# A Type VI Secretion System trans- kingdom effector is required for the delivery of a novel antibacterial toxin in Pseudomonas aeruginosa

Benjamin Berni, Chantal Soscia, Sarah Djermoun, Bérengère Ize, Sophie Bleves

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## To cite this version:

Benjamin Berni, Chantal Soscia, Sarah Djermoun, Bérengère Ize, Sophie Bleves. A Type VI Secretion System trans- kingdom effector is required for the delivery of a novel antibacterial toxin in Pseudomonas aeruginosa. Frontiers in Microbiology, In press, 10.3389/fmicb.2019.01218. [hal-02145565](https://hal-02145565)

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Submitted on 3 Jun 2019

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# A Type VI Secretion System trans-kingdom effector is required for the delivery of a novel antibacterial toxin in Pseudomonas aeruginosa

Benjamin Berni<fnref n="1" />, Chantal Soscia<fnref n="1" />, Sarah Djermoun<fnref n="1" />, Berengere Ize<fnref n="1" />, Sophie Bleves<fnref n="1" />*

**Submitted to Journal:**

Frontiers in Microbiology

**Specialty Section:**

Microbial Physiology and Metabolism

**Article type:**

Original Research Article

**Manuscript ID:**

456358

**Received on:**

25 Feb 2019

**Revised on:**

29 Apr 2019

**Frontiers website link:**

www.frontiersin.org

<footnote n="1">¹Centre National de la Recherche Scientifique (CNRS), France</footnote>
**Conflict of interest statement**

The authors declare that the research was conducted in the absence of any commercial or financial
relationships that could be construed as a potential conflict of interest

**Author contribution statement**

B.B. and S.B. designed and conceived the experiments. B.B., C.S., and S.D. performed the experiments. S.B. supervised the execution of the experiments. B.B., C.S., B.I. and S.B. analysed and discussed the data. S.B. wrote the paper with contribution from B.B. and reading from B.I.

**Keywords**

Pseudomonas aeruginosa, antibacterial toxin, trans-kingdom effector, adaptor, T6SS, Chaperone, Virulence, Tle (Type 6 lipase effector), immunity protein

**Abstract**

Word count: 260

Pseudomonas aeruginosa has evolved multiple strategies to disarm and take advantage of its host. For this purpose this opportunist pathogen has particularly developed protein secretion in the surrounding medium or injection into host cells. Among this, the Type VI Secretion System (T6SS) is utilized to deliver effectors into eukaryotic host as well as target bacteria. It assembles into a contractile bacteriophage tail-like structure that functions like a crossbow, injecting an arrow loaded with effectors into the target cell. The repertoire of T6SS antibacterial effectors of P. aeruginosa is remarkably broad to promote environmental adaptation and survival in various bacterial communities, and presumably in the eukaryotic host too.

Here we report the discovery of a novel pair of antibacterial effector and immunity of P. aeruginosa, Tle3 and Tli3. Tli3 neutralizes the toxicity of Tle3 in the periplasm to protect from fratricide intoxication. The characterization of the secretion mechanism of Tle3 indicates that it requires a cytoplasmic adaptor, Tla3, to be targeted and loaded onto the VgrG2b spike and thus delivered by the H2-T6SS machinery. Tla3 is different from the other adaptors discovered so far, and defines a novel family among T6SS with a DUF2875.

Interestingly this led us to discover that VgrG2b that we previously characterized as an anti-eukaryotic effector possesses an antibacterial activity as well, as it is toxic towards Escherichia coli. Excitingly Tli3 can counteract VgrG2b toxicity. VgrG2b is thus a novel trans-kingdom effector targeting both bacteria and eukaryotes. VgrG2b represents an interesting target for fighting against P. aeruginosa in the environment and in the context of host infection.

**Contribution to the field**

We have previously shown that a T6SS of P. aeruginosa (H2-T6SS), facilitates the internalization of the bacterium into non-phagocytic cells (Sana 2012; 2013). We have found that the H2-T6SS effector VgrG2b is responsible for this phenotype by manipulating host microtubule cytoskeleton (Sana, 2015). Here we have discovered that VgrG2b, as a component of the H2-T6SS puncturing device this time, is required for the delivery of an antibacterial effector. We have characterized a novel pair of antibacterial toxin and immunity. We showed that Tle3 is toxic once delivered in the periplasm of prey bacteria and that Tli3 can neutralize it. By characterizing the secretion mechanism of Tle3, we found that it requires Tla3, a cytoplasmic adaptor of a unique family, to be targeted to the VgrG2b spike. Tla3 represents a novel family of T6SS adaptors. Very interestingly we also discovered that the C-terminal extension of VgrG2b is toxic towards Escherichia coli making VgrG2b a trans-kingdom effector of P. aeruginosa, able to target both bacteria and eukaryotic cells. In conclusion VgrG2b is a fascinating protein, a unique case of trans-kingdom toxin required for the secretion of another antibacterial toxin. To our knowledge this has never been reported in the literature so far.

**Funding statement**

This work was supported by recurrent funding from the CNRS and Aix-Marseille University. The project leading to this publication has received funding from the Excellence Initiative of Aix-Marseille University-A*Midex, a French "Investissements d'Avenir" program ("Emergence & Innovation" A-M-AAP-EI-17-139-170301-10.31-BLEVES-HLS).
**Ethics statements**

(Authors are required to state the ethical considerations of their study in the manuscript, including for cases where the study was exempt from ethical approval procedures)

Does the study presented in the manuscript involve human or animal subjects: No

**Data availability statement**

Generated Statement: All datasets generated for this study are included in the manuscript and the supplementary files.

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A Type VI Secretion System trans-kingdom effector
is required for the delivery of
a novel antibacterial toxin in *Pseudomonas aeruginosa*

Benjamin Berni¹, Chantal Soscia¹, Sarah Djermoun¹, Bérengère Ize¹ and Sophie Bleves¹*

¹LISM (Laboratoire d'Ingénierie des Systèmes Macromoléculaires-UMR7255), IMM (Institut de Microbiologie de la Méditerranée), Aix-Marseille Univ and CNRS, 31 chemin J. Aiguier, Marseille 13402 Cedex 20, France.

*Correspondance Pr Sophie Bleves

bleves@imm.cnrs.fr
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**Abstract**

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*Pseudomonas aeruginosa* has evolved multiple strategies to disarm and take advantage of its host. For this purpose this opportunist pathogen has particularly developed protein secretion in the surrounding medium or injection into host cells. Among this, the Type VI Secretion System (T6SS) is utilized to deliver effectors into eukaryotic host as well as target bacteria. It assembles into a contractile bacteriophage tail-like structure that functions like a crossbow, injecting an arrow loaded with effectors into the target cell. The repertoire of T6SS antibacterial effectors of *P. aeruginosa* is remarkably broad to promote environmental adaptation and survival in various bacterial communities, and presumably in the eukaryotic host too.

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Here we report the discovery of a novel pair of antibacterial effector and immunity of *P. aeruginosa*, Tle3 and Tli3. Tli3 neutralizes the toxicity of Tle3 in the periplasm to protect from fratricide intoxication. The characterization of the secretion mechanism of Tle3 indicates that it requires a cytoplasmic adaptor, Tla3, to be targeted and loaded onto the VgrG2b spike and thus delivered by the H2-T6SS machinery. Tla3 is different from the other adaptors discovered so far, and defines a novel family among T6SS with a DUF2875.

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Interestingly this led us to discover that VgrG2b that we previously characterized as an anti-eukaryotic effector possesses an antibacterial activity as well, as it is toxic towards *Escherichia coli*. Excitingly Tli3 can counteract VgrG2b toxicity. VgrG2b is thus a novel trans-kingdom effector targeting both bacteria and eukaryotes. VgrG2b represents an interesting target for fighting against *P. aeruginosa* in the environment and in the context of host infection.

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**Keywords**

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Virulence, Type VI secretion system, bacterial competition, VgrG, Tle, immunity, adaptor, chaperone

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## 53 Introduction

Pseudomonas aeruginosa is one of the most virulent opportunistic pathogens, being responsible for various diseases such as acute infections of lungs and burned skin that can lead to septicemia more particularly in immunocompromized patients, or broncho-alveolar colonization in Cystic Fibrosis sufferers. P. aeruginosa has been classified in 2017 as critical by the WHO in the top three list of antibiotic resistant bacteria (WHO publishes list of bacteria for which new antibiotics are urgently needed). P. aeruginosa has developed various pathogenicity strategies among which protein secretion or protein delivery into target cells is key. Indeed this pathogen possesses 5 of the 6 secretion systems so far identified among Gram-negative bacteria, if we exclude the T9SS (Type IX Secretion System) restricted to one phylum, and remarkably in several copies for most of them (Bleves et al., 2010).

The T6SS (Type VI Secretion System) was first discovered in the context of eukaryotic host infection (Mougous et al., 2006)(Pukatzki et al., 2006) and later during bacterial competition (Hood et al., 2010), which seems to be its primary function (Cianfanelli et al., 2016b). The T6SS confers a fitness advantage (i) in environmental niches against rival bacteria (inter- and intraspecies competitiveness have been described) and (ii) in the eukaryotic host towards commensal bacteria (Cianfanelli et al., 2016b). Indeed recent studies have highlighted a novel role for T6SS-dependent antibacterial responses in interbacterial competition in the mammalian gut (Sana et al., 2016b)(Hecht et al., 2016), suggesting that T6SSs may be important not only in shaping microbial community composition, but also in governing interactions between the microbiota and invading pathogens. Interestingly, several T6SSs are also known to target both cell type genus such as the T6SS of Vibrio cholera (Pukatzki et al., 2006)(MacIntyre et al., 2010) and P. aeruginosa (Sana et al., 2012)(Russell et al., 2013). Even more remarkably, three T6SS effectors of P. aeruginosa, namely PldA (also called Tle5a), PldB (Tle5b) and TplE (Tle4) (Jiang et al., 2014, 2016) have been called "trans-kingdom effectors"
since these toxins can target both prokaryotic and eukaryotic cells (Bleves, 2016). Indeed toxins are usually directed against eukaryotic cells (like AB toxins or RTX pore-forming toxins) or against rival bacteria (like bacteriocins).

The T6SS functions as a dynamic contractile phage tail-like structure anchored in the bacterial cell envelope that delivers effector proteins directly into the target cell in a one-step manner. T6SS includes a contractile sheath that cover a nanotube of stacked Hcp topped with a membrane-puncturing spike made of VgrG and PAAR (proline-alanine-alanine-arginine repeat) proteins (Basler, 2015). The sheath can contract and inject the arrow loaded with effectors into the target cell. Characterizing the repertoire of effectors delivered by the T6SS has highlighted a great diversity in terms of effector activities, host cell targets, and mode of recruitment by the T6SS machinery. In brief, there are two broad effector categories: the "specialized" effectors fused to components of the machinery (evolved VgrG, evolved PAAR and evolved Hcp have been described so far) and the "cargo" effectors (Cianfanelli et al., 2016b). The later are addressed to the T6SS machinery by binding directly one of the arrow components (VgrG, PAAR and Hcp) or by being targeted through cytoplasmic adaptor proteins also called chaperones. To date three families of adaptor have been described, the first one harboring a DUF4123 (Liang et al., 2015; Miyata et al., 2013; Salomon et al., 2014; Unterweger et al., 2015), the second a DUF1795 (Alcoforado Diniz and Coulthurst, 2015; Whitney et al., 2015) and the third one a DUF2169 (Bondage et al., 2016). In line with this, many effector-encoding genes are found in close proximity to *vgrG*, *hcp*, *paar* or adaptor genes. Finally to protect themselves from self-intoxication or from antibacterial toxins injected by neighboring sibling cells, bacteria always synthesize immunity proteins, which are encoded by adjacent genes (Benz and Meinhart, 2014).

*P. aeruginosa* encodes three distinct T6SS loci, H1- to H3-T6SS. While H1-T6SS has only been involved in antibacterial activity so far (Sana et al., 2016a)(LaCourse et al., 2018),
H2-T6SS and H3-T6SS can target both bacterial and eukaryotic cells possessing even as said earlier trans-kingdom effectors (Burkinshaw et al., 2018; Jiang et al., 2014, 2016; Russell et al., 2013; Sana et al., 2012, 2015). We discovered the anti-eukaryotic function of the H2-T6SS machinery that promotes the uptake of *P. aeruginosa* by non-phagocytic cells (Sana et al., 2012). The two phospholipases D mentioned earlier, PldA (Tle5a) and PldB (Tle5b), delivered respectively by H2-T6SS and H3-T6SS machineries, participate in the host kinase pathway highjacking that facilitates further entry of *P. aeruginosa* (Jiang et al., 2014). The evolved VgrG2b effector (Sana et al., 2015) is delivered by H2-T6SS into epithelial cells where it targets the γ-tubulin ring complex, a microtubule-nucleating multiprotein complex to promote a microtubule-dependent internalization of *P. aeruginosa*. Finally TplE (Tle4), which is secreted by the H2-T6SS machinery, promotes autophagy in epithelial cells once localized to the endoplasmic reticulum (Jiang et al., 2016). Interestingly PldA (Tle5a), PldB (Tle5b) and TplE (Tle4) have also been identified as antibacterial phospholipases of the Tle (type VI lipase effectors) family (Russell et al., 2013). They work by affecting membrane integrity of the rival bacteria (Jiang et al., 2014, 2016; Russell et al., 2013). More precisely, PldA degrades the major constituent of bacterial membranes, the phosphatidylethanolamine (Russell et al., 2013).

In the present study we have discovered a novel antibacterial toxin, Tle3 and its cognate immunity, Tli3, whose genes are encoded downstream of *vrgG2b*. By characterizing the secretion mechanism of Tle3 by H2-T6SS, we showed that it requires Tla3, a cytoplasmic adaptor of a unique family, to be targeted to the VgrG2b spike. Interestingly we also found that the C-terminal extension of VgrG2b is toxic towards *Escherichia coli* making VgrG2b a new trans-kingdom effector of *P. aeruginosa*.

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126 **Materials and Methods**

127 **Bacterial strains, growth conditions and plasmid construction**
All *P. aeruginosa* and *E. coli* strains used in this study are described in Supplementary Table 1. Briefly, the *E. coli* K-12 DH5α and CC118λPir were used for cloning procedures. The BL21(DE3)pLysS and BTH101 were used for protein production and BACTH analyses respectively. Strains were grown in LB or in TSB medium (for *P. aeruginosa*) at 37°C or 30°C. Specific growth conditions are specified in the text when necessary. Recombinant plasmids were introduced into *P. aeruginosa* by triparental mating using the conjugative properties of the helper plasmid pRK2013 (Supplementary Table 1). Plasmids were maintained by the addition of ampicillin (50 µg/mL), kanamycin (50 µg/mL), chloramphenicol (30 µg/mL), streptomycin (30 µg/mL for *E. coli*, 2000 µg/mL for *P. aeruginosa*) or gentamicin (30 µg/mL for *E. coli*, 115 µg/mL for *P. aeruginosa*). Expression of genes from pT7 in BL21(DE3)pLysS was blocked with 0.4% of glucose and induced in exponential phase (OD<sub>600</sub>= 0.4-0.6) for 3 hours with 1mM of IPTG. Cloning procedures were described in [26]. The plasmids used and constructed are described in Supplementary Table 1, the list of oligonucleotides (synthesized by Eurogentec or IDT) is given in Supplementary Table 2.

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## Cloning procedures for *P. aeruginosa* mutants

To generate *P. aeruginosa* mutants, 500 bp upstream and 500 bp downstream of the gene to be deleted were amplified by overlapping PCR with Q5 high fidelity DNA polymerase (NEB) using primers listed in Supplementary Table 1. The PCR product was cloned in pKNG101 suicide vector by one-step sequence and ligation-independent cloning (SLIC) (Jeong et al., 2012), which was then sequenced. pKNG101 derivatives, maintained in the *E. coli* CC118λpir strain, were mobilized in *P. aeruginosa* strains. The mutants, in which the double recombination events occurred, were confirmed by PCR analysis.

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## Heterologous toxicity assays
E. coli BL21(DE3)pLysS containing plasmids producing cytoplasmic or periplasmic targeted proteins were grown overnight at 37°C in LB with 0.4% of glucose. 10 µL drops of bacterial suspensions serially diluted were spotted onto LB agar plates containing 0.1 mM IPTG or 0.4 % glucose and cells were grown for 16 h at 37°C.

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158 Bacterial two-hybrid assay

Protein-protein interactions were assessed with the adenylate cyclase-based two-hybrid technique using protocols published previously (Battesti and Bouveret, 2012; Karimova et al., 1998). Briefly, the proteins to be tested were fused to the isolated T18 and T25 catalytic domains of the *Bordetella* adenylate cyclase. After introduction of the two plasmids producing the fusion proteins into the reporter BTH101 strain, plates were incubated at 30°C for 24 h. Three independent colonies for each transformation were inoculated into 600 µl of LB medium supplemented with ampicillin (50 µg/mL), kanamycin (50 µg/mL), and IPTG (0.5 mM). After overnight growth at 30 °C, 5 µl of each culture was spotted onto LB agar plates supplemented with ampicillin, kanamycin, IPTG, and 5-bromo-4-chloro-3-indonyl-D-galactopyrannoside (X-gal, 40µg/mL) and incubated for 16 h at 30 °C.

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170 Protein purification by affinity chromatography

Escherichia coli BL21(DE3)plysS cells carrying the pRSFDUET-1 and pETDUET-1 derivates were grown at 37°C in LB to an OD₆₀₀ ~ 0.5 and the expression of the PA0262, PA0261, PA0260 or PA0259 genes was induced with IPTG (1 mM) for 3 h at 37°C. Cells were harvested by centrifugation at 1914 × g for 30 min at 4°C. The cell pellet was resuspended in Tris-HCl 50 mM pH 8.0, NaCl 150 mM, Triton X-100 0.1%, lysozyme 0.5 mg/mL and EDTA 1 mM and stored at -80°C. Cells were supplemented with DNase (20 µg/mL), MgCl₂ and phenylmethylsulfonyl fluoride 1mM and cells were lysed by three passages at the Emulsiflex-
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C5 (Avestin), and lysates were clarified by centrifugation at 16000 × g for 30 min. The supernatant was loaded onto a 5-mL StrepTrap HP (GE Healthcare) column and then washed with 50 mM Tris-HCl pH 8.0, 150 mM NaCl at 4°C. The fusion protein was eluted in the affinity buffer supplemented with 2.5 mM desthibiotin. Peak fractions were pooled and loaded onto a Superose 200 10/300 column (GE Healthcare) equilibrated in 50 mM Tris-HCl pH 8.0, 50 mM NaCl.

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## Fractionation of *P. aeruginosa*

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Fractionation of cells into spheroplasts (cytoplasm and membranes) and periplasmic fractions were done as previously described (Ize et al., 2014). Proteins corresponding to the cytoplasm and periplasm fractions or to insoluble material were resuspended in loading buffer.

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## Protein secretion

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*P. aeruginosa* strains were grown at 25°C in TBS for 24 hours. Cells corresponding to 10 units DO₆₀₀ and extracellular medium were separated by centrifugation at 2000 x g for 10 min at room temperature. 2/3 of the supernatants were collected and centrifuged at 13 000 x g for 5 min at room temperature. Proteins contained in the supernatant were precipitated with tricholoro-acetic acid (TCA, 15%) for 3 h at 4°C. Samples were centrifuged at 13 000 x g for 30 min at 4°C, pellets washed with 90% acetone and resuspended in loading buffer.

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## SDS-PAGE and western-blot

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Protein samples derived from equivalent amounts of culture (i.e. optical density equivalents) resuspended in loading buffer were boiled and separated by SDS-PAGE. Proteins were then stained by Coomassie-blue or immunodetected as described before (Sana et al., 2015) using primary polyclonal antibodies directed against His6 epitope-tag (Penta His, Qiagen,
dilution 1:1000), V5 epitope-tag (Bethyl Laboratories, dilution 1:1000), Strep epitope-tag (IBA
StrepMAB Classic, dilution 1:000), DsbA (kindly gifted by K.E. Jaeger – university of
Heinrich-Heine, dilution 1:25000), or monoclonal antibodies directed against EF-Tu (Hycult-
biotech, dilution 1:20000), XcpY (laboratory collection, dilution 1:5000), TolB (laboratory
collection, dilution 1/500). Peroxidase-conjugated anti-Mouse or anti-Rabbit IgGs (Sigma,
dilution 1:5000) were used as secondary antibodies. Nitrocellulose membranes were revealed
with homemade enhanced chemiluminescence and were scanned using ImageQuant LAS4000
analysis software (GE Healthcare Life sciences).

Protein samples equivalent to 0.1 OD₆₀₀ units were loaded for whole cell and
spheroplasts analysis while protein samples equivalent to 0.2 OD₆₀₀ units were used for
cytoplasm or periplasm analysis and protein samples equivalent to 1 OD₆₀₀ units were used for
extracellular medium analysis.

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216 Bacterial Competition assays

Intraspecific competition assays between *P. aeruginosa* strains were performed as
previously described (Jiang et al., 2014) with modifications. The prey cells carry pJN105 vector
(GmR) to allow counterselection. Overnight cultures of *P. aeruginosa* attacker and prey cells
were mixed in a 5 : 1 (attacker : prey) ratio and harvested by centrifugation at 3724 x g for 5
min. The pellet was resuspended in 200 µL of PBS 1X and spotted onto 0.45-µm nitrocellulose
membranes overlaid on a 1 % bactoagar plate. After 24 hours of incubation at 37°C, cells were
resuspended in 2 mL of PBS 1X, normalized to an OD₆₀₀nm of 0.5 and 10 µL of bacterial serially
diluted (10⁻¹ to 10⁻⁶) were spotted onto selective LB agar plates containing gentamicin (125
µg/mL). Significant growth difference of the prey bacteria for each competition assay was
computed by one-way ANOVA (Stat Plus) and unpaired Student's Test (Excel).

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## 228 Results

### 229 Tle3 is a novel antibacterial toxin of P. aeruginosa

The analysis of the *vgrG2b* genetic environment revealed the presence of the PA0260 gene encoding a protein with a α/β hydrolase domain (PF00561) and a putative Ser-Asp-His catalytic triad used by various esterase enzymes and has thus been classified in the Tle3 family of antibacterial Tle toxins (Russell et al., 2013) (Fig. 1A). The immunities of Tle proteins, which are lipolytic toxins active in the periplasm of the prey bacterium, are localized in or exposed to the periplasm where they neutralize the cognate toxin (Flaugnatti et al., 2016; Jiang et al., 2014, 2016; Russell et al., 2013). The two genes surrounding *tle3*, PA0259 and PA0261, are good candidates as Tle3 immunity. Indeed sequence comparisons of PA0259 and PA0261 showed that PA0261 is homologous to Tsi6, the immunity protein of a H1-T6SS effector called Tse6, and PA0259 to TplEi (Tli4), the immunity of TlpE (Tle4), a H2-T6SS effector (Jiang et al., 2016; Lu et al., 2014). We used the SignalP 4.1 server (Nielsen, 2017) to predict the cellular localization of the two immunity candidates. While a Sec signal sequence is predicted at the N-terminal extremity of PA0261, the analysis of the PA0259 sequence did not reveal any. However three upstream ATG can be found in frame with the annotated ATG of PA0259 (Fig. 1 sup). The sequence of the proteins synthesized from the two first ATG (ATG1 et ATG2) presents then a N-terminal signal peptide, while the protein synthesized from the last codon (ATG3) does not. Moreover a RBS (Ribosome Binding Sequence) can be found only upstream of ATG1 and with a significant Kolaskar score that indicates a strong probability to be used as an initiation codon (Kolaskar and Reddy, 1985). Altogether these data tend to indicate an incorrect annotation of the start codon of PA0259 and that ATG1 should be considered for the initiation of PA0259 translation. Such a correction has been already seen for Tli5 (PA3488) the immunity protein of PldA (Tle5a) of P. aeruginosa (Russell et al., 2013). In conclusion of this
252 *in silico* analysis, the two putative immunities may harbor a Sec signal peptide that suggests a
253 periplasmic localization in agreement with Tle3 activity in this compartment.

254 To demonstrate the antibacterial activity of Tle3 and to identify its immunity protein,
255 we developed a heterologous toxicity assay in *E. coli* on the basis that Tle3 should be toxic
256 when produced in the periplasm of *E. coli* and that it should be counteracted by the co-
257 production of its immunity protein. In order to artificially address Tle3 to the periplasm of *E.
258 coli* the *tle3* sequence has been cloned in frame with the sequence coding the PelB signal peptide
259 on the pET22b vector under a *P*<sub>T7</sub> promoter. PA0259 from ATG<sub>1</sub> and PA0261 were respectively
260 cloned in pRSF-DUET, a higher copy-number vector, to allow a maximal co-expression with
261 *ss-tle3* in the *E. coli* BL21(DE3) pLysS strain. The correct production and localization of all
262 the recombinant proteins in *E. coli* has been verified by western blot after cell fractionation (Fig.
263 2 sup). The results presented in Figure 1B indicate that whereas the cytoplasmic production of
264 Tle3 was not toxic, Tle3 targeting to the periplasm led to *E. coli* killing. Moreover while
265 PA0259 had no effect, the coproduction of PA0261 in the periplasm protected the cells against
266 the toxicity of Tle3. We verified that the sole overproduction of PA0259 and PA0261 was not
267 toxic to *E. coli*. As PA0261 neutralized Tle3 toxicity, we called it “Tli3” for Type VI lipase
268 immunity (Fig. 1A). During this study, we observed that the protection conferred by Tli3
269 coproduction could be sometimes partial and we solved this issue by cloning in tandem *tli3* and
270 *tle3* on the same plasmid like they are organized on *P. aeruginosa* genome (Fig. 1B, line 8).
271 The importance of this genetic link is the proof of the close connection between these two
272 proteins as a pair of toxin-antitoxin.

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274 Tli3 (PA0261) is the immunity protein of Tle3

275 As the immunities bind specifically their effector, which is suggested by the release of
276 Tle3 toxicity by Tli3, we tested the physical interaction between both proteins by co-
purification with affinity chromatography (Fig. 2A). A cytoplasmic Strep-tagged version of Tli3 was engineered by fusing the tag to the mature domain of Tli3, lacking its signal peptide. The recombinant protein was coproduced in *E. coli* BL21(DE3) pLysS with the cytoplasmic His₁₀-tagged Tle3. The bacterial lysate was loaded to a StrepTactin matrix (see Materials and Methods section), and Tli3CStrep was eluted with desthibiotin. The presence of Tle3CHis was controlled in the elution fraction with anti-His antibodies. As shown in Fig. 2A, Tle3CHis was found in the eluted fraction only upon coproduction with Tli3CStrep (left panel). Indeed when produced alone in *E. coli* Tle3CHis was not purified by affinity chromatography (right panel). As expected for an immunity protein, Tli3 directly interacts with Tle3.

To go further into Tli3 characterization we chose to determine its cellular localization in *P. aeruginosa*. All the immunity proteins identified so far for Tle proteins are localized in the periplasm or associated to the periplasmic side of the outer membrane (Flaugnatti et al., 2016; Jiang et al., 2014, 2016; Russell et al., 2013) in order to counteract their cognate toxin. A chromosomally encoded Tli3V5 translational fusion was engineered in order to specifically immunodetect the protein in *P. aeruginosa* (Supplementary Table 1). After fractionation of *P. aeruginosa* (Fig. 2B), Tli3 was readily observed in the same fraction as DsbA that catalyzes intrachain disulfide bond formation as peptides emerge into the periplasm. This indicates a periplasmic localization for Tli3 in *P. aeruginosa* in agreement with the presence of a Sec signal peptide and our working hypothesis suggesting an immunity function.

## Tle3 protein-protein interaction network

To further characterize Tle3, we performed a bacterial two-hybrid (BACTH) assay with the other gene products of the vgrG2b operon hypothesizing that a genetic link could reflect protein-protein interaction. The sequences coding PA0259 and Tli3 (PA0261) after their signal sequences and Tle3 were cloned downstream and upstream the T18 or T25 domains of the
302 *Bordetella adenylate* cyclase. Because of the high molecular weight of VgrG2b and since the 303 interaction of another Tle with a VgrG in entero-aggregative *E. coli* (EAEC) was previously 304 delimitated to the C-terminal domain of VgrG (Flaugnatti et al., 2016), we cloned the sequences 305 encoding the C-terminal extension (domains 1, 2 and 3) and a truncated version harboring only 306 the DUF2345 and the transthyretin-like (TTR) domains (domains 1 and 2) of VgrG2b 307 downstream the T18 and T25 domains (Fig. 3A).

308 Unexpectedly the sole interaction revealed by the BACTH assay for Tle3 was with 309 PA0259 (Fig. 3B) since only the T18/T25-Tle3 and T18/T25-PA0259 fusion proteins 310 coproduction activated the expression of the reporter gene. This assay did not confirm the 311 interaction between Tle3 and Tli3 observed by copurification, and did not evidence an 312 interaction with VgrG2b. Interestingly Tle3 and PA0259 did not interact anymore when they 313 were fused upstream the T18 and T25 domains suggesting that both proteins interact via their 314 C-terminal domains (Fig. 3A Sup). To go further in characterizing the interaction between Tle3 315 and PA0259, we constructed truncated variants taking into account their domain organization 316 (Fig. 3 A). We further delimitated the interacting domain of Tle3 to its extreme C-terminus (the 317 DUF3274 domain) since only the truncated T18-Tle3D2 construct still interacts with T25- 318 PA0259 (Fig. 3C). We did not find the domain of interaction within PA0259 as none of the 319 DUF2875 domains alone interacts with Tle3 or this may suggest that both of them are required 320 for the interaction (Fig. 3C).

321 Next, we took advantage of all the constructs we made to test other interactions. The 322 BACTH assay also showed that PA0259 interacts with both forms of the VgrG2b C-terminal 323 extension (Fig. 3B). The domain of interaction on VgrG2b is thus at least constituted by the 324 DUF2345 and TTR domains. We also observed that PA0259 at least dimerizes since all the 325 PA0259 constructs interact with each other whatever the orientation of PA0259 (Fig. 3B, 3D,
Fig. 3B Sup). VgrG2b and Tli3 (PA0261) are also able to homomultimerize since all the constructs interact with each other (Fig. 3D, Fig. 3B Sup).

Taking into account the interactions revealed by the BACTH assay, we propose that the Tle3 toxin can be addressed to the H2-T6SS machinery VgrG2b component via PA0259. We thus named PA0259 “Tla3” for Type VI lipase adaptor protein.

331

## Tla3 (PA0259) characterization

To gain insight into the role of Tla3 during Tle3 secretion, we first validated the interactions of Tla3 with Tle3 and VgrG2b by a complementary approach of co-purification by affinity chromatography. Two different tagged versions of Tla3 were engineered by fusing a Strep-tag or a 10His-tag to the mature domain of Tla3 this leading to cytoplasmic tagged Tla3 proteins. The recombinant $Tla3^C_{Strep}$ was coproduced in *E. coli* BL21(DE3) pLysS with $Tle3^C_{His}$, and the $Tla3^C_{His}$ with 3 recombinant forms of Strep-tagged VgrG2b consisting in the full-length VgrG2b, or VgrG2b truncated for the extreme C-terminus (deletion of domain 3 in Fig. 3A) or VgrG2b truncated for the extreme C-terminus and the TTR domain (deletion of domains 2 and 3 in Fig. 3A). We initially tried with His-tagged VgrG2b but a problem of protein instability led us to shift for Strep-tagged VgrG2b. The bacterial lysates were loaded on a StrepTactin matrix, and $Tla3^C_{Strep}$ or the three recombinant $VgrG2b_{Strep}$ were eluted with desthibiotin. The presence of $Tle3^C_{His}$ and of $Tla3^C_{His}$ was visualized in the elution fractions with anti-His antibodies. As shown in Fig. 4A and 2A right panel, $Tle3^C_{His}$ was only found in the eluted fraction upon coproduction with $Tla3^C_{Strep}$. We observed that $Tla3^C_{His}$ is copurified only upon coproduction with the full length VgrG2b (Fig. 4B) or the VgrG2b truncated for the extreme C-terminus (Fig. 4C). Indeed when produced alone (Fig. 4B) or with VgrG2b truncated for the extreme C-terminus and the TTR domain (Fig. 4D), $Tla3^C_{His}$ was not purified by affinity chromatography. Since VgrG2b truncated for the extreme C-terminus still copurified with Tla3
we can exclude that this domain is required for the interaction. This is in line with the BACTH assay that showed an interaction between Tla3 and a truncated VgrG2b consisting in only the DUF2345 and TTR domains (domains 1 and 2, Fig. 3A). Moreover, the deletion of the TTR domain affecting the copurification, one can conclude that this domain is key for the interaction. Taken together these data confirmed a direct interaction of Tla3 with Tle3 on one side and with VgrG2b on the other side. By taking into account the BACTH data and the copurification with two truncated forms of VgrG2b, the domain of interaction of VgrG2b with Tla3 can be delimited to the TTR domain.

We then analyzed the cellular localization of Tla3 in *P. aeruginosa* that, according to its interactions with Tle3 and VgrG2b, should be cytoplasmic. As for Tli3, we engineered a chromosomally encoded Tla3<sub>V5</sub> translational fusion (Supplementary Table 1). Tla3 was indeed immunodetected in the cytoplasmic fraction (Fig. 5A). One could note that in contrast with our first hypothesis suggesting an incorrect start codon for *tla3* and our observation of the recombinant protein in the periplasm of *E. coli* (Fig. 1 Sup), Tla3 was totally absent from the periplasmic fraction of *P. aeruginosa*. To strengthen this result, each putative ATG was individually mutated on the chromosome of the PAO1 strain encoding the Tla3<sub>V5</sub> translational fusion (Fig. 5B). In agreement with its cytoplasmic localization in *P. aeruginosa*, Tla3 was only produced if the fourth ATG was intact. Accordingly the Tla3 protein synthesized from this ATG is not predicted to possess a N-terminal signal peptide (Fig. 1 Sup). In conclusion Tla3 is a cytoplasmic protein synthesized from the annotated translation start (pseudomonas.com) and this localization is in agreement with a role in the targeting of the toxin to the secretion machinery.

Next, we asked whether Tla3 is specific for Tle3 or if it can be required for the secretion of other substrates of the H2-T6SS machinery. Since Hcp secretion is the hallmark of a functional secretion system, we studied the secretion of Hcp2b whose gene is upstream *vgrG2b*
376 (Fig. 1A). Like Allsopp and colleagues (2017) we deleted the *rsmA* gene to enable Hcp2b
377 production and thus secretion by a PAO1 strain encoding a Hcp2b<sub>His</sub> translational fusion (Fig.
378 5C, compare line 1 and 2). RsmA is a posttranscriptional regulator known to repress all three
379 T6SS clusters of *P. aeruginosa* (Allsopp et al., 2017). This results in a massive secretion since
380 Hcp2b<sub>His</sub> can be observed in the extracellular protein samples by Coomassie-blue staining (Fig.
381 5C, lower panel). While Hcp2b<sub>His</sub> secretion was abolished in a *rsmA* *clpV2* mutant, a H2-T6SS
382 mutant, we observed that Hcp2b<sub>His</sub> is still secreted in the absence of Tla3 (Fig. 5C, compare
383 line 3 with line 4), suggesting that Tla3 is specific for the secretion of Tle3 but not for other
384 H2-T6SS proteins. In line with this specific adaptor-toxin pair, Tla3<sup>CStrep</sup> did not copurify TplE
385 (Tle4), another antibacterial phospholipase delivered by the H2-T6SS machinery (Fig. 5D).

386 Finally as the interaction with a VgrG can suggest that Tla3 is itself a T6SS effector, we
387 studied whether Tla3 is secreted by *P. aeruginosa*. To this end the *rsmA* gene was deleted from
388 the PAO1 strain encoding a Tla3<sub>V5</sub> translational fusion. Whereas Tla3<sub>V5</sub> was better produced
389 upon *rsmA* deletion (Fig. 5E), this did not lead to immunodetection of Tla3<sub>V5</sub> in the extracellular
390 medium although Hcp2b was readily observed by Coomassie-blue staining of the same samples.
391 Tla3 is thus not an effector *per se*.

392 In conclusion the cytoplasmic localization of Tla3 in *P. aeruginosa* is appropriate with
393 a recruiting role of Tle3 to the H2-T6SS machinery through an interaction with the TTR domain
394 of VgrG2b. Tla3 is not co-secreted with Tle3. Tla3 role seems specific to Tle3 since it is not
395 required for a functional H2-T6SS machinery and does not interact with TplE (Tle4), another
396 H2-T6SS effector.

397

398
Tle3 secretion mechanism by the H2-T6SS machinery of P. aeruginosa

399
In order to study the antibacterial role and the secretion of Tle3 by *P. aeruginosa*, we
400
performed intra-species bacterial competition assays between *P. aeruginosa* strains. This
consists in studying the survival of prey bacteria lacking the *tli3* immunity gene (by CFU counting of antibiotic resistant bacteria) co-cultivated 24 hours at 37°C on plate with various attackers. As immunity genes are essential genes (protection from fratricide intoxication), both toxin and immunity genes have been deleted in order to construct a viable Δ*tli3Δtle3* mutant strain (Supplementary Table 1). The figure 6 first confirms the antibacterial function of Tle3 and the immunity role of Tli3 since the growth of the immunity mutant was affected by the WT strain because it cannot resist Tle3 toxicity, while the Δ*tle3* mutant had no effect (Fig. 6 compare line 1 with line 2). The use of the Δ*clpV2* strain, a H2-T6SS mutant and of Δ*vgrG2b* and Δ*tla3* mutants allowed to demonstrate that Tle3 is delivered to prey bacteria through the H2-T6SS machinery and confirms that VgrG2b and Tla3 participate in Tle3 targeting to the H2-T6SS machinery (Fig. 6 compare line 1 with lines 4, 5 and 6). Indeed Δ*clpV2*, Δ*vgrG2b* and Δ*tla3* mutants had no effect on the immunity mutant growth since they cannot deliver Tle3 into prey bacteria. The complementation in cis of *tle3* and *tla3* deletions (the mutants have been constructed for this study) restored a WT phenotype (Fig. 6 compare line 2 with line 3, and line 6 with line 7) this demonstrating no polar effect on downstream genes. Furthermore the introduction of a wild-type copy of *tli3* at the *attB* site on *P. aeruginosa* chromosome of the Δ*tli3Δtle3* mutant restores wild-type competition capacity to this strain (Fig. 4A Sup). This confirms that the absence of the immunity was responsible of the phenotypes observed for the Δ*tli3Δtle3* strain (Fig. 6). Finally, the Δ*tla3* mutant has been used as a prey and was not affected by the WT strain or any of the mutants excluding definitively a role of immunity as proposed by its annotation (Pseudomonas.com) and confirming its adaptor function (Fig. 4B Sup).

Taken together these results demonstrated that Tle3 is an effective H2-T6SS-dependent antibacterial toxin loaded onto the VgrG2b puncturing device via Tla3 and neutralized by Tli3 in resistant prey bacteria.

425
426 **VgrG2b is a trans-kingdom toxin**

427 A putative neutral zinc metallopeptidase domain has been predicted at the extreme C-terminus
428 of VgrG2b by Pukatzki and colleagues (Pukatzki et al., 2007) (Fig. 3A). This motif (Prosite
429 PS00142, PFAM04298) consists in a metal-binding consensus motif HExxH, the two histidine
430 residues being ligands of the catalytic Zn²⁺ and the glutamic acid residue involved in
431 nucleophilic attack. As an effector with a protease activity can target both eukaryotic and
432 bacterial proteins, we searched for an antibacterial activity of the VgrG2b C-terminal extension.
433 To do this we performed the same heterologous toxicity assay in *E. coli* as with Tle3. As shown
434 in figure 7, whereas the production of VgrG2bCter in the cytoplasm did not impact *E. coli* growth,
435 its periplasmic production killed *E. coli* (Fig. 7, compare line 2 and 5). Moreover substitution
436 of the histidine in position 935 and of the glutamic acid 936 for an alanine relieves VgrG2bCter
437 toxicity this showing that VgrG2b is a novel antibacterial protease active in the periplasm.

438 Finally because of their genetic proximity, we tested whether Tli3 can be the immunity
439 of VgrG2bCter. Indeed the coproduction of Tli3 counteracts VgrG2bCter toxicity whether it is
440 coproduced from a second plasmid (Fig. 7, line 6) or from the same transcript (Fig. 7, line 7).
441 In conclusion Tli3 serves two toxins and protects also from the antibacterial activity of VgrG2b.

442

443 **Discussion**

444 Here we report the existence of a novel pair of antibacterial effector and immunity of
445 the H2-T6SS of *P. aeruginosa*, Tle3 (PA0260) and Tli3 (PA0261), and we propose a
446 chronology of Tle3 secretion process that includes a cytoplasmic adaptor protein, Tla3 (PA0259)
447 to load the toxin onto the VgrG2b spike (a model is proposed in Fig. 8). Through heterologous
448 toxicity assay and bacterial competition, we show that Tle3 was toxic once delivered in the
449 periplasm of prey bacteria and that Tli3 can neutralize the toxin in this compartment.
Interestingly this led us to discover that VgrG2b that we previously recognized as an anti-eukaryotic effector possesses an antibacterial activity as well.

The VgrG-recruitment of cargo effectors has been previously evidenced for several antibacterial effectors among them two toxins of the Tle family, TseL (Tle2) of *V. cholerae* (Dong et al., 2013; Liang et al., 2015; Unterweger et al., 2015) and Tle1 of EAEC (Flaugnatti et al., 2016). In both cases the *tle* genes were just downstream the *vgrG* genes like the organization of *vgrG2b* and *tle3* of *P. aeruginosa*. TseL and Tle1 have been shown to directly bind a dedicated VgrG, VgrG3 and VgrG1 respectively (Dong et al., 2013; Flaugnatti et al., 2016). The domain of interaction within EAEC VgrG1 has been delimited to the TTR domain and may also include the DUF2345, both of which are present in the *P. aeruginosa* VgrG2b. In line with this, and despite Tle3 requiring an adaptor to be targeted to VgrG2b, we have shown that Tle3 interacts with the TTR domain of VgrG2b. Taken together these data demonstrate that TTR domains of VgrGs are involved in recruitment and transport of Tle effectors, directly or through adaptor. Likewise C-terminal extensions of VgrG1 and VgrG2 of *Agrobacterium tumefaciens* were identified as specifically required for the delivery of each cognate DNAse toxins, named Tde1 and Tde2 respectively (Bondage et al., 2016). C-terminal domains of VgrGs can thus be considered more generally as specificity determinants for T6SS effector loading and transport.

Interestingly TseL of *V. cholerae* requires also Tap-1 (Tec) as an adaptor protein to be delivered to another VgrG, called VgrG1 (Liang et al., 2015; Unterweger et al., 2015), showing that a sole toxin can be targeted directly and indirectly to two different VgrG proteins. Tap-1 (Tec) belongs to the DUF4123 family of adaptor proteins that contains also VasW of *V. cholerae* (Miyata et al., 2013) and several uncharacterized gene products linked to effector genes with a MIX (marker for type VI effectors) motif in *Proteus mirabilis* or *B. thailandensis* for instance (Salomon et al., 2014). Interestingly TecT, a DUF4123 adaptor of *P. aeruginosa*,
has been shown to require a co-adaptor, called co-TecT, to deliver the TseT effector to the PAAR4 protein (Burkinshaw et al., 2018). This is the first example of an adaptor-co-adaptor module. Taken together these data suggest a conserved role for DUF4123 adaptors in the recruitment of a number of T6SS effectors. Remarkably Tla3 of *P. aeruginosa* does not belong to the DUF4123 adaptor family, or to that of the two other unrelated families, the DUF1795 adaptor family, reported with EagR (effector-associated gene with Rhs) in *Serratia marcescens* (Alcoforado Diniz and Coulthurst, 2015) or EagT6 in *P. aeruginosa* (Whitney et al., 2015), and the DUF2169 adaptor family reported with Atu3641 in *A. tumefaciens* (Bondage et al., 2016). Nor Tla3 is a PAAR protein, the last class of effector targeting mode to a VgrG (Bondage et al., 2016; Burkinshaw et al., 2018; Cianfanelli et al., 2016a; Shneider et al., 2013; Whitney et al., 2014). Instead we find that Tla3 harbors two DUF2875 domains (Fig. 3A) that are both required for the interaction with the toxin. Moreover genes coding DUF2875 containing proteins can be found at the vicinity of *tle*, *tli*, *vgrG* or PAAR genes, but they are restricted to α and β proteobacteria (Fig. Sup 5). We thus hypothesize that DUF2875 might assist in T6SS-mediated effector delivery. Like the three other adaptor families (DUF4123, DUF1795, DUF2169), we have observed that Tla3 is not required for the H2-T6SS functionality since the *Δtla3* mutant still secretes Hcp2b and can compete with a WT strain, and that Tla3 is specific for the Tle3 toxin since it did not interact with another H2-T6SS effector, TplE (Tle4). Finally as we did not detect Tla3 secretion under constitutive H2-T6SS condition, we propose that Tla3 hands over Tle3 to VgrG2b in the cytoplasm prior to its loading to the baseplate and further recruitment to the central Hcp tube in preparation (Fig. 8). Overall, the existence of various modes of effector recruitment, further refined with adaptors, likely explains how the T6SS is able to deliver numerous and structurally diverse proteins.

Five families of Tle, Tle1-5, have been identified among Gram-negative bacteria (Russell et al., 2013) and four Tle have been studied in *P. aeruginosa* so far. Our demonstration
of the activity of Tle3 in the periplasm is consistent with the observations that the heterologous
periplasmic production of PldA (Tle5a) (Jiang et al., 2014; Russell et al., 2013), PldB (Tle5b)
(Jiang et al., 2014), Tle1 (Hu et al., 2014) and TplE (Tle4) (Jiang et al., 2016) is toxic. The
reason of the periplasmic activity of Tle proteins is still unclear although several hypothesis
have been proposed (Flaugnatti et al., 2016), the most likely being an activation of the toxin in
this compartment. Very recently this has nicely been exemplified with the hijacking of DsbA
in the target cells of *Serratia macedensis* for the activation of incoming effectors (Mariano et al.,
2018). No member of the Tle3 family has yet been enzymatically characterized. Our attempts
to efficiently purify Tle3 from *E. coli* or from *P. aeruginosa* have been unsuccessful, even if
we have noticed that the presence of the Tla3 adaptor stabilized Tle3, it still formed inclusion
bodies. In the future we will decipher the enzymatic activity of Tle3, which is presumably active
on membrane phospholipids as our preliminary data of thin-layer chromotography tend to show.

The periplasmic activity of Tle toxins is counteracted by the synthesis of a cognate
immunity protein that is usually a periplasmic soluble protein, as we showed for Tli3 in *P. aeruginosa*, or a membrane-anchored lipoprotein (Russell et al., 2013). Interestingly the genetic
organization of the *tli3* gene upstream of the *tle3* gene observed in *E. coli*, *K. pneumoniae*, *B. cenocepacia* or *R. solanacearum* (Russell et al., 2013) is conserved in *P. aeruginosa*. The fact
that the two genes are co-transcribed (the immunity being the first) is key for the protection
against toxicity. Indeed we have observed systematic protection against the periplasmic toxicity
of Tle3 in *E. coli* when the two genes were expressed from the same promoter under the same
plasmid whereas it was not as efficient when the genes were on two plasmids. This genetic link
reinforces the connection within the toxin/immunity pair. This has previously been noticed with
a T7SS antibacterial toxin and its immunity in *Staphylococcus aureus* (Cao et al., 2016). Other
immunities of Tle characterized so far have been shown to inhibit the action of the effector by
direct protein-protein contacts (Flaugnatti et al., 2016; Jiang et al., 2014; Russell et al., 2013).
Our copurification assay in *E. coli* demonstrates a direct interaction between Tle3 and Tli3 that was already suggested with the release of Tle3 toxicity upon coproduction of Tli3 in the periplasm. A crystal structure of the *P. aeruginosa* TplE (Tle4) effector in complex with its immunity protein TplEI (Tli4) revealed that the immunity uses a grasp mechanism to prevent the interfacial activation of the toxin (Lu et al., 2014).

Like two other H2-T6SS related orphan *vgrG* loci, the *vgrG4b* cluster encoding PldA (Tle5a) and the *vgrG2a* encoding TplE (Tle4), we show here that the *vgrG2b* cluster has both antibacterial activities (through Tle3 and VgrG2b) and anti-eukaryotic (through VgrG2b; Sana et al., 2015)). Interestingly VgrG2b is thus (i) a structural component of the H2-T6SS puncturing device since our bacterial competition showed its requirement for Tle3 delivery, (ii) an anti-eukaryotic effector through an interaction with the microtubule nucleating complex (Sana et al., 2015) and (iii) an antibacterial effector as suggested by our toxicity assay in *E.coli*. We have shown that two conserved residues of the putative metallopeptidase motif (an histidine and a glutamic acid) are essential for the VgrG2b antibacterial activity. This is consistent with an antibacterial protease activity for VgrG2b that will be, to our knowledge, the first case in the T6SS effector literature. The discovery of the VgrG2b immunity is even more exciting since we observed that Tli3 also relieves VgrG2bCter toxicity. To our knowledge, Tli3 is the first example of an T6SS immunity serving two different proteins. But one can cite the chaperones of the type III secretion translocators that form a pore in the eukaryotic target cell membrane (Neyt and Cornelis, 1999). Indeed the chaperone SycD from *Yersinia* enterocolitica and its homologues from other Gram negative bacteria bind the two translocators to prevent their premature interaction in the inner membrane and avoid further toxicity.

547

548 Acknowledgments
We thank V. Tutagata for pVT1, pVT8 and pVT9 constructs, B. Douzi for all the advices during protein purification and members of B.B. PhD committee for helpful discussion and support. We are grateful to M. Ba, I. Bringer, A. Brun and O. Uderso for technical assistance. B.B. was financed with a PhD fellowship from the French Research Ministry. This work was supported by recurrent funding from the CNRS and Aix-Marseille University. The project leading to this publication has received funding from the Excellence Initiative of Aix-Marseille University-A*Midex, a French "Investissements d'Avenir" program ("Emergence & Innovation" A-M-AAP-EI-17-139-170301-10.31-BLEVES-HLS).

## Author contributions statement

B.B. and S.B. designed and conceived the experiments. B.B., C.S., and S.D. performed the experiments. S.B. supervised the execution of the experiments. B.B., C.S., B.I. and S.B. analyzed and discussed the data. S.B. wrote the paper with contribution from B.B. and reading from B.I.

## Conflict of Interest Statement

The authors declare no conflict of interest.

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629 Jiang, F., Waterfield, N. R., Yang, J., Yang, G., and Jin, Q. (2014). A Pseudomonas aeruginosa type VI secretion phospholipase D effector targets both prokaryotic and eukaryotic cells. *Cell Host Microbe* 15, 600–610. doi:10.1016/j.chom.2014.04.010.

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635 Kolaskar, A. S., and Reddy, B. V. (1985). A method to locate protein coding sequences in DNA of prokaryotic systems. *Nucleic Acids Res.* 13, 185–194.

637 LaCourse, K. D., Peterson, S. B., Kulasekara, H. D., Radey, M. C., Kim, J., and Mougous, J. D. (2018). Conditional toxicity and synergy drive diversity among antibacterial effectors. *Nat Microbiol* 3, 440–446. doi:10.1038/s41564-018-0113-y.

640 Liang, X., Moore, R., Wilton, M., Wong, M. J. Q., Lam, L., and Dong, T. G. (2015). Identification of divergent type VI secretion effectors using a conserved chaperone domain. *Proc. Natl. Acad. Sci. U.S.A.* 112, 9106–9111. doi:10.1073/pnas.1505317112.

643 Lu, D., Zheng, Y., Liao, N., Wei, L., Xu, B., Liu, X., et al. (2014). The structural basis of the Tle4-Tli4 complex reveals the self-protection mechanism of H2-T6SS in Pseudomonas aeruginosa. *Acta Crystallogr. D Biol. Crystallogr.* 70, 3233–3243. doi:10.1107/S1399004714023967.

647 MacIntyre, D. L., Miyata, S. T., Kitaoka, M., and Pukatzki, S. (2010). The Vibrio cholerae type VI secretion system displays antimicrobial properties. *Proc. Natl. Acad. Sci. U.S.A.* 107, 19520–19524. doi:10.1073/pnas.1012931107.

650 Mariano, G., Monlezun, L., and Coulthurst, S. J. (2018). Dual Role for DsbA in Attacking and Targeted Bacterial Cells during Type VI Secretion System-Mediated Competition. *Cell Rep* 22, 774–785. doi:10.1016/j.celrep.2017.12.075.

653 Miyata, S. T., Unterweger, D., Rudko, S. P., and Pukatzki, S. (2013). Dual expression profile of type VI secretion system immunity genes protects pandemic Vibrio cholerae. *PLoS Pathog.* 9, e1003752. doi:10.1371/journal.ppat.1003752.

656 Mougous, J. D., Cuff, M. E., Raunser, S., Shen, A., Zhou, M., Gifford, C. A., et al. (2006). A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. *Science* 312, 1526–1530. doi:10.1126/science.1128393.
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664 Pukatzki, S., Ma, A. T., Revel, A. T., Sturtevant, D., and Mekalanos, J. J. (2007). Type VI
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668 Pukatzki, S., Ma, A. T., Sturtevant, D., Krastins, B., Sarracino, D., Nelson, W. C., et al. (2006).
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675 Salomon, D., Kinch, L. N., Trudgian, D. C., Guo, X., Klimko, J. A., Grishin, N. V., et al. (2014).
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678 Sana, T. G., Baumann, C., Merdes, A., Soscia, C., Rattei, T., Hachani, A., et al. (2015).
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682 Sana, T. G., Berni, B., and Bleves, S. (2016a). The T6SSs of Pseudomonas aeruginosa Strain
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685 Sana, T. G., Flaugnatti, N., Lugo, K. A., Lam, L. H., Jacobson, A., Baylot, V., et al. (2016b).
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689 Sana, T. G., Hachani, A., Bucior, I., Soscia, C., Garvis, S., Termine, E., et al. (2012). The second
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693 Shneider, M. M., Buth, S. A., Ho, B. T., Basler, M., Mekalanos, J. J., and Leiman, P. G. (2013).
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696 Unterweger, D., Kostiuk, B., Ötjengerdes, R., Wilton, A., Diaz-Satizabal, L., and Pukatzki, S.
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702 Whitney, J. C., Quentin, D., Sawai, S., LeRoux, M., Harding, B. N., Ledvina, H. E., et al. (2015). An interbacterial NAD(P)(+) glycohydrolase toxin requires elongation factor Tu for delivery to target cells. *Cell* 163, 607–619. doi:10.1016/j.cell.2015.09.027.

705 WHO publishes list of bacteria for which new antibiotics are urgently needed Available at: https://www.who.int/news-room/detail/27-02-2017-who-publishes-list-of-bacteria-for-which-new-antibiotics-are-urgently-needed [Accessed January 21, 2019].

708

709

710

In review
711 **Figure legends**

712 **Fig. 1: vgrG2b island organization (A)** The genes are labeled with the given name (i.e., *hcp2b*)
713 and are indicated by their annotation number (e.g., PA0263). The Sec signal peptide of Tli3
714 (PA0261) and the two DU2875 of Tla3 (PA0259) are represented with stripped boxes. **The Tle3**
715 **periplasmic toxicity is counteracted by Tli3 (PA0261) (B).** Serial dilutions (from non-diluted
716 to 10<sup>-7</sup>) of normalized cultures of *E. coli* BL21(DE3)pLysS producing the wild-type Tle3 in
717 the cytoplasm, called Tle3<sup>c</sup> (from pVT1, a pETDuet-1 derivate) or in the periplasm, called Tle3<sup>p</sup>
718 (from pSBC81, a pET22b(+) derivate yielding a fusion of Tle3 with a Sec signal peptide) were
719 spotted on LB agar plates supplemented (left panel) with 0.4% glucose or (right panel) with
720 0.1mM IPTG. Glucose and IPTG allow respectively repression and induction of the gene
721 encoding the T7 RNA polymerase. When indicated Tli3 (PA0261) or Tla3 (PA0259) were
722 produced in the periplasm from pVT8, pSBC107 and pVT9 respectively, pRSFDuet-1 derivates.
723 Line 1: pET22b(+) and pRSFDuet-1, line 2: pVT1 and pRSFDuet-1, line 3: pSBC81 and
724 pRSFDuet-1, line 4: pSBC81 and pVT8, line 5: pSBC81 and pVT9, line 6: pVT8 and
725 pET22b(+), line 7: pVT9 and pET22b(+) line 8: pSBC107 is a pRSFDuet-1 derivate producing
726 Tli3 (PA0261) and Tle3<sup>p</sup> from the same transcript.

727

728 **Fig. 2: Tli3 (PA0261) interacts with Tle3 (A)** Copurification assay on StrepTactin column of
729 Tli3cSTREP with Tle3c10HIS produced in *E. coli* BL21(DE3)pLysS produced from pBB26
730 and pVT1 respectively. The unbound (U) and eluted (E) fractions were collected and subjected
731 to SDS-PAGE (10.5 %) and Western blot analyses using anti-His antibody (Upper) and anti-
732 streptavidin antibody (Lower). The position of the proteins and the molecular mass markers (in
733 kDa) are indicated. **Tli3 (PA0261) is a periplasmic protein in P. aeruginosa (B)** Cells of *P.
734 aeruginosa* PAO1 tli3V5 were subjected to fractionation and immunoblotting using antibodies
735 directed against the V5 tag, XcpY and DsbA. XcpY and DsbA were used as membrane and
periplasmic controls respectively. T: whole cell, C: cytoplasm, Mb: total membrane, P:
periplasm. The position of the proteins and the molecular mass markers (in kDa) are indicated.

738

739 **Fig. 3: Tla3 (PA0259) interacts with the Tle3 toxin and with VgrG2b. Domain**
740 **organization of VgrG2b, Tle3 and Tla3 (PA0259) (A).** The first 581 residues of VgrG2b
741 carry the VgrG domain homologous to gp27 and gp5 phage tail proteins and consisting of three
742 sub-domains. This is followed by the C-terminal extension, composed of a conserved domain
743 of uncharacterized proteins (DUF2345, PF10106) (2bCt1), a TTR (transthyretin-like region)
744 (2bCt2), and a putative zinc-dependent metallopeptidase pattern (LFIHEMTHVW signature,
745 PS00142 (2bCt3). Tle3 architecture consists of an α β hydrolase fold domain (Tle3D1) followed
746 by a DUF3274 (Tle3D2), Tla3 of a tandem of DUF2875 (Tla3D1 and Tla3D2). **Bacterial two-**
747 **hybrid assay (B, C and D).** BTH101 reporter cells producing the indicated proteins or domains
748 fused to the T18 or T25 domain of the *Bordetella* adenylate cyclase were spotted on X-gal
749 indicator plates. The blue color of the colony reflects the interaction between the two proteins.
750 TolB and Pal are two proteins known to interact but unrelated to the T6SS. The experiment was
751 performed in triplicate and a representative result is shown.

752

753 **Fig. 4: Tla3 interaction with Tle3 (A) and VgrG2b (B-D) Copurification assay on**
754 StrepTactin column of Tla3<sup>cSTREP</sup> with Tle3<sup>cHis</sup> produced in *E. coli* BL21(DE3)pLysS from
755 pBB27 and pVT1 respectively (A) or of Tla3<sup>cHis</sup> with VgrG2b<sup>FLSTREP</sup> (B) or VgrG2b<sup>FLD3STREP</sup>
756 (C) or VgrG2b<sup>FLD2-3STREP</sup> (D) produced from pBB28 and pBB25 (B) or pBB41(C) or pBB42 (D).
757 The unbound (U) and eluted (E) fractions were collected and subjected to SDS-PAGE (10.5%)
758 and Western blot analysis using anti-His antibody (Upper) and anti-streptavidin antibody
759 (Lower). The dashed line separates lanes from non-adjacent part of the same gel. The position
760 of the proteins and the molecular mass markers (in kDa) are indicated.
Fig. 5: **Tla3(PA0259) is a cytoplasmic protein of P. aeruginosa (A)** Cells of P. aeruginosa PAO1 *tla3v5* were subjected to fractionation and immunoblotting using antibodies directed against the V5 tag, XcpY and DsbA. XcpY and DsbA were used as membrane and periplasmic controls respectively. T: whole cell, C: cytoplasm, Mb: total membrane, P: periplasm. **The annotated ATG drives the initiation of translation of tla3 in P. aeruginosa (B).** Immunodetection of Tla3v5 with anti-V5 antibodies produced in a WT background or in strains in which one of the four predicted ATG in *tla3* have been substituted in ATA. The number followed by a star indicates which ATG from Fig. 1Supp., ATG4 corresponding to the annotated ATG. **Tla3 is not required for Hcp2b secretion (C).** Immunodetection of Hcp2b<sub>6His</sub> with anti-His antibodies produced in a WT background (line 1) or in strains deleted for *rsmA* (lines 2 to 4) and *clpV2* (line 3) or *tla3* (line 4). The strains were grown at 25°C for 24 h and total bacteria were separated from extracellular medium. Anti-EF-Tu is used as a lysis control. The extracellular medium proteins were also stained with Coomassie-blue. **Tla3 doesn't interact with Tle4 (D).** Copurification assay on StrepTactin column of Tla3<sup>cSTREP</sup> with Tle4<sub>His</sub> produced in *E. coli* BL21(DE3)pLysS from pBB27 and pVT3 respectively. Legend as in Fig. 4. **Tla3 is not secreted (E).** Immunodetection of Tla3v5 with anti-HV5 antibodies produced in a WT background (line 1) or in strain deleted for *rsmA* (line 2). The strains were grown at 25°C for 24 h and total bacteria were separated from extracellular medium. Anti-EF-Tu is used as a lysis control. The extracellular medium proteins were also stained with Coomassie-blue. (A-E) The position of the proteins and the molecular mass markers (in kDa) are indicated.

Fig. 6: **P. aeruginosa growth competition.** The *P. aeruginosa* prey strain (Δ*tli3Δtle3*) was incubated with various *P. aeruginosa* attacker strains as indicated in the figure for 24 h at 37°C. The number of recovered prey bacteria is indicated in CFU/ml. “comp” stand for *cis* complementation of the corresponding mutation with a wild-type copy inserted at the *attB* site
on *P. aeruginosa* chromosome. Error bars represent ± SEM (n = 3); ***p < 0.001, ns not significant.

788

**Fig. 7: The protease domain of VgrG2b is required for its antibacterial activity.** Serial dilutions (from non-diluted to 10⁻⁵) of normalized cultures of *E. coli* BL21(DE3)pLys producing the wild-type C-terminal domain of VgrG2b in the cytoplasm, called CterVgrG2bC (from pBB43, a pETDuet-1 derivate, line 5) or in the periplasm, called CterVgrG2bP (from pBB44, a pET22b(+) derivate yielding a fusion of the C-terminal domain with a Sec signal peptide, line 2) or two variants CterVgr2bP<sup>H935A</sup> (from pBB45, line 3) and CterVgr2bP<sup>E936A</sup> (from pBB46, line 4) were spotted on LB agar plates supplemented (left panel) with 0.4% glucose or (right panel) with 0.1mM IPTG. In line 6, the CterVgrG2bP (from pBB44) was coproduced with Tli3 (from pVT8) and in line 7, the CterVgrG2bP and Tli3 were coproduced from the same transcript (from pSBC110). Glucose and IPTG allow respectively repression and induction of the gene encoding the T7 RNA polymerase.

800

**Fig. 8: Working model: Tle3 targeting to the H2-T6SS machinery (left panel) Tle3 (in pink) is taken in charge in the cytoplasm by Tla3 (in orange) that binds VgrG2b (green). Upon Hcp2b (in dark grey) assembly into the growing sheath (light grey), the VgrG2b loaded with the Tle3 is placed at the tip of the Hcp arrow. Tle3 and VgrG2bCter delivery into bacteria (right panel).** The sheath contraction in the cytoplasm propels the Hcp arrow towards the target bacterium. Tle3 associated with this expelled structure is thus translocated into target cells, as well as VgrG2bCter (green circle). The prey is killed (PreyS) except if it has the Tli3 immunity (in blue) in the periplasm (PreyR). The mechanism of resistance to VgrG2bCter is still unknown. OM: outer membrane, PG: peptidoglycan, IM: inner membrane, BP: base plate, MC: membrane complex. PreyS: sensitive prey, PreyR: resistant prey.
811

812

In review
<figure><img src="image_4.png" /><figcaption>**Figure 1**</figcaption></figure>

+ glucose 0.4%

+ IPTG 0.1 mM

E. coli

empty vectors

Tle3<sup>c</sup><sub>10His</sub>

Tle3<sup>p</sup><sub>10His</sub>

Tle3<sup>p</sup><sub>10His</sub> & Tli3 (PA0261)

Tle3<sup>p</sup><sub>10His</sub> & Tla3 (PA0259<sub>ATG1</sub>)

Tli3 (PA0261)

Tla3 (PA0259<sub>ATG1</sub>)

Tle3<sup>p</sup><sub>10His</sub> & Tli3 (PA0261)

from the same transcript

Cytoplasm

Periplasm
<figure><img src="image_5.png" /><figcaption>**Figure 2**</figcaption></figure>

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>Figure 3</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Figure 4</figcaption></figure>

<figure><img src="image_9.png" /></figure>
<figure><img src="image_10.png" /><figcaption>Figure 5</figcaption></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>
<caption>Figure 6.TIF</caption>

<figure><img src="image_15.png" /><figcaption>Figure 6</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Figure 7**</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>Figure 8</figcaption></figure>