up described here. To analyze homogeneity of protein translocation into eukaryotic cells, we infected HeLa cells with the YopE 1-138 -Myc encoding strain and stained the Myc tag by IF ( FIGS. 2  A and B). While in the beginning only the bacteria were stained, at 30 min post infection (p.i.) cell outlines start to be visible, which is enhanced upon increased infection time ( FIG. 2  B). This trend is well reflected by the Myc tag staining intensity inside HeLa cells ( FIGS. 2  A and B). The YopE 1-138 -Myc can be detected everywhere in the cells ( FIG. 2  A), except in the nuclei [58]. Remarkably, most if not all cells were reached by this approach in a comparable way. As  Y. enterocolitica  is known to infect many different cell types [59], we followed YopE 1-138 -Myc delivery into various cell lines. The same homogenous anti-Myc IF staining was observed in infected murine fibroblasts, Jurkat cells and HUVECs ( FIG. 11 ). Even more, tuning the MOI up or down allows modulating the protein amount delivered ( FIG. 2  C), while still most of the cells remain targeted. A low bacterial number will not result in few cells with lots of delivered protein but rather with most cells containing a low amount of delivered protein ( FIG. 2  C). 
     Redirection of T3SS Delivered Proteins to the Nucleus 
     As YopE itself localized to the cytoplasm ( FIG. 2  A), it is of special interest to test if the YopE 1-138  fragment hampers localization of nuclear fusion proteins. We therefore added the SV40 NLS to the C-terminus (and N-terminus, similar results) of YopE 1-138 -EGFP (SEQ ID No. 39 and SEQ ID No. 38, respectively). While YopE 1-138 -EGFP (SEQ ID No. 37) led to a weak cytoplasmic staining, YopE 1-138 -EGFP-NLS gave rise to a stronger nuclear EGFP signal in HeLa cells infected ( FIG. 3 ). This indicates that the YopE 1-138  fragment is compatible with the use of an NLS. While mCherry had already been used in plant pathogens [60], this represents a successful delivery of a GFP-like protein via human or animal pathogenic bacteria encoding a T3SS. This validates the SycE and YopE 1-138  dependent strategy to be very promising for delivery of many proteins of choice. 
     Removal of the YopE 1-138  Appendage after Translocation of the Fusion Protein to the Eukaryotic Cell 
     While for bacterial delivery the YopE 1-138  fragment is of great benefit, it might hamper the fusion proteins function and/or localization. Therefore, its removal after protein delivery would be optimal. To this end, we introduced two TEV cleavage sites (ENLYFQS) [61-63] in between YopE 1-138  and a fusion partner (the transcriptional regulator ET1-Myc (SEQ ID No. 36 and 41) [64] and human INK4C (SEQ ID No. 40 and SEQ ID No. 43)). To keep the advantages of the presented method, we further fused the TEV protease (S219V variant; [65]) to YopE 1-138  (SEQ ID No. 42) in another  Y. enterocolitica  strain. HeLa cells were infected with both strains at once. To allow analysis of the translocated fraction of proteins only, infected HeLa cells were lysed at 2 h p.i. ( FIG. 4 ) with Digitonin, which is known not to lyse the bacteria ([66]; see  FIG. 12  for control). Western blot analysis revealed the presence of the YopE 1-138 -2×TEV-cleavage-site-ET1-Myc or YopE 1-138 -2×TEV-cleavage-site-Flag-INK4C-Myc only when cells had been infected with the corresponding strain ( FIGS. 4  A and C). Upon overnight digestion of this cell-lysate with purified TEV protease, a shifted band could be observed ( FIGS. 4  A and C). This band corresponds to ET1-Myc ( FIG. 4  C) or Flag-INK4C ( FIG. 4  A) with the N-terminal remnants of the TEV cleavage site, most likely only one Serine. Upon coinfection of cells with the strain delivering the TEV protease, the same cleaved ET1-Myc or Flag-INK4C fragment became visible, indicating that the TEV protease delivered via T3SS is functional and that single cells had been infected by both bacterial strains ( FIGS. 4  A and C). While cleavage is not complete, the majority of translocated protein is cleaved already 2 h post infection and even over-night digestion with purified TEV protease did not yield better cleavage rates ( FIG. 4  B). As reported, TEV protease dependent cleavage might need optimization dependent on the fusion protein [67,68]. TEV protease dependent removal of the YopE 1-138  appendage after translocation hence provides for the first time a T3SS protein delivery of almost native heterologous proteins, changing the amino acid composition by only one N-terminal amino acid. 
     An alternative approach to the TEV protease dependent cleavage of the YopE fragment consisted in incorporating Ubiquitin into the fusion protein of interest. Indeed, Ubiquitin is processed at its C-terminus by a group of endogenous Ubiquitin-specific C-terminal proteases (Deubiquitinating enzymes, DUBs). As the cleavage is supposed to happen at the very C-terminus of Ubiquitin (after G76), the protein of interest should be free of additional amino acid sequence. This method was tested on the YopE1-138-Ubiquitin-Flag-INK4C-MycHis fusion protein. In control cells infected by YopE1-138-Flag-INK4C-MycHis-expressing bacteria, a band corresponding to YopE1-138-Flag-INK4C-MycHis was found, indicative of efficient translocation of the fusion protein ( FIG. 24 ). When cells were infected for 1 h with YopE1-138-Ubiquitin-Flag-INK4C-MycHis-expressing bacteria, an additional band corresponding to the size of Flag-INK4C-MycHis was visible, indicating that part of the fusion protein was cleaved. This result shows that the introduction of Ubiquitin into the fusion protein enables to cleave off the YopE1-138 fragment without a need for an exogenous protease. 
     Translocation of Type III and Type IV Bacterial Effectors 
     SopE from  Salmonella enterica  is a well-characterized guanine nucleotide exchange factor (GEF) that interacts with Cdc42, promoting actin cytoskeletal remodeling [69]. Whereas the translocation of YopE 1-138 -Myc into HeLa cells has no effect, translocated YopE 1-138 -SopE (SEQ ID No. 5 and 135) induced dramatic changes in the actin network ( FIG. 5  A). Similar results were obtained with another GEF effector protein, IpgB1 from  Shigella flexneri  (SEQ ID No. 4). Remarkably, first changes in the actin cytoskeleton were observed as fast as 2 min p.i. ( FIG. 5  A). Therefore, one can conclude that T3SS dependent protein delivery happens immediately after infection is initiated by centrifugation. To proof strict T3SS dependent transport, one of the T3SS proteins forming the translocation pore into the eukaryotic cell membrane was deleted (YopB, see [70]) ( FIG. 12 ). 
     During  Salmonella  infection, SopE translocation is followed by translocation of SptP, which functions as a GTPase activating protein (GAP) for Cdc42 [71]. Whereas the translocation of YopE 1-138 -SopE-Myc (SEQ ID No. 135) alone triggered massive F-actin rearrangements, the co-infection with YopE 1-138 -SptP (SEQ ID No. 8) expressing bacteria abolished this effect in a dose dependent manner ( FIG. 5  B). An anti-Myc staining indicated that this inhibition was not due to a reduced level of YopE 1-138 -SopE-Myc translocation ( FIG. 5  B). Together these results showed that the co-infection of cells with two bacterial strains is a valid method to deliver two different effectors into single cells to address their functional interaction. 
     The  S. flexneri  type III effector OspF functions as a phosphothreonine lyase that dephosphorylates MAP kinases p38 and ERK [72]. To test the functionality of translocated YopE 1-138 -OspF (SEQ ID No. 7), we monitored the phosphorylation of p38 after stimulation with TNFα. In uninfected cells or in cells infected with YopE 1-138 -Myc expressing bacteria, TNFα induced p38 phosphorylation. In contrast, after translocation of YopE 1-138 -OspF, TNFα-induced phosphorylation was abolished, showing that the delivered OspF is active towards p38 ( FIG. 6  A). 
     During  Salmonella  infection, the type III effector SopB protects epithelial cells from apoptosis by sustained activation of Akt [73]. Whereas the translocation of YopE 1-138 -Myc or YopE 1-138 -SopE had no effect on Akt, the translocation of YopE 1-138 -SopB (SEQ ID No. 6) induced a strong phosphorylation of Akt at T308 and S473, reflecting the active form ( FIG. 6  B). Similar results were obtained with the SopB-homolog from  S. flexneri  (IpgD, SEQ ID No. 9). Altogether, our results show that the YopE 1-138 -based delivery system functions for all T3S effectors tested so far, and that it allows investigating proteins involved in the control of central cellular functions including the cytoskeleton, inflammation and cell survival. 
     A number of bacteria, including  Agrobacterium tumefaciens, Legionella pneumophila  and  Bartonella henselae , use type IV secretion to inject effectors into cells. We tested whether the type IV effector BepA from  B. henselae  could be translocated into HeLa cells using our tool. Full length BepA (SEQ ID No. 10) and BepA E305-end  (SEQ ID No. 11) containing the C-terminal Bid domain, were cloned and cells were infected with the respective strains. As BepA was shown to induce the production of cyclic AMP (cAMP) [74], the level of cAMP in HeLa cells was measured after infection. Whereas the translocation of the Bid domain of the  B. henselae  effector BepG (SEQ ID No. 136) failed to induce cAMP, full length BepA and BepA E305-end  triggered cAMP production in expected amounts [74] ( FIG. 6  C). This result shows, that type IV effectors can also be effectively delivered by the YopE 1-138 -based delivery system into host cell targets and that they are functional. 
     Translocation of Eukaryotic Proteins into Epithelial Cells 
     To show that human proteins can translocate via type III secretion we fused human apoptosis inducers for delivery by  Y. enterocolitica  to YopE 1-138  or for delivery by  S. enterica  to SteA 1-20 , SteA, SopE 1-81  or SopE 1-105 . We then monitored the translocation of the human BH3 interacting-domain death agonist (BID, SEQ ID No. 24), which is a pro-apoptotic member of the Bcl-2 protein family. It is a mediator of mitochondrial damage induced by caspase-8 (CASP8). CASP8 cleaves BID, and the truncated BID (tBID, SEQ ID No. 25) translocates to mitochondria where it triggers cytochrome c release. The latter leads to the intrinsic mode of caspase 3 (CASP3) activation during which it is cleaved into 17 and 12 kDa subunits [75]. Whereas infection for 1 h with YopE 1-138 -Myc or YopE 1-138 -BID expressing  Y. enterocolitica  failed to induce apoptosis, the translocation of human tBID triggered cell death in larger extend than the well-characterized apoptosis inducer staurosporin ( FIGS. 7  A and C). As expected, the translocation of tBID lead to the production of CASP3 p17 subunit, even in larger amounts as with staurosporin ( FIG. 7  A). To be able to compare translocated protein amounts to endogenous Bid, HeLa cells were lysed with Digitonin and analyzed by Western blotting using an anti Bid antibody ( FIG. 7  B). T3SS delivered YopE 1-138 -tBID reached about endogenous Bid levels in HeLa cells, while delivered YopE 1-138 -BID was present in even higher quantities (2.5 fold) ( FIG. 7  B). A deep proteome and transcriptome mapping of HeLa cells estimated 4.4 fold 10 5  copies of BID per single cell [76]. Therefore, one can conclude that T3SS dependent human protein delivery reaches 10 5  to 10 6  proteins per cell. These numbers fit the copies per cell of nanobodies translocated via  E. coli  T3SS [4]. Assuming a levelling of a factor of 10 for the MOI and for the duration of the infection, a factor of 3.2 for the time-point of antibiotic addition and for the culture time at 37° C. before infection, the delivered protein copies/cell can be tuned from some 1000 copies/cell up to some 10 6  copies/cell Altogether, these results indicated that translocated tBID was functional and delivered at relevant levels. This validated the translocation tool to study the role of proteins in the regulation of apoptosis, a central aspect of cell biology. 
     We further fused murine tBID (codon optimized for  Y. enterocolitica ; SEQ ID No. 194) or the BH3 domains of murine tBID or murine BAX (in both cases codon optimized for  Y. enterocolitica ; SEQ ID No. 138 and 139) to YopE 1-138  for delivery by  Y. enterocolitica . Whereas infection for 2.5 h with  Y. enterocolitica  ΔHOPEMT asd delivering no protein or YopE 1-138 -Myc failed to induce apoptosis, the translocation of murine tBID (codon optimized to  Y. enterocolitica , SEQ ID No. 194) triggered cell death in B16F10 ( FIG. 16 ), D2A1 ( FIG. 17 ), HeLa ( FIG. 18 ) and 4T1 ( FIG. 19 ) cells. The translocation of the BH3 domain of murine BID codon optimized for  Y. enterocolitica  (SEQ ID 138) or murine BAX codon optimized for  Y. enterocolitica  (SEQ ID 139) were as well found to induce massive cell death in B16F10 ( FIG. 16 ), D2A1 ( FIG. 17 ), HeLa ( FIG. 18 ) and 4T1 ( FIG. 19 ) cells. 
     Whereas infection for 4 h with  S. enterica  aroA bacteria failed to induce apoptosis, the translocation of murine tBID triggered apoptosis, as the translocation of murine tBID lead to the production of CASP3 p17 subunit ( FIGS. 20 and 21 ). The extent of apoptosis induction for SopE fusion proteins was larger when using SpiI T3SS inducing conditions ( FIG. 20 ), which reflects the transport of SopE exclusively by SpiI T3SS. SteA 1-20  fused murine tBID failed to induce apoptosis, very likely because the secretion signal within the 20 N-terminal amino acids of SteA is not sufficient to allow delivery of a fusion protein ( FIGS. 20 and 21 ). Murine tBID fused to full length SteA lead to apoptosis induction in HeLa cells ( FIGS. 20 and 21 ), both in SpiI and SpiII T3SS inducing conditions, reflecting the ability of SteA to be transported by both T3SS. It has to be noted that even under SpiII T3SS inducing conditions, a partial activity of the SpiI T3SS is expected as seen by the activity of SopE fusion proteins in SpiII T3SS inducing conditions ( FIG. 21 ). 
     Besides the here functionally elaborated translocated eukaryotic proteins, several other eukaryotic proteins have been secreted using the here-described tool. This includes for delivery by  Y. enterocolitica  ( FIGS. 13, 14 and 23 ) proteins from cell cycle regulation (Mad2 (SEQ ID No. 15), CDK1 (SEQ ID No. 14), INK4A (SEQ ID No. 16), INK4B (SEQ ID No. 17) and INK4C (SEQ ID No. 18)) as well as parts thereof (INK4A 84-103 (SEQ ID No. 158), p107 657-662 (SEQ ID No. 159), p21 141-160 (SEQ ID No. 160), p21 145-160 (SEQ ID No. 161), p21 17-33 (SEQ ID No. 162) and cyclin D2 139-147 (SEQ ID No 163)), apoptosis related proteins (Bad (SEQ ID No. 29), FADD (SEQ ID No. 28), and Caspase 3 p17 (SEQ ID No. 22) and p12 (SEQ ID No. 23), zebrafish Bid (SEQ ID No. 19) and t-Bid (SEQ ID No. 20)) as well as parts thereof (tBid BH3 (SEQ ID No.138), Bax BH3 (SEQ ID No.139)), signalling proteins (murine TRAF6 (SEQ ID No. 12), TIFA (SEQ ID No. 13)), GPCR Gα subunit (GNA12, shortest isoform, (SEQ ID No. 30)), nanobody (vhhGFP4, (SEQ ID No. 31)) and nanobody fusion constructs for targeted protein degradation (Slmb-vhhGFP4; (SEQ_ID Nos. 32, 33, 34) [77]) ( FIGS. 13 and 14 ) as well as small GTPases (Rac1 Q61E (SEQ ID No. 26 and 137) and RhoA Q63L (SEQ ID No. 27) and Pleckstrin homology domain from human Akt (SEQ ID No. 35). Besides the functionally elaborated apoptosis related proteins (murine tBid, SEQ ID No. 144-147), this further includes for delivery by  S. enterica  ( FIG. 22 ) proteins from cell cycle regulation (Mad2 (SEQ ID No. 168-169), CDK1 (SEQ ID No. 170-171), INK4A (SEQ ID No. 164-165) and INK4C (SEQ ID No. 166-167)). While those proteins have not been functionally validated, the possibility of T3SS dependent secretion of diverse eukaryotic proteins in combination with the possible removal of the YopE appendage opens up new vistas on the broad applicability of T3SS in cell biology and therapeutic applications. 
     In Vivo Translocation of Truncated Bid in Zebrafish Embryos Induces Apoptosis 
     An interesting feature of this bacterial tool is the potential use in living animals. Zebrafish in their embryonic state can be kept transparent allowing fluorescent staining and microscopy [44,78,79]. Few zebrafish apoptosis inducers have been described in detail, whereof z-BIM is the most potent [80]. Therefore, we decided to clone z-BIM into our system. Even if weakly homolgous to human BIM, we assayed the potency of apoptosis induction of YopE 1-138 -z-BIM (SEQ ID No. 21) in human epithelial cells. HeLa cells infected for 1 h with the strain translocating YopE 1-138 -z-BIM showed clear signs of cell death. We then performed in-vivo experiments with 2 days post fertilization (dpf) zebrafish embryos, using a localized infection model via microinjection of bacteria into the hindbrain [44]. After infection for 5.5 h the fish were fixed, permeabilized and stained for presence of CASP3 p17. Upon infection with the YopE 1-138 -Myc expressing strain, bacteria were visible in the hindbrain region (staining “b”,  FIG. 8  A I) but no induction of apoptosis around the bacteria was detected (staining “c”,  FIG. 8  A I). In contrast, upon infection with the strain delivering YopE 1-138 -z-BIM a strong increase in presence of cleaved CASP3 was observed in regions surrounding the bacteria ( FIG. 8  A II). Automated image analysis on maximum intensity z projections confirms that YopE 1-138 -z-BIM translocating bacteria induce apoptosis in nearby cells by far more than control bacteria do ( FIG. 8  B). This indicates that z-BIM is functional in zebrafish upon bacterial translocation. These results further validate the use of T3SS for eukaryotic protein delivery in living animals. 
     Phosphoproteomics Reveal the Global Impact of Translocated Proteins on Protein Phosphorylation 
     Phosphorylation is a wide-spread post-translational modification which can either activate or inactivate biological processes and is therefore a suitable target to study signaling events [81,82]. Despite this, no systems-level analysis of phosphorylation in apoptosis is available today. To analyze the impact of human tBid delivered into HeLa cells, we used a label-free phosphoproteomic approach by LC-MS/MS. In three independent experiments, cells were either left untreated, infected with ΔHOPEMT asd+YopE 1-138 -Myc or with ΔHOPEMT asd+YopE 1-138 -tBid for 30 minutes. Cells were lysed, followed by enzymatic digestion, phosphopeptide enrichment and quantification and identification of individual phosphopeptides. We compared cells infected with ΔHOPEMT asd+YopE 1-138 -Myc to cells infected with ΔHOPEMT asd+YopE 1-138 -tBid, allowing us to identify 363 tBid dependent phosphorylation events. 286 phosphopeptides showed an increase in phosphorylation whereas 77 were less phosphorylated upon tBid delivery, corresponding to 243 different proteins, which we defined as the tBid phosphoproteome. The STRING database was used to create a protein-protein interaction network of the tBid phosphoproteome [83] ( FIG. 9  A). Additionally 27 proteins known to be related to mitochondrial apoptosis were added to the network, building a central cluster. Interestingly, only few proteins from the tBid phosphoproteome are connected to this central cluster indicating that many proteins undergo a change in phosphorylation that were so far not directly linked to apoptotic proteins. To characterize the biological functions covered by the tBid phosphoproteome, we performed a gene ontology analysis using the functional annotation tool of the Database for Annotation, Visualization, and Integrated Discovery (DAVID, http://david.abcc.ncifcrf.ov) [84,85]. Identified biological functions show that diverse cellular processes are affected by tBid. Many proteins involved in chromatin rearrangement and the regulation of transcription undergo a change in phosphorylation (i.e. CBX3, CBX5, TRIM28, HDAC1). HDAC1 for example is a histone deacetylase playing a role in regulation of transcription. It has been shown that HDAC1 can modulate transcriptional activity of NF-kB, a protein also participating in apoptosis. We additionally identified a cluster of proteins involved in RNA processing which has previously been shown to play an important role in the regulation of apoptosis [86]. HNRPK for instance mediates a p53/TP53 response to DNA damage and is necessary for the induction of apoptosis [87]. Furthermore, the phosphorylation of proteins involved in protein translation is also affected. Several eukaryotic initiation factors (i.e. EIF4E2, EIF4B, EIF3A, EIF4G2) undergo a change in phosphorylation, which is in line with the observation that overall protein synthesis is decreased in apoptotic cells. Interestingly, the phosphorylation of many proteins involved in cytoskeleton remodeling (e.g. PXN, MAP1B9 are altered upon tBid delivery. This is in concordance with the observation that the morphology of cells changes dramatically upon tBid delivery ( FIG. 9  B). Cells shrinkage and loss of contact is reflected by the fact that we observe phosphorylation of adhesion related proteins like ZO2 and Paxillin. Similarly, shrinkage of the nuclei is accompanied by phosphorylation of laminar proteins like LaminA/C and Lamin B1. Altogether, tBID delivery induces a rapid apoptotic response also indicated by rupture of the mitochondrial integrity ( FIG. 9  B). We showed that tBid induced apoptosis affects hundreds of phosphorylation events participating in diverse cellular processes. While many identified proteins have been related to apoptosis, only few were known to be phosphorylated upon apoptosis induction. The phosphoproteomic approach thus provides a useful resource for further studies on apoptosis. 
     Translocation of Eukaryotic Heterologous Fusion Proteins Consisting of Repeated Identical or Variable Protein Domains into Epithelial Cells 
     To show that heterologous fusion proteins consisting of repeated identical or variable protein domains can translocate via type III secretion we fused murine apoptosis inducers for delivery by  Y. enterocolitica  to YopE 1-138 . As control, we fused murine tBID (codon optimized for  Y. enterocolitica ; SEQ ID No. 194) or the BH3 domains of murine tBID or murine BAX (in both cases codon optimized for  Y. enterocolitica ; SEQ ID No. 200 and 201) to YopE 1-138  for delivery by  Y. enterocolitica . The heterologous fusion protein consisted in one case of murine BH3 domain of tBID fused to itself, resulting in YopE 1-138 -(tBID-BH3) 2  (SEQ ID No. 202). In a second case, the heterologous fusion proteins consisted of murine BH3 domain of tBID fused to murine BH3 domain of BAX, resulting in YopE 1-138 -(tBID-BH3)-(BAX-BH3) (SEQ ID No. 203). In the case of murine tBID and murine BAX the codon was optimized for  Y. enterocolitica . Schematic representation of repeated identical domains or combination of different protein domains is shown in  FIG. 25 . 
     Whereas infection for 4 h with  Y. enterocolitica  ΔHOPEMT asd delivering YopE 1-138 -Myc failed to induce apoptosis, the translocation of murine BH3 domain tBID (codon optimized to  Y. enterocolitica , SEQ ID No. 194) triggered cell death in B16F10 and 4T1 cells ( FIGS. 26 and 27 ), with a clear dose-response effect upon increasing multiplicity of infection (MOI). Surprisingly, delivered YopE 1-138 -(tBID-BH3)-(BAX-BH3) or YopE 1-138 -(tBID-BH3) 2  were found more active than YopE 1-138 -(tBID-BH3) at lower MOI ( FIGS. 26 and 27 ). This indicates that upon delivery of repeated identical domains or combination of different protein domains, the impact on a desired cellular pathway as apoptosis can be enlarged. 
     Generation of Enhanced Pro-Apoptotic Bacteria 
     In above mentioned experiments it is shown that the T3SS-based delivery of pro-apoptotic proteins (e.g. t-BID (SEQ ID No. 25) or BIM (SEQ ID No. 21)) efficiently induces cell death in both murine and human cells, including cancerous cells, and that this effect could be increased when using murine t-BID optimized to the bacterial codon usage (SEQ ID No. 138). This increased cell killing very likely reflects increased amount of protein production and following delivery via T3SS due to optimal codons used. 
     In order to optimize the delivery or pro-apoptotic proteins, strains transformed with different pro-apoptotic proteins have been generated according to Table IV. 
     
       
         
           
               
             
               
                 TABLE IV 
               
             
            
               
                   
               
               
                 Strains transformed with different pro-apoptotic proteins 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Protein to 
                   
                 Resulting 
                   
                   
               
               
                   
                 Background 
                 be delivred 
                 Backbone 
                 plasmid 
                 Primers. 
               
               
                 Strain Name 
                 strain 
                 by T3SS 
                 plasmid 
                 name 
                 Si_Nr.: 
                 resistances 
               
               
                   
               
               
                 YopE1-138- 
                 
                   Y. enterocolitica 
                 
                 YopE1-138- 
                 pBad_Si_2 
                 pSi_353 
                 725/726 
                 Nal Amp 
               
               
                 ( Y. enterocolitica   
                 ΔyopH, O, P, 
                 
                   Y. enterocolitica 
                 
               
               
                 codon optimized 
                 E, M, T Δasd 
                 codon optimized 
               
               
                 murine tBid 
                   
                 murine tBid 
               
               
                 BH3 extended 
                   
                 BH3 extended 
               
               
                 part) 
                   
                 (by 4 Aa) 
               
               
                 YopE1-138- 
                 
                   Y. enterocolitica 
                 
                 YopE1-138- 
                 pBad_Si_2 
                 pSi_354 
                 727/728 
                 Nal Amp 
               
               
                 10 Aa linker- 
                 ΔyopH, O, P, 
                 10 Aa linker- 
               
               
                 ( Y. enterocolitica   
                 E, M, T Δasd 
                 
                   Y. enterocolitica 
                 
               
               
                 codon optimized 
                   
                 codon optimized 
               
               
                 murine tBid 
                   
                 murine tBid 
               
               
                 BH3 part) 
                   
                 BH3 
               
               
                 YopE1-(138- 
                 
                   Y. enterocolitica 
                 
                 YopE1-138- 
                 pSi_357 
                 pSi_374 
                 736/737 
                 Nal Amp 
               
               
                 
                   Y. enterocolitica 
                 
                 ΔyopH, O, P, 
                 
                   Y. enterocolitica 
                 
               
               
                 codon optimized 
                 E, M, T Δasd 
                 codon optimized 
               
               
                 murine Bax 
                   
                 murine Bax BH3-. 
               
               
                 BH3 part- 
                   
                 
                   enterocolitica 
                 
               
               
                 
                   Y. enterocolitica 
                 
                   
                 codon optimized 
               
               
                 codon optimized 
                   
                 murine tBid 
               
               
                 murine tBid 
                   
                 BH3 
               
               
                 BH3 part 
               
               
                   
               
            
           
         
       
     
     Shortening the delivered proteins to the essential domains required for signaling (SEQ ID No. 138 or 200)) could increase the efficiency of cell killing ( FIG. 28 ). Without being bound by theory, this increase in efficacy is likely to be related to increased amount of protein production and following delivery via T3SS due to smaller size of the delivered protein. Introduction of a linker between the YopE part and the BH3 domain of tBID (SEQ ID No. 218) decreased efficacy, as well as extending the BH3 domain by 4 further amino acids (SEQ ID No. 217) ( FIG. 28 ). 
     Additionally, synthetic cargos with repeats of such essential domains (e.g. the BH3 domain oft-BID (SEQ ID No. 202))) or combinations of these essential domains (e.g. the BH3 domain of t-BID and the BH3 domain of BAX (SEQ ID No. 203 and 219)) were generated. Surprisingly, tandem repeats of the same or different BH3 domains were found to result in enhanced apoptosis induction on cancerous cell lines (including 4T1 and B16F10 cells,  FIG. 28 ). The IC50 (half maximal inhibitory concentration), referring to the number of bacteria per eukaryotic cell (MOI) needed in order to kill 50% of such cells, was found to be decreased upon delivery of tandem repeats of tBID BH3 domain as compared to a single tBID BH3 domain ( FIG. 28 ). 
     This finding was surprising, as the protein size is increased by fusing as second BH3 domain of t-BID. Due to this, decreased expression and delivery levels of YopE 1-138 -(tBID BH3) 2  (SEQ ID No. 202) as compared to YopE 1-138 -tBID BH3 (SEQ ID No. 138 or 200) would be expected, and might maximally reach equivalent levels. In order to reach an increase in cell killing activity, the fused tBID BH3 domains must simultaneously act side by side upon delivery by the T3SS into eukaryotic cells. In case only one tBID BH3 domain in the YopE 1-138 -(tBID BH3) 2  construct would be functional, at best the same efficiency as with YopE 1-138 -tBID BH3 might be expected. 
     In order to increase the genetic stability of YopE 1-138 -(tBID BH3) 2  (SEQ ID No. 202) for in vivo studies, we cloned YopE 1-138 -(tBID BH3) 2  (SEQ ID No. 202) by homologous recombination on the  Yersinia  virulence plasmid pYV at the native site of YopE and under the native YopE promoter (using mutator plamids pSI_408 and pSI_419). Such mutators contain the DNA sequence coding for the desired protein, flanked by 200-250 bp of sequences on both sides corresponding to the site of the respective gene, where the integration shall take place. These plasmids are transformed into  E. coli  Sm10λ pir, from where plasmids were mobilized into the corresponding  Y. enterocolitica  strain. Mutants carrying the integrated vector were propagated for several generations without selection pressure. Then sucrose was used to select for clones that have lost the vector. Finally mutants were identified by colony PCR. The endogenous proteins for the transport by the T3SS (called “ Yersinia  outer proteins”, Yops) are encoded by  Y. enterocolitica  on this 70 kb plasmid, named plasmid of  Yersinia  Virulence (pYV), which further encodes the T3SS apparatus.  Yersinia  strains encoding YopE 1-138 -(tBID BH3) (SEQ ID No. 138 or 200) or YopE 1-138 -(tBID BH3) 2  (SEQ ID No. 202) on the  Yersinia  virulence plasmid pYV at the native site of YopE and under the native YopE promoter were assessed for their capacity of inducing apoptosis in cancerous cells (including 4T1 and B16F10 cells,  FIG. 29 ). The IC50 (half maximal inhibitory concentration), referring to the number of bacteria per eukaryotic cell (MOI) needed in order to kill 50% of such cells, was found to be decreased upon delivery of tandem repeats of tBID BH3 domain as compared to a single tBID BH3 domain, when both proteins are encoded on the  Yersinia  virulence plasmid pYV at the native site of YopE and under the native YopE promoter ( FIG. 29 ). This is in agreement with findings from expression plasmid borne delivery of these proteins ( FIG. 28 ). Again, this finding was surprising, as the protein size is increased by fusing a second BH3 domain of t-BID. Due to this, decreased expression and delivery levels of YopE 1-138 -(tBID BH3) 2  (SEQ ID No. 202) as compared to YopE 1-138 -tBID BH3 (SEQ ID No. 138 or 200) would be expected, and might maximally reach equivalent levels. In order to reach an increase in cell killing activity, the fused tBID BH3 domains must simultaneously act side by side upon delivery by the T3SS into eukaryotic cells. In case only one tBID BH3 domain in the YopE 1-138 -(tBID BH3) 2  construct would be functional, at best the same efficiency as with YopE 1-138 -tBID BH3 might be expected. Furthermore,  Yersinia  strains encoding YopE 1-138 -(tBID BH3) 2  (SEQ ID No. 202) on the  Yersinia  virulence plasmid pYV at the native site of YopE and under the native YopE promoter were compared for their capacity of inducing apoptosis in cancerous cells to expression plasmid (pBad-MycHisA based) derived delivery of YopE 1-138 -(tBID BH3) 2 . In agreement with the higher copy number of pBad-MycHisA (20-25 copies) as compared to the pYV (1-6 copies are reported), pBad-MycHisA based delivery of YopE 1-138 -(tBID BH3) 2  (SEQ ID No. 202) resulted in a slightly decreased IC50 value on 4T1 and B16F10 cells ( FIG. 29 ). 
     Validation of Tumor Specific Growth In Vivo Up to Day 14 Post Bacterial Administration 
     The experiment of tumor colonization by genetically modified  Y. enterocolitica  was repeated in a syngeneic murine allograft model (4T1 breast cancer model) and bacterial colonization was followed over two weeks. This time, mice were infected with 1*10 6  colony forming units (CFU) of  Y. enterocolitica  ΔyopH,O,P,E,M,T. While obtaining similar results to the B16F10 model at early days post infection, we could further show that the tumor colonization is consistently found at day 8 and up to day 14 after infection ( FIG. 30 ). Furthermore, the colonization remains highly specific with only low counts of bacteria detected in all other organs assessed ( FIG. 31 ). These findings indicate that  Y. enterocolitica  ΔyopH,O,P,E,M,T is able to establish a persistent colonization of the tumor thereby preventing clearance by the immune system. 
     Efficacy of  Y. enterocolitica  ΔHOPEMT in Delaying Tumor Progression 
     In order to assess the impact of YopE 1-138 -(tBID BH3) 2  (SEQ ID No. 202) delivered to tumor cells in vivo, we performed studies in wildtype Balb/C mice allografted s.c. with 4T1 breast cancer cells. We aimed at assessing the  Y. enterocolitica  ΔHOPEMT strain encoding YopE 1-138 -(tBID BH3) 2  (SEQ ID No. 202) on the  Yersinia  virulence plasmid pYV at the native site of YopE and under the native YopE promoter. Mice were i.v. injected with PBS or 1*10 7    Y. enterocolitica  ΔHOPEMT pYV-YopE 1-138 -(tBID BH3) 2 , once the tumor had reached a size of 150-250 mm3. The day of the i.v. injection of bacteria was defined as day 0. Tumor volume was measured over the following days (day 0 to day 9 post i.v. injection of bacteria) with calipers. The tumor volume was normalized to the tumor volume at day 0 to compensate for any initial heterogeneity in tumor size. Treatment with  Y. enterocolitica  ΔHOPEMT pYV-YopE 1-138 -(tBID BH3) 2  showed an impact on tumor volume progression, with statistically significant tumor reduction at day 8, 9 and 10 post bacterial administration ( FIG. 32 ). Importantly,  Y. enterocolitica  ΔHOPEMT alone was found not to impact tumor progression in the 4T1 murine cancer model ( FIG. 33 ). These findings highlight that such bacteria and their T3SS can be employed for interference with tumor progression. 
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