BACKGROUND
within the genus yersinia there are three human-pathogenic species; y. pestis, the causative agent of plague, and two enteric pathogens, y. pseudotuberculosis and y. enterocolitica.
despite the differences in disease, y. pestis and y. pseudotuberculosis are very closely related at the genetic level.
y. pestis is believed to have evolved from y. pseudotuberculosis between  <dig> - <dig>  years ago  <cit> .
thus, in a remarkably short length of evolutionary time, y. pestis has evolved from an enteropathogen, to a blood-borne pathogen with an insect vector  <cit> .
genome sequencing of several y. pseudotuberculosis and y. pestis strains, revealed that y. pestis has accumulated a large number of pseudogenes since its divergence.
by the "use it or lose it" paradigm, this is suggestive of the decay of those genes that are no longer required for function as y. pestis adapts to a new lifestyle  <cit> .
gene disruption may also result in pathoadaptive mutation, whereby loss of gene function results in an increase in virulence  <cit> .
this has been demonstrated in several pathogenic bacteria including shigella spp.
and escherichia coli  <cit> .
pathoadaptive mutations have previously been identified in y. pestis, with the negative regulators of biofilm formation, rcsa and ngha, being disrupted, resulting in the ability of y. pestis to form biofilms within the flea vector  <cit> .
pseudogenes in y. pestis that are known to be essential for the enteric lifestyle of y. pseudotuberculosis, include the adhesins yada and invasin  <cit> .
invasin was one of the first bacterial virulence factors identified, when it was observed that the inv gene alone was sufficient to convert benign non-invasive laboratory e. coli strains, to being capable of invading tissue culture cells  <cit> .
invasin is a  <dig> kda protein that is capable of binding to β <dig> integrins on the host cells, promoting internalisation of the bacterium  <cit> .
during early infection, invasin specifically binds β <dig> integrins on the apical surface of m cells, which facilitates efficient translocation to the underlying peyer's patches  <cit> .
the invasin protein is composed of a short n-terminal transmembrane domain, four structural bacterial immunoglobulin domains  and a c-type lectin-like domain  <cit> .
the last big domain and the c-type lectin-like domain comprise the functional β <dig> integrin binding region  <cit> .
in the same family of bacterial adhesion proteins as invasin, is intimin, an important adhesin expressed by enteropathogenic  and enterohaemorrhagic  e. coli on the lee pathogenicity island  <cit> .
intimin is a  <dig> kda outer membrane protein that is also found in citrobacter freundii and hafnia alvei  <cit> .
the functional binding domain of intimin is located in the  <dig> amino acid c-terminal region, and consists of two big domains and a c-type lectin-like domain, which are structurally similar to invasin  <cit> .
when epec and ehec colonise the small intestine mucosa, a characteristic attaching and effacing lesion is formed.
first there is localised destruction  of the microvilli, which leads to intimate attachment of the bacterium to the host cell  <cit> .
epec and ehec encode a specific intimin receptor, translocated intimin receptor .
this receptor is translocated directly into the host cells via a type iii secretion system, where it becomes expressed on the cell surface  <cit> .
intimin binds to tir leading to its activation, which results in actin polymerisation within the host cell and the formation of a pedestal, facilitating tighter adherence between the host cell and the bacterium  <cit> .
other eukaryotic receptors have been suggested for intimin, including nucleolin and some β <dig> integrins, but as yet it is unknown if these interactions have a role in vivo  <cit> .
there is considerable sequence variation between the intimins from different e. coli strains and they have been categorised into different subtypes, each with a high affinity for its own cognate tir  <cit> .
however, despite this diversity, it has been found that within the c-terminal binding domain there are four tryptophan residues and two cysteine residues, which are conserved between all subtypes  <cit> .
the two cysteines are also conserved in similar locations within the y. pseudotuberculosis invasin.
in both invasin and intimin a disulphide bond is formed, which is essential for the structure of the c-terminal binding domain and therefore required for full functionality  <cit> .
in the instance of invasin, disruption of either cysteine results in an inability to bind to integrin, and therefore is defective for invasion  <cit> .
analysis of y. pseudotuberculosis strain ip <dig> sequence data identified a gene encoding a protein with significant amino acid similarity to invasin and intimin, which has not been previously investigated.
we have termed this protein ifp  and intriguingly it has been mutated to a pseudogene in all seven y. pestis genomes sequenced to date.
examination of the amino acid sequence of ifp revealed that three of the four tryptophans and both of the cysteine residues that are important in intimin function are conserved.
however, no tir orthologue can be identified in the ip <dig> genome sequence.
given the amino acid similarity of ifp to both invasin and intimin, coupled with it being putatively non-functional in y. pestis, we postulated that ifp may be an adhesin.
we demonstrate that ifp is a functional adhesin that binds to distinct foci on host cells.
expression occurs in late log or early stationary phase at 37°c only and coincides with a decline in the expression of invasin at this temperature.
methods
strains used and culture conditions
all y. pseudotuberculosis strains were cultured in luria-bertani  broth miller  or on lb agar  at 28°c unless otherwise stated.
the retention of the virulence plasmid  was screened by plating y. pseudotuberculosis strains on crmox plates  <cit> .
antibiotics were used at the following concentrations where appropriate, ampicillin , kanamycin , chloramphenicol  and trimethoprim .
e. coli strains were cultured at 37°c overnight in lb broth miller or on lb agar unless otherwise stated.
see table  <dig> for a list of the strains and plasmids used in this study.
construction of lux reporter strains
pcr primers  were designed to amplify  <dig> bp and  <dig> bp fragments between yptb <dig> and yptb <dig> and between yptb <dig> and yptb <dig> respectively using y. pseudotuberculosis strain ip <dig> genomic dna as a template.
these regions contain the putative promoter and regulatory sequences for ifp  and inv .
these pcr products were cloned into the pgem-t easy vector .
kpni and spei restriction sites had been incorporated into the primer sequences to enable the luxcdabe operon from pbluelux  <cit>  to be inserted downstream of each promoter region.
the entire promoter-lux construct was excised from pgem-t easy then re-cloned into the pdm <dig> suicide plasmid using transformax ec100d pir+ e. coli  for selection and screening.
the resulting promoter fusions 1572lux and 1668lux in pdm <dig> were then electroporated into the ip <dig> strain of y. pseudotuberculosis and screened for single crossover event into the genome by chloramphenicol resistance.
this crossover event resulted in a functional gene of interest, with the lux cassette with native promoter inserted upstream of the gene on the chromosome.
restriction sites are in bold and position of mutated cysteine to glycine residue is underlined.
bioluminescence and optical density of lux constructs
bioluminescence and optical density were monitored simultaneously in 96-well microtitre plates using the anthos lucy <dig> combined photometer and luminometer controlled by the stingray software .
overnight 30°c cultures of 1572lux and 1668lux were diluted 1: <dig> in fresh lb with antibiotics and grown for 3- <dig> h at 30°c.
the optical density of the culture was adjusted to a starting od <dig> of  <dig>  and  <dig> μl was added to the wells of the  <dig> well plate.
assays were performed at 24°c, 28°c and 37°c.
luminescence and od at  <dig> nm of the cultures was automatically determined every  <dig> min for  <dig> h and presented as relative light units per unit of od <dig> .
for every promoter fusion assay each sample was assayed in triplicate on the 96-well plate, and each experiment was carried out in duplicate.
construction of maltose binding protein  fusion proteins
primers yptbintmbp- <dig> and yptbintmbp- <dig>  were used to pcr amplify the 3' end  of the ifp coding sequence.
this pcr product was cloned into the pmal-p2x vector  using ecori and xbai enzyme restriction sites which had been incorporated into the primer design.
in order to generate an mbp-ifp fusion with the terminal cysteine  mutated to a glycine , primer yptbintmbp- <dig> was substituted for yptbmbp- <dig>  primer yptbintmbp- <dig> contained an alternative sequence  to mutate the terminal cysteine to a glycine.
mbp-fusion proteins were then expressed in tb <dig> e. coli.
purification of mbp-ifp and mbp-ifpc337g
e. coli transformed with the mbp-fusion plasmids were cultured for  <dig> h at 37°c using  <dig> ml of an overnight culture in  <dig> ml lb broth with  <dig> mm glucose and ampicillin until a culture growth of od <dig>  <dig>  was reached.
expression of the fusion protein was induced with  <dig>  mm isopropyl-β-d-thiogalactoside  then the culture was incubated for further  <dig> hours at 37°c.
the cultures were centrifuged and pellets stored overnight at -20°c then resuspended in column buffer .
the cells were lysed by sonication using a bioruptor sonicator .
insoluble proteins were removed by centrifugation and the supernatant was applied to  <dig> ml columns of amylose resin .
after washing with  <dig> ml column buffer, proteins were eluted with  <dig> ml column buffer/ <dig> mm maltose.
proteins were concentrated using amicon ultra  <dig> kda columns , and then confirmed by coomassie staining and western blotting with anti-mbp .
protein concentrations were determined by pierce bca protein assay kit  according to the manufacturer's protocol.
analysis of mbp-fusion protein binding to hep- <dig> cells by fluorescence microscopy
binding of mbp-tagged ifp was determined by fluorescence microscopy as described previously  <cit> .
hep- <dig> cells were cultured overnight on glass coverslips in 24-well plates at  <dig>  ×  <dig> cells/well in  <dig> ml tissue culture media , 10% fetal calf serum ,  <dig> mm l-glutamine,  <dig> u penicillin ml- <dig>   <dig> μg streptomycin ml- <dig>  mem non-essential amino acids).
the cells were blocked for  <dig> minutes at 37°c/5% co <dig> in  <dig> μl binding solution ,  <dig>  mm maltose,  <dig> mm l-glutamine in rpmi media), then incubated for  <dig> minutes at 37°c/5% co <dig> with  <dig> μl mbp-ifp, mbp-ifpc337g or mbp protein alone  at  <dig> μg ml- <dig> in binding solution.
the cells were washed  <dig> times with  <dig> ml pbs/1% bsa  and incubated for  <dig> minutes at 37°c/5% co <dig> in  <dig> μl of a 1: <dig> dilution of rabbit anti-mbp antibody  in binding solution used.
cells were washed  <dig> times with  <dig> ml pbs/1% bsa  and incubated for  <dig> minutes at 37°c/5% co <dig> in  <dig> μl of a 1: <dig> dilution of goat anti-rabbit igg alexafluor  <dig>  in binding solution.
cells were washed  <dig> times with  <dig> ml pbs/1% bsa  and fixed for  <dig> minutes at -20°c in  <dig> μl of 95% ethanol-5% acetic acid .
the cover slips were removed from the wells, washed in milli q h2o and mounted onto glass slides with vectashield-dapi  mounting medium.
the coverslips were examined using an axiovert 200m  confocal microscope.
experiment was performed on three independent occasions and at least  <dig> cells were examined per experiment.
facscan analysis of mbp-fusion protein binding to hep- <dig> cells
a similar methodology was used as for the fluorescence microscopy as described previously  <cit> , with the following modifications.
the cells were grown directly in 6-well plates at  <dig> ×  <dig> cells/well.
the alexafluor  <dig> anti-rabbit igg antibody was diluted to 1: <dig> in pbs/1% bsa .
cells were resuspended in pbs/ <dig> % edta  and transferred to bd falcon  <dig> ml tubes .
cells were washed once with pbs/1% bsa  and centrifuged, then were fixed for  <dig> minutes on ice in 2% paraformaldehyde/pbs .
the cells were washed once with pbs/1% bsa , centrifuged and then were resuspended in  <dig> μl pbs/1% bsa/ <dig> % edta .
the fluorescence was measured using a facscan machine .
experiment was performed on two independent occasions and  <dig>  cells were examined for fluorescence from each sample.
analysis of co-localisation of mbp-fusion protein and the receptors cd <dig> and β <dig> integrin on hep- <dig> cells by fluorescence microscopy
a similar methodology was used as for the fluorescence microscopy described above with the following modifications.
after the mbp-fusion protein incubation the cells were washed  <dig> times with pbs then incubated with  <dig> μl of a 1: <dig> dilution rabbit anti-ifp  and 1: <dig> dilution of mouse anti-cd <dig>  or a 1: <dig> dilution of mouse anti-β <dig> integrin in binding solution for  <dig> minutes at 37°c/5% co <dig>  the cells were washed  <dig> times with  <dig> ml pbs then incubated with  <dig> μl of a 1: <dig> dilution of goat anti-rabbit igg alexafluor  <dig>  and 1: <dig> dilution of goat anti-mouse igg alexafluor  <dig>  for  <dig> minutes at 37°c/5% co <dig>  the cells were washed  <dig> times with  <dig> ml pbs then fixed for  <dig> minutes at 4°c with  <dig> μl 2% paraformaldehyde .
the coverslips were removed from the wells, washed with pbs then mounted onto glass slides with vectashield-dapi mounting medium .
the slides were examined using an axiovert  <dig> m confocal microscope .
at least three areas of approximately  <dig> cells each were examined per sample and the experiment was performed on three independent occasions.
construction of ifp and inv insertional mutants
an ifp knockout mutant was generated in the y. pseudotuberculosis strain ip <dig>  after initially constructing an ifp mutant in strain ypiii.
briefly,  <dig> bp of ifp was amplified with inta and intb primers, digested with saci and sphi then ligated into the cloning vector pgem-t easy.
the plasmid was digested with bglii to linearise and allow for the ligation of the kanamycin cassette within the ifp sequence.
pcr with primers inta and intb was undertaken on the plasmid to create linear fragments of kanamycin cassette flanked by ifp sequence.
this pcr product was electroporated into ypiii previously transformed with pkobeg, which contains the λ red recombinase operon.
the temperature sensitive pkobeg plasmid was then lost from putative mutants by growth at 37°c, whilst the presence of the pyv plasmid was maintained by the addition of  <dig>  mm cacl <dig>  southern blot analysis confirmed correct mutation.
genomic dna from this ypiiiΔifp was used as a template for pcr amplification of the kanamycin cassette flanked by two ~ <dig> bp regions of gene-specific dna.
the primers inta and intb  were used to amplify a  <dig>  kbp product.
this was purified using a qiagen pcr purification kit, precipitated, and then resuspended in  <dig> μl milliq h2o.
strain ip <dig> containing the mutagenesis plasmid pajd <dig>  <cit>  was grown in lb broth containing  <dig> μg trimethoprim ml- <dig> and  <dig> % arabinose  for  <dig> hours at 28°c in order to induce the expression of the λ-red genes from the pajd <dig> plasmid.
these cells were electroporated with the purified pcr product and kanamycin resistant colonies were screened by pcr and southern blot to confirm the correct insertion.
the pajd <dig> plasmid was then removed by incubation overnight at 37°c in the presence of  <dig>  mm cacl <dig>  colonies were screened to confirm the loss of the pajd <dig> plasmid and the presence of the virulence plasmid .
a similar method was used for the construction of the inv mutant except primers yptb1668chlor <dig> and yptb1668chlor <dig> , were designed to amplify the chloramphenicol resistant cassette from pbad <dig> flanked by  <dig> bp gene-specific regions.
this pcr product was then used as described above to generate an insertional mutant of the inv gene  and a double ifp and inv insertional mutant , by electroporation into ip <dig> wt or mutated ifp  strains.
colonies were screened as above with inv specific primers and the presence of pyv and loss of pajd <dig> was confirmed.
construction of ifp complement in pbad <dig> plasmid
the ifp gene including native promoter was amplified by pcr using specific primers intprom <dig> + intprom <dig> .
after ligation into pgem-t easy vector  the construct was transformed into xl2-blue e. coli .
the construct was screened by pcr and sequenced, before the ifp gene with promoter was digested from the pgem-t easy vector with kpni and sphi and purified by gel extraction using a gen elute purification kit .
this insert was cloned into a pbad <dig> plasmid  <cit> , also digested with kpni and sphi and transformed into top <dig> e. coli .
these colonies were again screened by pcr and by digestion with ecorv to confirm the correct insert and orientation within the pbad <dig> vector.
ipΔifp cells were made competent by washing  <dig> times in  <dig> ml ice cold h2o and electroporated with pbad33ifp  plasmid to generate an ifp mutant with a complemented ifp gene .
these were screened by pcr and were dna sequenced again to confirm the presence of the correct complemented gene.
plasmid cured strains
wild type, defined mutants and ifp complemented mutant strains lacking the pyv plasmid were generated by culturing strains overnight at 37°c the selecting for white colonies on crmox plates  <cit> .
loss of pyv was verified by pcr and repeated screening on crmox.
adhesion and invasion of hep- <dig> cells
hep- <dig> cells were cultured overnight at 37°c 5% co <dig> on coverslips in 24-well plates at  <dig> ×  <dig> cells/well in  <dig> ml tissue culture medium.
the  <dig> ml lb broth cultures of ip <dig> wild type , defined mutants  and mutant with complemented ifp , were incubated at 37°c for  <dig> hours with appropriate antibiotics and  <dig>  mm cacl <dig>  the cells were washed  <dig> times with  <dig> ml pbs and then, at a multiplicity of infection  of 70: <dig>  incubated for  <dig> hour with  <dig> ml of bacterial culture in mem media at 37°c, 5% co <dig>  inoculum was plated on lb agar to determine number of colony forming units .
the cells were washed  <dig> times with  <dig> ml pbs and then fixed with 2% paraformaldehyde  for  <dig> minutes at 4°c, before being washed again  <dig> times with  <dig> ml pbs.
the coverslips were incubated with a 1: <dig> dilution of anti-yersinia pseudotuberculosis antibody  in pbs for  <dig> minutes at room temperature.
the coverslips were washed with pbs then incubated with a 1: <dig> dilution of anti-rabbit igg alexafluor  <dig>   in pbs for  <dig> minutes at room temperature.
after washing with pbs the cells were permeabilised with  <dig> % triton x100-pbs  for  <dig> minutes at room temperature.
the coverslips were washed with pbs and incubated with 1: <dig> dilution of anti-y.
pseudotuberculosis antibody  in pbs for  <dig> minutes at room temperature, before being washed again with pbs.
anti-rabbit igg alexafluor  <dig>   was diluted 1: <dig> in pbs and the coverslips were incubated for  <dig> minutes at room temperature.
the coverslips were washed with pbs and mounted onto slides with vectorshield-dapi mounting media .
slides were examined by axiovert  <dig> m  confocal microscope.
three coverslips per strain with  <dig> hep- <dig> cells per coverslip were counted, utilising the z stack images to gain a 3d representation of the cell.
adhesion and invasion were quantified by counting invaded  and adhered  bacteria and calculating percentage adhesion or invasion per hep- <dig> cell based on known moi.
this adhesion and invasion assay was performed on at least three independent occasions.
detection of the presence of invasin by western blot
strains were cultured for  <dig> hours at 28°c and 37°c, the od <dig> nm measured and cultures adjusted so all strains had equal quantities of bacteria/ml.
strains were run on a sds-page 12% bis-tris gel, blotted onto nitrocellulose membrane and blocked with 5% milk-pbs- <dig> % tween <dig>  invasin was visualised by staining with anti-invasin monoclonal antibody  <cit>  at 1: <dig> and anti-rabbit igg peroxidase conjugate  secondary antibody at 1: <dig>
galleria mellonella model of infection
g. mellonella larvae were purchased from livefood uk ltd .
larvae were infected with  <dig> cfu y. pseudotuberculosis ip32953wt, ipΔifp, ipΔinv, ipΔifpΔinv or ipΔifppifp in  <dig> μl inocula by micro-injection  in the right foremost leg.
pbs and no injection controls were used.
the larvae were incubated at 37°c and survival at  <dig> hours post-infection was recorded.
larvae were scored as dead when the colouration changed from normal pale cream to brown and failed to respond even after gentle manipulation with a pipette tip.
RESULTS
identification of an intimin and invasin-like protein in y. pseudotuberculosis
the genome sequence of y. pestis strain co <dig> first revealed the potential presence of an intimin-like protein  <cit> .
however, in this sequence and all other subsequently sequenced y. pestis strains, the predicted coding sequence for the intimin-like protein is disrupted by an is <dig> element, or in the instance of strain  <dig>  a premature stop codon.
by contrast, this gene is intact in all four y. pseudotuberculosis strains sequenced to date, with at maximum, only six amino acid differences between these strains .
alignments with the european bioinformatics institute  emboss pairwise alignment tool  <cit>  revealed that the translated full-length coding sequence of ip <dig> ifp has  <dig> % amino acid identity  to y. pseudotuberculosis ip <dig> invasin, and  <dig> % amino acid identity  to the α-subtype of intimin  from e2348/ <dig> e. coli, therefore this gene was termed ifp.
using the clustalw program  alignment of the c terminal domains of invasin, intimin and ifp, reveals the presence of two cysteine residues in ifp, which are important for the function of invasin and intimin .
in addition three of the four tryptophans, which are present in all intimin subtypes  <cit> , are also present in ifp and invasin .
thermoregulated temporal expression of ifp and inv
the expression of ifp  and inv  at 24°c, 28°c and 37°c, were monitored using lux-based promoter fusions 1572lux and 1668lux in y. pseudotuberculosis ip <dig>  with the resultant luminescence read in a lucy <dig> combined photometer and luminometer .
the expression was determined as relative light units/optical density  therefore the growth phase could also be determined, based on these od readings .
inv was maximally expressed during log phase after  <dig> hours at 24°c and 28°c, but after only  <dig>  hours at 37°c, suggesting that mammalian body temperature is important in the induction of inv and confirms the observation of isberg et al.
<cit> .
in contrast, ifp expression remains low at 24°c and 28°c throughout the time course, whereas at 37°c there was little expression in first  <dig>  hours, after which expression increases to a peak at  <dig> hours .
ifp binds to localised foci on hep- <dig> cells
invasin and intimin are able to bind directly to specific receptors on the surface of mammalian cells.
we therefore investigated the ability of ifp to bind directly to hep- <dig> cells using a mbp tagged ifp purified protein .
in addition, to determine if the terminal cysteine was as important in ifp functionality , a mbp-ifp recombinant protein with the terminal cysteine mutated to a glycine  was constructed and tested.
utilising flow cytometry, facscan analysis showed a shift in the peak of fluorescence of hep- <dig> cells which had been incubated with mbp-ifp .
this shift was not seen with cells incubated with mbp-ifpc337g or mbp alone, indicating not only that this is a specific binding of mbp-ifp, but also that the terminal cysteine is important in the functional binding of ifp to hep- <dig> cells.
however, mbp-ifp only appears to bind to a subset of cells and to differing levels, as shown by the width of the shifted peak.
in order to investigate this further, we used confocal microscopy to visualise the binding of mbp-ifp to hep- <dig> cells .
again no specific binding is seen with the mbp control and binding is greatly reduced with mbp-ifpc337g, whilst the mbp-ifp fusion protein binds to individual cells with significant levels of fluorescence visible.
of  <dig> cells examined ~40% showed mbp-ifp adherence, with only ~15% showing mbp-ifpc337g adhesion.
of those showing mbp-ifpc337g adherence, fewer fluorescing spots were observed per cell compared to mbp-ifp, and these spots were smaller.
interestingly, this binding appears to be localised to specific foci on the cell surface, rather than a random scattering of fluorescence across the entire cell surface.
this suggests that the protein is binding to specific receptors on the cell surface which are localised in foci.
in order to investigate if a putative receptor was localised in cholesterol and sphingolipid-enriched plasma membrane micro-domains , we used co-localisation assays.
in this instance the gpi-anchored protein cd <dig>  which is known to localise to these microdomains  <cit> , was used as a marker for the position of the lipid rafts.
confocal microscopy revealed that there is co-localisation between cd <dig> and mbp-ifp bound on the cell surface, indicating that there is a putative receptor for ifp present within these lipid rafts .
however, as there is binding of mbp-ifp which does not co-localise, and as invasin is known to bind to β <dig> integrin, co-localisation between mbp-ifp and β <dig> integrin was also investigated .
no co-localisation was observed between mbp-ifp and β <dig> integrin.
adhesion and invasion assays
in order to confirm the role of ifp as an adhesin, we constructed an insertion mutant in the ifp coding sequence of y. pseudotuberculosis strain ip <dig> .
for comparative purposes, we also constructed an insertion mutant in the inv gene , and a double insertion mutant  in the same strain.
to determine the level of adhesion and invasion of these defined mutants, in comparison to wild type, a differential staining assay  <cit>  was utilised.
figure  <dig> shows that there is a significant decrease in the level of adhesion of ipΔifp compared to wild type , which could be restored by complementation with the wild type ifp gene  .
the inv mutant did not show as great a reduction in adhesion as ipΔifp, but the double mutant showed comparable levels to the ifp single mutant.
a significant decrease in invasion of ipΔifp compared to wild type is observed .
both ipΔinv and ipΔifpΔinv show significant decreases in invasion compared to wild type; however, it was beyond the sensitivity of this assay to determine slight differences between these two strains.
the average ratio of intracellular:extracellular bacteria for each of the strains associated with the hep- <dig> cells was as follows; ipwt 1:8; ipΔifp 1:8; ipΔinv 1:176; ipΔifpΔinv 1: <dig> and ipΔifppifp 1: <dig>  to determine the role of the virulence factors of the pyv in the adhesion and invasion still seen in these assays, the strains were cured of the pyv plasmid and the differential staining assay repeated .
invasion levels were all below the sensitivity of this assay, but a significant difference was observed between wild type and ipΔifp, ipΔinv and ipΔifpΔinv for adhesion.
although the expression analysis suggested the invasin should not be expressed at the time point used for these experiments, as there was a significant difference between wild type and inv mutants, presence of invasin was examined by western blot .
invasin was found to still be present at 37°c although at a reduced level compared to 28°c  <dig> hour cultures.
no invasin was observed in ipΔinv and ipΔifpΔinv which confirms the mutation of the inv gene in these strains.
galleria model of infection
galleria mellonella has been used as an infection model for several bacterial pathogens because it possesses an innate immune system with structural and functional homology to the mammalian immune system.
consequently it can be utilised as a less labour intensive and more cost effective model, in comparison to the traditional murine model of in vivo infection  <cit> .
this model has been used recently for infection studies with y. pseudotuberculosis  <cit> .
therefore, in addition to the adhesion and invasion assays, the ability of the mutants to infect and kill the wax moth larvae g. mellonella was examined.
bacteria, which had been cultured overnight at 37°c, were injected into the foreleg of the g. mellonella at  <dig> colony forming units  per  <dig> μl injection.
after  <dig> hours at 37°c the number of dead g. mellonella were enumerated ; larvae were scored as dead if they had become melanised and ceased moving  <cit> .
both ipΔifp  and ipΔinv  mutants showed modest if not significant attenuation in the g. mellonella model, compared to wild type ip <dig> .
ipΔifppifp shows similar levels of virulence to ipwt .
average survival of 75% was recorded in larvae infected with the double mutant, which showed a significant difference to the wild type  when analysed by non-parametric t-test .
discussion
in this study we investigated the role of a novel y. pseudotuberculosis adhesin , which shows similarity to both invasin and the intimin adhesin of epec and ehec .
as invasin and intimin are well characterised virulence determinants, the discovery of a new member in the same family of outer membrane adhesins, is intriguing.
the predicted coding sequence for ifp is disrupted in all seven currently sequenced strains of y. pestis, although it is intact in the four y. pseudotuberculosis strains sequenced.
this disruption is due to an insertion element  in all y. pestis strains, with the exception of the atypical  <dig> y. pestis microtus strain, where it is disrupted by a nonsense mutation  <cit> .
this may suggest that the disruption in this gene in y. pestis occurred early in the divergence of y. pestis from y. pseudotuberculosis and may have been a potentially important step in this evolutionary process.
the inv gene of y. pseudotuberculosis is also disrupted by an insertion element  in y. pestis  <cit> .
the reason for the loss of function of invasin and ifp in y. pestis is unknown, but given that it does not have an enteric phase in its lifecycle, it is likely to be not required.
this follows the "use it or lose it" concept, whereby genes which no longer provide a selective advantage to the bacteria become pseudogenes  <cit> .
the amino acid similarity of ifp to both invasin and intimin, coupled with its retention in y. pseudotuberculosis, suggests a putative role for ifp in adhesion to cells and that ifp is a new member in the family of surface adhesins together with invasin and intimin.
the c-terminal  <dig> amino acids of intimin are the functional domain in this adhesin  <cit> , and two cysteine  and four tryptophan  residues are conserved between all intimin subtypes.
an alignment of the c-terminal region amino acid sequences of α-subtype intimin from epec, y. pseudotuberculosis invasin and ifp, revealed that both cysteine and three out of the four tryptophan residues were found to be conserved in both ifp and invasin .
only w <dig> was not conserved in ifp and invasin.
these cysteine and tryptophan residues are involved in receptor binding by intimin  <cit>  and therefore may have a role in ifp receptor binding.
we demonstrated direct binding of ifp using both flow cytometry and fluorescence microscopy, where the mbp-ifp fusion proteins could bind to hep- <dig> cells.
by contrast the mbp alone did not bind and the specific cysteine residue mutant mbp-ifpc337g showed greatly reduced levels of binding .
this reduced level of binding with the mbp-ifpc337g shows that the cysteine residue is important to allow ifp binding.
the same cysteine residue is known to be important in both intimin and invasin, through the formation of a disulphide bond  <cit> , therefore it may be that the cysteine has a similar role in ifp.
the width of the facs fluorescence intensity graph suggests that mbp-ifp does not bind uniformly to all cells .
this was confirmed by confocal microscopy, where the cells which were exposed to the mbp-ifp fusion protein, showed a pattern of fluorescence of intensely stained localised areas, instead of scattered fluorescence across the cell surface.
a similar pattern of adherence was observed when hep- <dig> cells were incubated with mbp fusion proteins of intimin and invasin  <cit> .
as invasin is known to bind to β <dig> integrins and it has been suggested that intimin can bind to β <dig> integrin  <cit>  co-localisation of ifp to β <dig> integrin was investigated .
as no co-localisation was observed it shows that ifp binds to alternative receptors on the cell surface.
lipid rafts are sphingolipid and cholesterol rich regions of the plasma membrane, into which tir is thought to be transferred  <cit> .
additionally uropathogenic e. coli are known to invade via lipid rafts  <cit> , and salmonella and shigella sp.
use a type three secretion system to translocate effectors, by binding to cholesterol within lipid rafts  <cit> .
to investigate if mbp-ifp was binding to a component of these lipid rafts, co-localisation of mbp-ifp to cd <dig> was undertaken by confocal microscopy.
cd <dig> was selected as it is known to localise to these micro-domains and could therefore act as a marker.
the results show co-localisation of ifp and cd <dig>  which was reduced with mbp-ifpc337g , suggesting that there is a putative receptor for ifp within these lipid rafts.
the ifp receptor within these lipid rafts has yet to be determined, but as not all of the mbp-ifp co-localised, no conclusions can currently be made as to the exact receptor of ifp.
inv is differentially thermoregulated with lower levels being expressed at 37°c compared to 28°c  <cit> .
in comparison, yada shows maximal expression at 37°c in exponential phase culture, conditions where inv expression is repressed  <cit> .
yada is a virulence plasmid  encoded adhesin, known to be involved during the infection of y. pseudotuberculosis  <cit> .
the pattern of inv expression was confirmed by this study, where inv was expressed both at 28°c and 37°c during lag and early log phase culture, although at a greater degree at 28°c .
the ifp gene appears to be expressed at 37°c at a later time point in the late log or early stationary phase, when inv expression is reduced.
as ifp and yada are expressed at similar time points and at the same temperature, ifp may have a similar role to yada during the infection of y. pseudotuberculosis  <cit> .
although inv expression is decreased at a later time point, it still appears to have an effect on the invasion of y. pseudotuberculosis ; this is despite using stationary phase cultures which had been grown at 37°c.
the western blot analysis for presence of invasin under these conditions , confirmed that although inv may no longer be actively expressed, invasin was still present in the cell and could therefore have a role in invasion of hep- <dig> cells.
the invasion and adhesion assays confirmed the microscopy and flow cytometry results, in demonstrating a role for ifp as an adhesin, as the levels of adhesion were reduced with ipΔifp in comparison to wild type .
the inv mutant did not show as great a decrease in adhesion as the ifp mutant, but the double mutant showed similar if not a marginally greater reduction in adhesion as ipΔifp, in comparison to the wild type.
although levels of invasion were significantly affected by ipΔifp, this may be due to reduced adhesion, suggesting that ifp is an adhesin.
any differences between ipΔinv and ipΔifpΔinv were beyond the detection capability of this assay, but it appeared that invasin was the dominant protein involved in the invasion of the hep- <dig> cells.
removal of the pyv and therefore the yada and yop virulence factors allowed greater distinction of the role of ifp.
without these extra virulence determinants compensating for the mutation of ifp, the ipΔifp mutant showed a statistically significant reduction in adhesion compared to ipwt .
this confirms the role of ifp as an adhesin of y. pseudotuberculosis.
as g. mellonella possesses an innate immune system with structural and functional similarities to the mammalian innate immune system, it is a useful alternative to the traditional murine yersiniosis infection model, to examine virulence in vivo, especially as unlike the c. elegans model, g. mellonella can be incubated at 37°c  <cit> .
previous studies with y. pseudotuberculosis comparing g. mellonella and the murine model, showed that g. mellonella could reflect infection in mammals and therefore could be useful as a higher throughput screen of mutants, before a more in depth analysis was undertaken in the murine model  <cit> .
in this study the g. mellonella model demonstrated a role for ifp in the pathogenesis of y. pseudotuberculosis, in particular in concert with invasin, as the double mutant showed a significant increase in survival compared to the wild type .
there also appeared to be mild attenuation in virulence with both of the single mutants.
this suggests that ifp, together with invasin, does have a role in virulence of y. pseudotuberculosis in this infection model.
CONCLUSIONS
we have shown the presence of a novel functional adhesin in y. pseudotuberculosis that has been mutated with an is element and is presumably non-functional in y. pestis.
ifp is expressed during late log to early stationary phase at 37°c and demonstrates an ability to bind to hep- <dig> cells in vitro, which can be disrupted by mutation of the gene, or even a single cysteine residue.
together with invasin and intimin, ifp is a new member of a family of outer membrane adhesins that is activated at 37°c and may act at a later stage than invasin during infection.
authors' contributions
all authors read and approved the manuscript and contributed to experimental design.
ps and bw contributed to manuscript preparation.
supplementary material
additional file 1
amino acid alignment of ifp from the four currently sequenced genomes of y. pseudotuberculosis.
utilising the clustalw program, the amino acid sequences of y. pseudotuberculosis strains ip <dig>  ip <dig>  pb <dig> and ypiii were aligned.
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additional file 2
growth curves from the temporal expression of ifp and invasin assay.
within the anthos lucy <dig> combined photometer and luminometer, od readings at  <dig> nm were taken at  <dig> minute intervals and used to construct these growth curves.
cultures were incubated at  24°c  28°c and  37°c.
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acknowledgements
we are grateful to g. frankel, imperial college, london, uk for the intimin advice; e. carniel, institut pasteur, paris, france for the y. pseudotuberculosis strain ip <dig> and the pkobeg vector; a. darwin, nyu school of medicine, new york, usa for the pajd <dig> plasmid; and r. isberg, tufts university, boston, usa for the gift of the anti-invasin monoclonal antibody.
we thank dstl for financial support for this project.

 within the genus yersinia there are three human-pathogenic species; y. pestis, the causative agent of plague, and two enteric pathogens, y. pseudotuberculosis and y. enterocolitica. despite the differences in disease, y. pestis and y. pseudotuberculosis are very closely related at the genetic level. y. pestis is believed to have evolved from y. pseudotuberculosis between - years ago. thus, in a remarkably short length of evolutionary time, y. pestis has evolved from an enteropathogen, to a blood-borne pathogen with an insect vector. genome sequencing of several y. pseudotuberculosis and y. pestis strains, revealed that y. pestis has accumulated a large number of pseudogenes since its divergence. by the "use it or lose it" paradigm, this is suggestive of the decay of those genes that are no longer required for function as y. pestis adapts to a new lifestyle. gene disruption may also result in pathoadaptive mutation, whereby loss of gene function results in an increase in virulence. this has been demonstrated in several pathogenic bacteria including shigella spp. and escherichia coli. pathoadaptive mutations have previously been identified in y. pestis, with the negative regulators of biofilm formation, rcsa and ngha, being disrupted, resulting in the ability of y. pestis to form biofilms within the flea vector. pseudogenes in y. pestis that are known to be essential for the enteric lifestyle of y. pseudotuberculosis, include the adhesins yada and invasin. invasin was one of the first bacterial virulence factors identified, when it was observed that the inv gene alone was sufficient to convert benign non-invasive laboratory e. coli strains, to being capable of invading tissue culture cells. invasin is a kda protein that is capable of binding to β integrins on the host cells, promoting internalisation of the bacterium. during early infection, invasin specifically binds β integrins on the apical surface of m cells, which facilitates efficient translocation to the underlying peyer's patches. the invasin protein is composed of a short n-terminal transmembrane domain, four structural bacterial immunoglobulin domains and a c-type lectin-like domain. the last big domain and the c-type lectin-like domain comprise the functional β integrin binding region. in the same family of bacterial adhesion proteins as invasin, is intimin, an important adhesin expressed by enteropathogenic and enterohaemorrhagic e. coli on the lee pathogenicity island. intimin is a kda outer membrane protein that is also found in citrobacter freundii and hafnia alvei. the functional binding domain of intimin is located in the amino acid c-terminal region, and consists of two big domains and a c-type lectin-like domain, which are structurally similar to invasin. when epec and ehec colonise the small intestine mucosa, a characteristic attaching and effacing lesion is formed. first there is localised destruction of the microvilli, which leads to intimate attachment of the bacterium to the host cell. epec and ehec encode a specific intimin receptor, translocated intimin receptor. this receptor is translocated directly into the host cells via a type iii secretion system, where it becomes expressed on the cell surface. intimin binds to tir leading to its activation, which results in actin polymerisation within the host cell and the formation of a pedestal, facilitating tighter adherence between the host cell and the bacterium. other eukaryotic receptors have been suggested for intimin, including nucleolin and some β integrins, but as yet it is unknown if these interactions have a role in vivo. there is considerable sequence variation between the intimins from different e. coli strains and they have been categorised into different subtypes, each with a high affinity for its own cognate tir. however, despite this diversity, it has been found that within the c-terminal binding domain there are four tryptophan residues and two cysteine residues, which are conserved between all subtypes. the two cysteines are also conserved in similar locations within the y. pseudotuberculosis invasin. in both invasin and intimin a disulphide bond is formed, which is essential for the structure of the c-terminal binding domain and therefore required for full functionality. in the instance of invasin, disruption of either cysteine results in an inability to bind to integrin, and therefore is defective for invasion. analysis of y. pseudotuberculosis strain ip sequence data identified a gene encoding a protein with significant amino acid similarity to invasin and intimin, which has not been previously investigated. we have termed this protein ifp and intriguingly it has been mutated to a pseudogene in all seven y. pestis genomes sequenced to date. examination of the amino acid sequence of ifp revealed that three of the four tryptophans and both of the cysteine residues that are important in intimin function are conserved. however, no tir orthologue can be identified in the ip genome sequence. given the amino acid similarity of ifp to both invasin and intimin, coupled with it being putatively non-functional in y. pestis, we postulated that ifp may be an adhesin. we demonstrate that ifp is a functional adhesin that binds to distinct foci on host cells. expression occurs in late log or early stationary phase at 37°c only and coincides with a decline in the expression of invasin at this temperature. methods strains used and culture conditions all y. pseudotuberculosis strains were cultured in luria-bertani broth miller or on lb agar at 28°c unless otherwise stated. the retention of the virulence plasmid was screened by plating y. pseudotuberculosis strains on crmox plates. antibiotics were used at the following concentrations where appropriate, ampicillin, kanamycin, chloramphenicol and trimethoprim. e. coli strains were cultured at 37°c overnight in lb broth miller or on lb agar unless otherwise stated. see table for a list of the strains and plasmids used in this study. construction of lux reporter strains pcr primers were designed to amplify bp and bp fragments between yptb and yptb and between yptb and yptb respectively using y. pseudotuberculosis strain ip genomic dna as a template. these regions contain the putative promoter and regulatory sequences for ifp and inv. these pcr products were cloned into the pgem-t easy vector. kpni and spei restriction sites had been incorporated into the primer sequences to enable the luxcdabe operon from pbluelux to be inserted downstream of each promoter region. the entire promoter-lux construct was excised from pgem-t easy then re-cloned into the pdm suicide plasmid using transformax ec100d pir+ e. coli for selection and screening. the resulting promoter fusions 1572lux and 1668lux in pdm were then electroporated into the ip strain of y. pseudotuberculosis and screened for single crossover event into the genome by chloramphenicol resistance. this crossover event resulted in a functional gene of interest, with the lux cassette with native promoter inserted upstream of the gene on the chromosome. restriction sites are in bold and position of mutated cysteine to glycine residue is underlined. bioluminescence and optical density of lux constructs bioluminescence and optical density were monitored simultaneously in 96-well microtitre plates using the anthos lucy combined photometer and luminometer controlled by the stingray software. overnight 30°c cultures of 1572lux and 1668lux were diluted 1: in fresh lb with antibiotics and grown for 3- h at 30°c. the optical density of the culture was adjusted to a starting od of and μl was added to the wells of the well plate. assays were performed at 24°c, 28°c and 37°c. luminescence and od at nm of the cultures was automatically determined every min for h and presented as relative light units per unit of od. for every promoter fusion assay each sample was assayed in triplicate on the 96-well plate, and each experiment was carried out in duplicate. construction of maltose binding protein fusion proteins primers yptbintmbp- and yptbintmbp- were used to pcr amplify the 3' end of the ifp coding sequence. this pcr product was cloned into the pmal-p2x vector using ecori and xbai enzyme restriction sites which had been incorporated into the primer design. in order to generate an mbp-ifp fusion with the terminal cysteine mutated to a glycine, primer yptbintmbp- was substituted for yptbmbp- primer yptbintmbp- contained an alternative sequence to mutate the terminal cysteine to a glycine. mbp-fusion proteins were then expressed in tb e. coli. purification of mbp-ifp and mbp-ifpc337g e. coli transformed with the mbp-fusion plasmids were cultured for h at 37°c using ml of an overnight culture in ml lb broth with mm glucose and ampicillin until a culture growth of od was reached. expression of the fusion protein was induced with mm isopropyl-β-d-thiogalactoside then the culture was incubated for further hours at 37°c. the cultures were centrifuged and pellets stored overnight at -20°c then resuspended in column buffer. the cells were lysed by sonication using a bioruptor sonicator. insoluble proteins were removed by centrifugation and the supernatant was applied to ml columns of amylose resin. after washing with ml column buffer, proteins were eluted with ml column buffer/ mm maltose. proteins were concentrated using amicon ultra kda columns, and then confirmed by coomassie staining and western blotting with anti-mbp. protein concentrations were determined by pierce bca protein assay kit according to the manufacturer's protocol. analysis of mbp-fusion protein binding to hep- cells by fluorescence microscopy binding of mbp-tagged ifp was determined by fluorescence microscopy as described previously. hep- cells were cultured overnight on glass coverslips in 24-well plates at × cells/well in ml tissue culture media, 10% fetal calf serum, mm l-glutamine, u penicillin ml- μg streptomycin ml- mem non-essential amino acids). the cells were blocked for minutes at 37°c/5% co in μl binding solution, mm maltose, mm l-glutamine in rpmi media), then incubated for minutes at 37°c/5% co with μl mbp-ifp, mbp-ifpc337g or mbp protein alone at μg ml- in binding solution. the cells were washed times with ml pbs/1% bsa and incubated for minutes at 37°c/5% co in μl of a 1: dilution of rabbit anti-mbp antibody in binding solution used. cells were washed times with ml pbs/1% bsa and incubated for minutes at 37°c/5% co in μl of a 1: dilution of goat anti-rabbit igg alexafluor in binding solution. cells were washed times with ml pbs/1% bsa and fixed for minutes at -20°c in μl of 95% ethanol-5% acetic acid. the cover slips were removed from the wells, washed in milli q h2o and mounted onto glass slides with vectashield-dapi mounting medium. the coverslips were examined using an axiovert 200m confocal microscope. experiment was performed on three independent occasions and at least cells were examined per experiment. facscan analysis of mbp-fusion protein binding to hep- cells a similar methodology was used as for the fluorescence microscopy as described previously, with the following modifications. the cells were grown directly in 6-well plates at × cells/well. the alexafluor anti-rabbit igg antibody was diluted to 1: in pbs/1% bsa. cells were resuspended in pbs/ % edta and transferred to bd falcon ml tubes. cells were washed once with pbs/1% bsa and centrifuged, then were fixed for minutes on ice in 2% paraformaldehyde/pbs. the cells were washed once with pbs/1% bsa, centrifuged and then were resuspended in μl pbs/1% bsa/ % edta. the fluorescence was measured using a facscan machine. experiment was performed on two independent occasions and cells were examined for fluorescence from each sample. analysis of co-localisation of mbp-fusion protein and the receptors cd and β integrin on hep- cells by fluorescence microscopy a similar methodology was used as for the fluorescence microscopy described above with the following modifications. after the mbp-fusion protein incubation the cells were washed times with pbs then incubated with μl of a 1: dilution rabbit anti-ifp and 1: dilution of mouse anti-cd or a 1: dilution of mouse anti-β integrin in binding solution for minutes at 37°c/5% co the cells were washed times with ml pbs then incubated with μl of a 1: dilution of goat anti-rabbit igg alexafluor and 1: dilution of goat anti-mouse igg alexafluor for minutes at 37°c/5% co the cells were washed times with ml pbs then fixed for minutes at 4°c with μl 2% paraformaldehyde. the coverslips were removed from the wells, washed with pbs then mounted onto glass slides with vectashield-dapi mounting medium. the slides were examined using an axiovert m confocal microscope. at least three areas of approximately cells each were examined per sample and the experiment was performed on three independent occasions. construction of ifp and inv insertional mutants an ifp knockout mutant was generated in the y. pseudotuberculosis strain ip after initially constructing an ifp mutant in strain ypiii. briefly, bp of ifp was amplified with inta and intb primers, digested with saci and sphi then ligated into the cloning vector pgem-t easy. the plasmid was digested with bglii to linearise and allow for the ligation of the kanamycin cassette within the ifp sequence. pcr with primers inta and intb was undertaken on the plasmid to create linear fragments of kanamycin cassette flanked by ifp sequence. this pcr product was electroporated into ypiii previously transformed with pkobeg, which contains the λ red recombinase operon. the temperature sensitive pkobeg plasmid was then lost from putative mutants by growth at 37°c, whilst the presence of the pyv plasmid was maintained by the addition of mm cacl southern blot analysis confirmed correct mutation. genomic dna from this ypiiiΔifp was used as a template for pcr amplification of the kanamycin cassette flanked by two ~ bp regions of gene-specific dna. the primers inta and intb were used to amplify a kbp product. this was purified using a qiagen pcr purification kit, precipitated, and then resuspended in μl milliq h2o. strain ip containing the mutagenesis plasmid pajd was grown in lb broth containing μg trimethoprim ml- and % arabinose for hours at 28°c in order to induce the expression of the λ-red genes from the pajd plasmid. these cells were electroporated with the purified pcr product and kanamycin resistant colonies were screened by pcr and southern blot to confirm the correct insertion. the pajd plasmid was then removed by incubation overnight at 37°c in the presence of mm cacl colonies were screened to confirm the loss of the pajd plasmid and the presence of the virulence plasmid. a similar method was used for the construction of the inv mutant except primers yptb1668chlor and yptb1668chlor, were designed to amplify the chloramphenicol resistant cassette from pbad flanked by bp gene-specific regions. this pcr product was then used as described above to generate an insertional mutant of the inv gene and a double ifp and inv insertional mutant, by electroporation into ip wt or mutated ifp strains. colonies were screened as above with inv specific primers and the presence of pyv and loss of pajd was confirmed. construction of ifp complement in pbad plasmid the ifp gene including native promoter was amplified by pcr using specific primers intprom + intprom. after ligation into pgem-t easy vector the construct was transformed into xl2-blue e. coli. the construct was screened by pcr and sequenced, before the ifp gene with promoter was digested from the pgem-t easy vector with kpni and sphi and purified by gel extraction using a gen elute purification kit. this insert was cloned into a pbad plasmid, also digested with kpni and sphi and transformed into top e. coli. these colonies were again screened by pcr and by digestion with ecorv to confirm the correct insert and orientation within the pbad vector. ipΔifp cells were made competent by washing times in ml ice cold h2o and electroporated with pbad33ifp plasmid to generate an ifp mutant with a complemented ifp gene. these were screened by pcr and were dna sequenced again to confirm the presence of the correct complemented gene. plasmid cured strains wild type, defined mutants and ifp complemented mutant strains lacking the pyv plasmid were generated by culturing strains overnight at 37°c the selecting for white colonies on crmox plates. loss of pyv was verified by pcr and repeated screening on crmox. adhesion and invasion of hep- cells hep- cells were cultured overnight at 37°c 5% co on coverslips in 24-well plates at × cells/well in ml tissue culture medium. the ml lb broth cultures of ip wild type, defined mutants and mutant with complemented ifp, were incubated at 37°c for hours with appropriate antibiotics and mm cacl the cells were washed times with ml pbs and then, at a multiplicity of infection of 70: incubated for hour with ml of bacterial culture in mem media at 37°c, 5% co inoculum was plated on lb agar to determine number of colony forming units. the cells were washed times with ml pbs and then fixed with 2% paraformaldehyde for minutes at 4°c, before being washed again times with ml pbs. the coverslips were incubated with a 1: dilution of anti-yersinia pseudotuberculosis antibody in pbs for minutes at room temperature. the coverslips were washed with pbs then incubated with a 1: dilution of anti-rabbit igg alexafluor in pbs for minutes at room temperature. after washing with pbs the cells were permeabilised with % triton x100-pbs for minutes at room temperature. the coverslips were washed with pbs and incubated with 1: dilution of anti-y. pseudotuberculosis antibody in pbs for minutes at room temperature, before being washed again with pbs. anti-rabbit igg alexafluor was diluted 1: in pbs and the coverslips were incubated for minutes at room temperature. the coverslips were washed with pbs and mounted onto slides with vectorshield-dapi mounting media. slides were examined by axiovert m confocal microscope. three coverslips per strain with hep- cells per coverslip were counted, utilising the z stack images to gain a 3d representation of the cell. adhesion and invasion were quantified by counting invaded and adhered bacteria and calculating percentage adhesion or invasion per hep- cell based on known moi. this adhesion and invasion assay was performed on at least three independent occasions. detection of the presence of invasin by western blot strains were cultured for hours at 28°c and 37°c, the od nm measured and cultures adjusted so all strains had equal quantities of bacteria/ml. strains were run on a sds-page 12% bis-tris gel, blotted onto nitrocellulose membrane and blocked with 5% milk-pbs- % tween invasin was visualised by staining with anti-invasin monoclonal antibody at 1: and anti-rabbit igg peroxidase conjugate secondary antibody at 1: galleria mellonella model of infection g. mellonella larvae were purchased from livefood uk ltd. larvae were infected with cfu y. pseudotuberculosis ip32953wt, ipΔifp, ipΔinv, ipΔifpΔinv or ipΔifppifp in μl inocula by micro-injection in the right foremost leg. pbs and no injection controls were used. the larvae were incubated at 37°c and survival at hours post-infection was recorded. larvae were scored as dead when the colouration changed from normal pale cream to brown and failed to respond even after gentle manipulation with a pipette tip.  identification of an intimin and invasin-like protein in y. pseudotuberculosis the genome sequence of y. pestis strain co first revealed the potential presence of an intimin-like protein. however, in this sequence and all other subsequently sequenced y. pestis strains, the predicted coding sequence for the intimin-like protein is disrupted by an is element, or in the instance of strain a premature stop codon. by contrast, this gene is intact in all four y. pseudotuberculosis strains sequenced to date, with at maximum, only six amino acid differences between these strains. alignments with the european bioinformatics institute emboss pairwise alignment tool revealed that the translated full-length coding sequence of ip ifp has % amino acid identity to y. pseudotuberculosis ip invasin, and % amino acid identity to the α-subtype of intimin from e2348/ e. coli, therefore this gene was termed ifp. using the clustalw program alignment of the c terminal domains of invasin, intimin and ifp, reveals the presence of two cysteine residues in ifp, which are important for the function of invasin and intimin. in addition three of the four tryptophans, which are present in all intimin subtypes, are also present in ifp and invasin. thermoregulated temporal expression of ifp and inv the expression of ifp and inv at 24°c, 28°c and 37°c, were monitored using lux-based promoter fusions 1572lux and 1668lux in y. pseudotuberculosis ip with the resultant luminescence read in a lucy combined photometer and luminometer. the expression was determined as relative light units/optical density therefore the growth phase could also be determined, based on these od readings. inv was maximally expressed during log phase after hours at 24°c and 28°c, but after only hours at 37°c, suggesting that mammalian body temperature is important in the induction of inv and confirms the observation of isberg et al.. in contrast, ifp expression remains low at 24°c and 28°c throughout the time course, whereas at 37°c there was little expression in first hours, after which expression increases to a peak at hours. ifp binds to localised foci on hep- cells invasin and intimin are able to bind directly to specific receptors on the surface of mammalian cells. we therefore investigated the ability of ifp to bind directly to hep- cells using a mbp tagged ifp purified protein. in addition, to determine if the terminal cysteine was as important in ifp functionality, a mbp-ifp recombinant protein with the terminal cysteine mutated to a glycine was constructed and tested. utilising flow cytometry, facscan analysis showed a shift in the peak of fluorescence of hep- cells which had been incubated with mbp-ifp. this shift was not seen with cells incubated with mbp-ifpc337g or mbp alone, indicating not only that this is a specific binding of mbp-ifp, but also that the terminal cysteine is important in the functional binding of ifp to hep- cells. however, mbp-ifp only appears to bind to a subset of cells and to differing levels, as shown by the width of the shifted peak. in order to investigate this further, we used confocal microscopy to visualise the binding of mbp-ifp to hep- cells. again no specific binding is seen with the mbp control and binding is greatly reduced with mbp-ifpc337g, whilst the mbp-ifp fusion protein binds to individual cells with significant levels of fluorescence visible. of cells examined ~40% showed mbp-ifp adherence, with only ~15% showing mbp-ifpc337g adhesion. of those showing mbp-ifpc337g adherence, fewer fluorescing spots were observed per cell compared to mbp-ifp, and these spots were smaller. interestingly, this binding appears to be localised to specific foci on the cell surface, rather than a random scattering of fluorescence across the entire cell surface. this suggests that the protein is binding to specific receptors on the cell surface which are localised in foci. in order to investigate if a putative receptor was localised in cholesterol and sphingolipid-enriched plasma membrane micro-domains, we used co-localisation assays. in this instance the gpi-anchored protein cd which is known to localise to these microdomains, was used as a marker for the position of the lipid rafts. confocal microscopy revealed that there is co-localisation between cd and mbp-ifp bound on the cell surface, indicating that there is a putative receptor for ifp present within these lipid rafts. however, as there is binding of mbp-ifp which does not co-localise, and as invasin is known to bind to β integrin, co-localisation between mbp-ifp and β integrin was also investigated. no co-localisation was observed between mbp-ifp and β integrin. adhesion and invasion assays in order to confirm the role of ifp as an adhesin, we constructed an insertion mutant in the ifp coding sequence of y. pseudotuberculosis strain ip. for comparative purposes, we also constructed an insertion mutant in the inv gene, and a double insertion mutant in the same strain. to determine the level of adhesion and invasion of these defined mutants, in comparison to wild type, a differential staining assay was utilised. figure shows that there is a significant decrease in the level of adhesion of ipΔifp compared to wild type, which could be restored by complementation with the wild type ifp gene. the inv mutant did not show as great a reduction in adhesion as ipΔifp, but the double mutant showed comparable levels to the ifp single mutant. a significant decrease in invasion of ipΔifp compared to wild type is observed. both ipΔinv and ipΔifpΔinv show significant decreases in invasion compared to wild type; however, it was beyond the sensitivity of this assay to determine slight differences between these two strains. the average ratio of intracellular:extracellular bacteria for each of the strains associated with the hep- cells was as follows; ipwt 1:8; ipΔifp 1:8; ipΔinv 1:176; ipΔifpΔinv 1: and ipΔifppifp 1: to determine the role of the virulence factors of the pyv in the adhesion and invasion still seen in these assays, the strains were cured of the pyv plasmid and the differential staining assay repeated. invasion levels were all below the sensitivity of this assay, but a significant difference was observed between wild type and ipΔifp, ipΔinv and ipΔifpΔinv for adhesion. although the expression analysis suggested the invasin should not be expressed at the time point used for these experiments, as there was a significant difference between wild type and inv mutants, presence of invasin was examined by western blot. invasin was found to still be present at 37°c although at a reduced level compared to 28°c hour cultures. no invasin was observed in ipΔinv and ipΔifpΔinv which confirms the mutation of the inv gene in these strains. galleria model of infection galleria mellonella has been used as an infection model for several bacterial pathogens because it possesses an innate immune system with structural and functional homology to the mammalian immune system. consequently it can be utilised as a less labour intensive and more cost effective model, in comparison to the traditional murine model of in vivo infection. this model has been used recently for infection studies with y. pseudotuberculosis. therefore, in addition to the adhesion and invasion assays, the ability of the mutants to infect and kill the wax moth larvae g. mellonella was examined. bacteria, which had been cultured overnight at 37°c, were injected into the foreleg of the g. mellonella at colony forming units per μl injection. after hours at 37°c the number of dead g. mellonella were enumerated; larvae were scored as dead if they had become melanised and ceased moving. both ipΔifp and ipΔinv mutants showed modest if not significant attenuation in the g. mellonella model, compared to wild type ip. ipΔifppifp shows similar levels of virulence to ipwt. average survival of 75% was recorded in larvae infected with the double mutant, which showed a significant difference to the wild type when analysed by non-parametric t-test. discussion in this study we investigated the role of a novel y. pseudotuberculosis adhesin, which shows similarity to both invasin and the intimin adhesin of epec and ehec. as invasin and intimin are well characterised virulence determinants, the discovery of a new member in the same family of outer membrane adhesins, is intriguing. the predicted coding sequence for ifp is disrupted in all seven currently sequenced strains of y. pestis, although it is intact in the four y. pseudotuberculosis strains sequenced. this disruption is due to an insertion element in all y. pestis strains, with the exception of the atypical y. pestis microtus strain, where it is disrupted by a nonsense mutation. this may suggest that the disruption in this gene in y. pestis occurred early in the divergence of y. pestis from y. pseudotuberculosis and may have been a potentially important step in this evolutionary process. the inv gene of y. pseudotuberculosis is also disrupted by an insertion element in y. pestis. the reason for the loss of function of invasin and ifp in y. pestis is unknown, but given that it does not have an enteric phase in its lifecycle, it is likely to be not required. this follows the "use it or lose it" concept, whereby genes which no longer provide a selective advantage to the bacteria become pseudogenes. the amino acid similarity of ifp to both invasin and intimin, coupled with its retention in y. pseudotuberculosis, suggests a putative role for ifp in adhesion to cells and that ifp is a new member in the family of surface adhesins together with invasin and intimin. the c-terminal amino acids of intimin are the functional domain in this adhesin, and two cysteine and four tryptophan residues are conserved between all intimin subtypes. an alignment of the c-terminal region amino acid sequences of α-subtype intimin from epec, y. pseudotuberculosis invasin and ifp, revealed that both cysteine and three out of the four tryptophan residues were found to be conserved in both ifp and invasin. only w was not conserved in ifp and invasin. these cysteine and tryptophan residues are involved in receptor binding by intimin and therefore may have a role in ifp receptor binding. we demonstrated direct binding of ifp using both flow cytometry and fluorescence microscopy, where the mbp-ifp fusion proteins could bind to hep- cells. by contrast the mbp alone did not bind and the specific cysteine residue mutant mbp-ifpc337g showed greatly reduced levels of binding. this reduced level of binding with the mbp-ifpc337g shows that the cysteine residue is important to allow ifp binding. the same cysteine residue is known to be important in both intimin and invasin, through the formation of a disulphide bond, therefore it may be that the cysteine has a similar role in ifp. the width of the facs fluorescence intensity graph suggests that mbp-ifp does not bind uniformly to all cells. this was confirmed by confocal microscopy, where the cells which were exposed to the mbp-ifp fusion protein, showed a pattern of fluorescence of intensely stained localised areas, instead of scattered fluorescence across the cell surface. a similar pattern of adherence was observed when hep- cells were incubated with mbp fusion proteins of intimin and invasin. as invasin is known to bind to β integrins and it has been suggested that intimin can bind to β integrin co-localisation of ifp to β integrin was investigated. as no co-localisation was observed it shows that ifp binds to alternative receptors on the cell surface. lipid rafts are sphingolipid and cholesterol rich regions of the plasma membrane, into which tir is thought to be transferred. additionally uropathogenic e. coli are known to invade via lipid rafts, and salmonella and shigella sp. use a type three secretion system to translocate effectors, by binding to cholesterol within lipid rafts. to investigate if mbp-ifp was binding to a component of these lipid rafts, co-localisation of mbp-ifp to cd was undertaken by confocal microscopy. cd was selected as it is known to localise to these micro-domains and could therefore act as a marker. the results show co-localisation of ifp and cd which was reduced with mbp-ifpc337g, suggesting that there is a putative receptor for ifp within these lipid rafts. the ifp receptor within these lipid rafts has yet to be determined, but as not all of the mbp-ifp co-localised, no conclusions can currently be made as to the exact receptor of ifp. inv is differentially thermoregulated with lower levels being expressed at 37°c compared to 28°c. in comparison, yada shows maximal expression at 37°c in exponential phase culture, conditions where inv expression is repressed. yada is a virulence plasmid encoded adhesin, known to be involved during the infection of y. pseudotuberculosis. the pattern of inv expression was confirmed by this study, where inv was expressed both at 28°c and 37°c during lag and early log phase culture, although at a greater degree at 28°c. the ifp gene appears to be expressed at 37°c at a later time point in the late log or early stationary phase, when inv expression is reduced. as ifp and yada are expressed at similar time points and at the same temperature, ifp may have a similar role to yada during the infection of y. pseudotuberculosis. although inv expression is decreased at a later time point, it still appears to have an effect on the invasion of y. pseudotuberculosis; this is despite using stationary phase cultures which had been grown at 37°c. the western blot analysis for presence of invasin under these conditions, confirmed that although inv may no longer be actively expressed, invasin was still present in the cell and could therefore have a role in invasion of hep- cells. the invasion and adhesion assays confirmed the microscopy and flow cytometry results, in demonstrating a role for ifp as an adhesin, as the levels of adhesion were reduced with ipΔifp in comparison to wild type. the inv mutant did not show as great a decrease in adhesion as the ifp mutant, but the double mutant showed similar if not a marginally greater reduction in adhesion as ipΔifp, in comparison to the wild type. although levels of invasion were significantly affected by ipΔifp, this may be due to reduced adhesion, suggesting that ifp is an adhesin. any differences between ipΔinv and ipΔifpΔinv were beyond the detection capability of this assay, but it appeared that invasin was the dominant protein involved in the invasion of the hep- cells. removal of the pyv and therefore the yada and yop virulence factors allowed greater distinction of the role of ifp. without these extra virulence determinants compensating for the mutation of ifp, the ipΔifp mutant showed a statistically significant reduction in adhesion compared to ipwt. this confirms the role of ifp as an adhesin of y. pseudotuberculosis. as g. mellonella possesses an innate immune system with structural and functional similarities to the mammalian innate immune system, it is a useful alternative to the traditional murine yersiniosis infection model, to examine virulence in vivo, especially as unlike the c. elegans model, g. mellonella can be incubated at 37°c. previous studies with y. pseudotuberculosis comparing g. mellonella and the murine model, showed that g. mellonella could reflect infection in mammals and therefore could be useful as a higher throughput screen of mutants, before a more in depth analysis was undertaken in the murine model. in this study the g. mellonella model demonstrated a role for ifp in the pathogenesis of y. pseudotuberculosis, in particular in concert with invasin, as the double mutant showed a significant increase in survival compared to the wild type. there also appeared to be mild attenuation in virulence with both of the single mutants. this suggests that ifp, together with invasin, does have a role in virulence of y. pseudotuberculosis in this infection model.  we have shown the presence of a novel functional adhesin in y. pseudotuberculosis that has been mutated with an is element and is presumably non-functional in y. pestis. ifp is expressed during late log to early stationary phase at 37°c and demonstrates an ability to bind to hep- cells in vitro, which can be disrupted by mutation of the gene, or even a single cysteine residue. together with invasin and intimin, ifp is a new member of a family of outer membrane adhesins that is activated at 37°c and may act at a later stage than invasin during infection. authors' contributions all authors read and approved the manuscript and contributed to experimental design. ps and bw contributed to manuscript preparation. supplementary material additional file 1 amino acid alignment of ifp from the four currently sequenced genomes of y. pseudotuberculosis. utilising the clustalw program, the amino acid sequences of y. pseudotuberculosis strains ip ip pb and ypiii were aligned. click here for file additional file 2 growth curves from the temporal expression of ifp and invasin assay. within the anthos lucy combined photometer and luminometer, od readings at nm were taken at minute intervals and used to construct these growth curves. cultures were incubated at 24°c 28°c and 37°c. click here for file acknowledgements we are grateful to g. frankel, imperial college, london, uk for the intimin advice; e. carniel, institut pasteur, paris, france for the y. pseudotuberculosis strain ip and the pkobeg vector; a. darwin, nyu school of medicine, new york, usa for the pajd plasmid; and r. isberg, tufts university, boston, usa for the gift of the anti-invasin monoclonal antibody. we thank dstl for financial support for this project.
in order to identify new virulence determinants in y. pseudotuberculosis a comparison between its genome and that of yersinia pestis was undertaken. this reveals dozens of pseudogenes in y. pestis, which are still putatively functional in y. pseudotuberculosis and may be important in the enteric lifestyle. one such gene, yptb in the y. pseudotuberculosis ip genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic and enterohaemorraghic escherichia coli. a new y. pseudotuberculosis adhesin has been identified and characterised. cysteine and tryptophan residues in the c-terminal region of intimin that are essential for function in epec and ehec are conserved in ifp. this ifp is a new member in the family of invasin/intimin outer membrane adhesins. protein binding occurred at distinct foci on the hep- cell surface and can be disrupted by mutation of a single cysteine residue at the c-terminus of the protein.
 in order to identify new virulence determinants in y. pseudotuberculosis a comparison between its genome and that of yersinia pestis was undertaken. this reveals dozens of pseudogenes in y. pestis, which are still putatively functional in y. pseudotuberculosis and may be important in the enteric lifestyle. one such gene, yptb in the y. pseudotuberculosis ip genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic and enterohaemorraghic escherichia coli.  we termed yptb ifp and show that it is able to bind directly to human hep- epithelial cells. cysteine and tryptophan residues in the c-terminal region of intimin that are essential for function in epec and ehec are conserved in ifp. protein binding occurred at distinct foci on the hep- cell surface and can be disrupted by mutation of a single cysteine residue at the c-terminus of the protein. temporal expression analysis using lux reporter constructs revealed that ifp is expressed at late log phase at 37°c in contrast to invasin, suggesting that ifp is a late stage adhesin. an ifp defined mutant showed a reduction in adhesion to hep- cells and was attenuated in the galleria mellonella infection model.  a new y. pseudotuberculosis adhesin has been identified and characterised. this ifp is a new member in the family of invasin/intimin outer membrane adhesins. 