YPTB3816 of Yersinia pseudotuberculosis strain IP32953 is a virulence-related metallo-oligopeptidase.

BACKGROUND: Although bacterial peptidases are known to be produced by various microorganisms, including pathogenic bacteria, their role in bacterial physiology is not fully understood. In particular, oligopeptidases are thought to be mainly involved in degradation of short peptides e.g. leader peptides released during classical protein secretion pathways. The aim of this study was to investigate effects of inactivation of an oligopeptidase encoding gene opdA gene of Yersinia pseudotuberculosis on bacterial properties in vivo and in vitro, and to test dependence of the enzymatic activity of the respective purified enzyme on the presence of different divalent cations. RESULTS: In this study we found that oligopeptidase OpdA of Yersinia pseudotuberculosis is required for bacterial virulence, whilst knocking out the respective gene did not have any effect on bacterial viability or growth rate in vitro. In addition, we studied enzymatic properties of this enzyme after expression and purification from E. coli. Using an enzyme depleted of contaminant divalent cations and different types of fluorescently labelled substrates, we found strong dependence of its activity on the presence of particular cations. Unexpectedly, Zn2+ showed stimulatory activity only at low concentrations, but inhibited the enzyme at higher concentrations. In contrast, Co2+, Ca2+ and Mn2+ stimulated activity at all concentrations tested, whilst Mg2+ revealed no effect on the enzyme activity at all concentrations used. CONCLUSIONS: The results of this study provide valuable contribution to the investigation of bacterial peptidases in general, and that of metallo-oligopeptidases in particular. This is the first study demonstrating that opdA in Yersinia pseudotuberculsosis is required for pathogenicity. The data reported are important for better understanding of the role of OpdA-like enzymes in pathogenesis in bacterial infections. Characterisation of this protein may serve as a basis for the development of novel antibacterials based on specific inhibition of this peptidase activity.

Molecular recognition of chymotrypsin by the serine protease inhibitor ecotin from Yersinia pestis.

Resistance to antibiotics is a problem not only in terms of healthcare but also biodefense. Engineering of resistance into a human pathogen could create an untreatable biothreat pathogen. One such pathogen is Yersinia pestis, the causative agent of plague. Previously, we have used a bioinformatic approach to identify proteins that may be suitable targets for antimicrobial therapy and in particular for the treatment of plague. The serine protease inhibitor ecotin was identified as one such target. We have carried out mutational analyses in the closely related Yersinia pseudotuberculosis, validating that the ecotin gene is a virulence-associated gene in this bacterium. Y. pestis ecotin inhibits chymotrypsin. Here, we present the structure of ecotin in complex with chymotrypsin to 2.74 Å resolution. The structure features a biologically relevant tetramer whereby an ecotin dimer binds to two chymotrypsin molecules, similar to what was observed in related serine protease inhibitor structures. However, the vast majority of the interactions in the present structure are distinctive, indicating that the broad specificity of the inhibitor for these proteases is based largely on its capacity to recognize features unique to each of them. These findings will have implications for the development of small ecotin inhibitors for therapeutic use.

Yersinia pseudotuberculosis mntH functions in intracellular manganese accumulation, which is essential for virulence and survival in cells expressing functional Nramp1.

Manganese has an important yet undefined role in the virulence of many bacterial pathogens. In this study we confirm that a null mutation in Yersinia pseudotuberculosis mntH reduces intracellular manganese accumulation. An mntH mutant was susceptible to killing by reactive oxygen species when grown under manganese-limited conditions. The mntH mutant was defective in survival and growth in macrophages expressing functional Nramp1, but in macrophages deficient in Nramp the bacteria were able to survive and replicate. In Galleria mellonella, the mntH mutant was attenuated. Taken together, these data suggest a role for manganese in Y. pseudotuberculosis during macrophage intracellular survival, protecting the bacteria from the antimicrobial products released during the respiratory burst.

Steps toward broad-spectrum therapeutics: discovering virulence-associated genes present in diverse human pathogens.

BACKGROUND: New and improved antimicrobial countermeasures are urgently needed to counteract increased resistance to existing antimicrobial treatments and to combat currently untreatable or new emerging infectious diseases. We demonstrate that computational comparative genomics, together with experimental screening, can identify potential generic (i.e., conserved across multiple pathogen species) and novel virulence-associated genes that may serve as targets for broad-spectrum countermeasures. RESULTS: Using phylogenetic profiles of protein clusters from completed microbial genome sequences, we identified seventeen protein candidates that are common to diverse human pathogens and absent or uncommon in non-pathogens. Mutants of 13 of these candidates were successfully generated in Yersinia pseudotuberculosis and the potential role of the proteins in virulence was assayed in an animal model. Six candidate proteins are suggested to be involved in the virulence of Y. pseudotuberculosis, none of which have previously been implicated in the virulence of Y. pseudotuberculosis and three have no record of involvement in the virulence of any bacteria. CONCLUSION: This work demonstrates a strategy for the identification of potential virulence factors that are conserved across a number of human pathogenic bacterial species, confirming the usefulness of this tool.

Predatory bacteria can protect SKH-1 mice from a lethal plague challenge.

With the rise of antimicrobial resistance, novel ways to treat bacterial infections are required and the use of predatory bacteria may be one such approach. Bdellovibrio species have been shown in vitro to predate on a wide range of other Gram-negative bacteria, including CDC category A/B pathogens such as Yersinia pestis. The data reported here show that treatment of SKH-1 mice with Bdellovibrio bacteriovorus HD100 provided significant protection from a lethal challenge of Yersinia pestis CO92. This is the first report of protection conferred by predation in vivo against a systemic pathogen challenge. However, this protective effect was not observed in a preliminary study with Balb/c mice. Therefore the effects of the predatory bacteria are complex and may be dependent on immune status/genetics of the host. Overall, predatory bacteria may have utility as a therapeutic modality but further work is required to understand the predator-host interaction.

Yersinia pseudotuberculosis is resistant to killing by human neutrophils.

The interaction between human neutrophils and the Gram negative gastrointestinal pathogen Yersinia pseudotuberculosis was investigated in vitro. Despite the wealth of data describing how Yersinia can affect the function of neutrophils, there are no published studies describing if neutrophil cells can affect the viability of Y. pseudotuberculosis. The wild-type IP32953 strain of Y. pseudotuberculosis was found to be resistant to killing by human neutrophils. Confocal examination and flow-cytometric analysis of this interaction revealed that bacteria were taken up.

Galleria mellonella as an alternative infection model for Yersinia pseudotuberculosis.

We report that larvae of the wax moth (Galleria mellonella) are susceptible to infection with the human enteropathogen Yersinia pseudotuberculosis at 37 degrees C. Confocal microscopy demonstrated that in the initial stages of infection the bacteria were taken up into haemocytes. To evaluate the utility of this model for screening Y. pseudotuberculosis mutants we constructed and tested a superoxide dismutase C (sodC) mutant. This mutant showed increased susceptibility to superoxide, a key mechanism of killing in insect haemocytes and mammalian phagocytes. It showed reduced virulence in the murine yersiniosis infection model and in contrast to the wild-type strain IP32953 was unable to kill G. mellonella. The complemented mutant regained all phenotypic properties associated with SodC, confirming the important role of this metalloenzyme in two Y. pseudotuberculosis infection models.