Stewart J. Hinchliffe

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.

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 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.

Insecticidal Toxins from the Photorhabdus and Xenorhabdus Bacteria

Insect pathogens are an excellent source of novel insecticidal agents with proven toxicity. In particular, bacteria from the genera Photorhabdus and Xenorhabdus are proving to be a genomic goldmine, encoding a multitude of insecticidal toxins. Some are highly specific in their target species, whilst others are more generalist, but all are of potential use in crop protection against insect pests. These astounding bacterial species are also turning out to be equipped to produce a vast range of anti-microbial compounds which could be of use to medical science. This review will cover the current knowledge of the lifecycles of the two genera and the potential role of the toxins in their biology, before a more in depth exploration of some of the best studied toxins and their potential use in agriculture.

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 insect toxin complex of Yersinia

Many members of the Yersinia genus encode homologues of insect toxins first observed in bacteria that are insect pathogens such as Photorhabdus, Xenorhabdus and Serratia entomophila. These bacteria secrete high molecular weight insecticidal toxins comprised of multiple protein subunits, termed the Toxin Complexes or Tcs. In Photorhabdus three distinct Tc subunits are required for full oral toxicity in insects, that include the [A], [B] and [C] types, although the exact stochiometry remains unclear. The genomes of Photorhabdus strains encode multiple tc loci, although only two have been shown to exhibit oral and injectable activity against the Hawk Moth, Manduca sexta. The exact role of the remaining homologues is unclear. The availability of bacterial genome sequences has revealed the presence of tc gene homologues in many different species. In this chapter we review the tc gene homologues in Yersinia genus. We discuss what is known about the activity of the Yersinia Tc protein homologues and attempt to relate this to the evolution of the genus and of the tca gene family.

Identification and characterisation of a novel adhesin Ifp in Yersinia pseudotuberculosis

BackgroundIn 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.ResultsWe 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.ConclusionA 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.

Separated at Birth? Microarray Analysis of Two Strikingly Similar Yersinia Species

The Black Death is possibly the most infamouspandemic in human history, which killed one-third of the European population and subsequentlyshaped Western civilization (reviewed in [5]). Epi-demics occurred in relentless cycles up to the sev-enteenth century, until severe depopulation causeda gradual decline in cases. All this was caused by asingle pathological agent, the Gram-negative bac-terium,