Identification and characterisation of a novel adhesin Ifp in Yersinia pseudotuberculosis.

BACKGROUND: 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, YPTB1572 in the Y. pseudotuberculosis IP32953 genome sequence, encodes a protein with similarity to invasin, a classic adhesion/invasion protein, and to intimin, the attaching and effacing protein from enteropathogenic (EPEC) and enterohaemorraghic (EHEC) Escherichia coli. RESULTS: We termed YPTB1572 Ifp (Intimin family protein) and show that it is able to bind directly to human HEp-2 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-2 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-2 cells and was attenuated in the Galleria mellonella infection model. CONCLUSION: 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.

YtxR acts as an overriding transcriptional off switch for the Yersinia enterocolitica Ysc-Yop type 3 secretion system.

The Yersinia enterocolitica YtxR protein is a LysR-type transcriptional regulator that induces expression of the ytxAB locus, which encodes a putative ADP-ribosylating toxin. The ytxR and ytxAB genes are not closely linked in the Y. enterocolitica chromosome, and whereas ytxR is present in all sequenced Yersinia spp., the ytxAB locus is not. These observations suggested that there might be other YtxR-regulon members besides ytxAB and prompted us to investigate coregulated genes and gene products by using transcriptional and proteomic approaches. Microarray and reverse transcription-PCR analysis showed that YtxR strongly activates expression of the yts2 locus, which encodes a putative type 2 secretion system, as well as several uncharacterized genes predicted to encode extracytoplasmic proteins. Strikingly, we also discovered that under Ysc-Yop type 3 secretion system-inducing conditions, YtxR prevented the appearance of Yop proteins in the culture supernatant. Microarray and lacZ operon fusion analysis showed that this was due to specific repression of ysc-yop gene expression. YtxR was also able to repress VirF-dependent Phi(yopE-lacZ) and Phi(yopH-lacZ) expression in a strain lacking the virulence plasmid, which suggested a direct repression mechanism. This was supported by DNase I footprinting, which showed that YtxR interacted with the yopE and yopH control regions. Therefore, YtxR is a newly identified regulator of the ysc-yop genes that can act as an overriding off switch for this critical virulence system.

Comparative phylogenomics of pathogenic bacteria by microarray analysis.

DNA microarrays represent a powerful technology that enables whole-scale comparison of bacterial genomes. This, coupled with new methods to model DNA microarray data, is facilitating the development of robust comparative phylogenomics analyses. Such studies have dramatically increased our ability to differentiate between bacteria, highlighting previously undetected genetic differences and population structures and providing new insight into virulence and evolution of bacterial pathogens. Recent results from such studies have generated insights into the evolution of bacterial pathogens, the levels of diversity and plasticity in the genome of a species, as well as the differences in virulence amongst pathogenic bacteria.

The importance of the Rcs phosphorelay in the survival and pathogenesis of the enteropathogenic yersiniae.

The human-pathogenic yersiniae represent an ideal species group to study the evolution of highly virulent bacteria, with Yersinia pestis having emerged from the enteropathogen Y. pseudotuberculosis an estimated 20 000 years ago. Sequence data reveal that the Y. pestis genome is in the early stages of decay and contains hundreds of non-functioning pseudogenes, some of which may be important in the enteric lifestyle of Y. pseudotuberculosis. Bioinformatic analysis of pseudogenes from seven Y. pestis genome sequences identified rcsD as a gene disrupted early in the evolution of this organism. This phosphotransfer protein is part the of the Rcs phosphorelay, a two-component system present in the Enterobacteriaceae which has been shown to regulate the expression of capsular polysaccharide and other virulence determinants in several species including Escherichia coli and Salmonella. Using microarray analysis, we determined that the Y. pseudotuberculosis Rcs phosphorelay regulates the expression of 136 coding sequences, of which 60 % are predicted to affect the cell envelope. Several putative virulence determinants were identified as being regulated by this phosphorelay, along with proteins involved in biofilm formation, motility, mammalian cell adhesion and stress survival. Phenotypic assays on defined mutants confirmed a role for the phosphorelay in these processes in both Y. pseudotuberculosis and Y. enterocolitica.

The Yersinia pseudotuberculosis and Yersinia pestis toxin complex is active against cultured mammalian cells.

The toxin complex (Tc) genes were first identified in the insect pathogen Photorhabdus luminescens and encode approximately 1 MDa protein complexes which are toxic to insect pests. Subsequent genome sequencing projects have revealed the presence of tc orthologues in a range of bacterial pathogens known to be associated with insects. Interestingly, members of the mammalian-pathogenic yersiniae have also been shown to encode Tc orthologues. Studies in Yersinia enterocolitica have shown that divergent tc loci either encode insect-active toxins or play a role in colonization of the gut in gastroenteritis models of rats. So far little is known about the activity of the Tc proteins in the other mammalian-pathogenic yersiniae. Here we present work to suggest that Tc proteins in Yersinia pseudotuberculosis and Yersinia pestis are not insecticidal toxins but have evolved for mammalian pathogenicity. We show that Tc is secreted by Y. pseudotuberculosis strain IP32953 during growth in media at 28 degrees C and 37 degrees C. We also demonstrate that oral toxicity of strain IP32953 to Manduca sexta larvae is not due to Tc expression and that lysates of Escherichia coli BL21 expressing the Yersinia Tc proteins are not toxic to Sf9 insect cells but are toxic to cultured mammalian cell lines. Cell lysates of E. coli BL21 expressing the Y. pseudotuberculosis Tc proteins caused actin ruffles, vacuoles and multi-nucleation in cultured human gut cells (Caco-2); similar morphology was observed after application of a lysate of E. coli BL21 expressing the Y. pestis Tc proteins to mouse fibroblast NIH3T3 cells, but not Caco-2 cells. Finally, transient expression of the individual Tc proteins in Caco-2 and NIH3T3 cell lines reproduced the actin and nuclear rearrangement observed with the topical applications. Together these results add weight to the growing hypothesis that the Tc proteins in Y. pseudotuberculosis and Y. pestis have been adapted for mammalian pathogenicity. We further conclude that Tc proteins from Y. pseudotuberculosis and Y. pestis display differential mammalian cell specificity in their toxicity.

The RovA regulons of Yersinia enterocolitica and Yersinia pestis are distinct: evidence that many RovA-regulated genes were acquired more recently than the core genome.

RovA is a transcriptional activator of Yersinia invasin, an outer membrane protein involved in bacterial attachment and invasion across the intestinal epithelium. In Y. enterocolitica, a rovA mutant is attenuated for virulence compared with either wild-type or inv mutant strains, indicating that RovA may regulate additional virulence factors. Here, we used microarray analysis to define the RovA regulon. Curiously, there was little overlap between the RovA regulons of Y. enterocolitica and Y. pestis despite the fact that RovA itself is highly conserved between the two species. Some of these differences are explained by the observation that a number of RovA-regulated loci in Y. enterocolitica do not have orthologues in Y. pestis and vice versa, suggesting that RovA established regulatory control over genetic material acquired after the divergence of the species. Electromobility shift assays demonstrated that 15 of these RovA-regulated loci directly interact with RovA, and 11 of these promoters had similar affinity as observed for the inv promoter. H-NS and YmoA are believed to form a transcriptional repression complex on the inv promoter, and several studies indicate that RovA and H-NS have overlapping DNA binding sites. H-NS and YmoA regulated a subset of the RovA-regulated loci. Furthermore, H-NS directly bound to 14 of the 15 promoters bound by RovA. From these data, we hypothesize that RovA generally behaves as an anti-H-NS factor to alleviate transcriptional repression in Y. enterocolitica. A number of recent studies have presented data and a model suggesting that H-NS functions as a transcriptional silencer of horizontally acquired genes. This repression can be selectively relieved by regulators such as RovA, and the observation that nearly all RovA-activated genes are repressed by H-NS is consistent with this model.

Coxsackie B viruses that use human DAF as a receptor infect pig cells via pig CAR and do not use pig DAF.

Coxsackie B viruses (CVB) are enteroviruses belonging to the family Picornaviridae. Serotypes 1, 3 and 5 of CVB bind to the human membrane complement regulator decay-accelerating factor (DAF) and the coxsackievirus/adenovirus receptor (CAR), using either or both as receptors. These viruses are known to infect pig cell lines, but the receptor(s) involved has not been identified. We have recently characterized the pig homologue of DAF and here explore the interactions of human DAF-binding CVB with pig homologues of DAF and CAR. CVB infection of three pig cell lines resulted in cytolysis, which could be not be blocked by anti-pig DAF antibodies. CVB bound to CHO cells transfected with human DAF, but not pig DAF. Modification of pig DAF by incorporation of the fourth short consensus repeat of human DAF did not confer CVB-binding capacity. CVB did bind CHO cells expressing pig or human CAR, and pre-incubation of pig cells with anti-CAR antibody blocked CVB infection.

Application of DNA microarrays to study the evolutionary genomics of Yersinia pestis and Yersinia pseudotuberculosis.

Yersinia pestis, the causative agent of plague, diverged from Yersinia pseudotuberculosis, an enteric pathogen, an estimated 1500-20,000 years ago. Genetic characterization of these closely related organisms represents a useful model to study the rapid emergence of bacterial pathogens that threaten mankind. To this end, we undertook genome-wide DNA microarray analysis of 22 strains of Y. pestis and 10 strains of Y. pseudotuberculosis of diverse origin. Eleven Y. pestis DNA loci were deemed absent or highly divergent in all strains of Y. pseudotuberculosis. Four were regions of phage origin, whereas the other seven included genes encoding a vitamin B12 receptor and the insect toxin sepC. Sixteen differences were identified between Y. pestis strains, with biovar Antiqua and Mediaevalis strains showing most divergence from the arrayed CO92 Orientalis strain. Fifty-eight Y. pestis regions were specific to a limited number of Y. pseudotuberculosis strains, including the high pathogenicity island, three putative autotransporters, and several possible insecticidal toxins and hemolysins. The O-antigen gene cluster and one of two possible flagellar operons had high levels of divergence between Y. pseudotuberculosis strains. This study reports chromosomal differences between species, biovars, serotypes, and strains of Y. pestis and Y. pseudotuberculosis that may relate to the evolution of these species in their respective niches.