It has previously been shown that the plasmid-encoded YopE protein of Yersinia pseudotuberculosis is a virulence determinant. In this study, HeLa cells, macrophages and mice were used as different model systems to determine the actual role of YopE in the virulence process. The YopE protein mediates a cytotoxic response on a confluent layer of HeLa cells. A prerequisite of this activity is that the pathogen binds to the cell surface. YopE also induces a cytotoxic response on mouse macrophages where it influences the ability of the pathogen to resist phagocytosis. Bacterial mutants defective in their ability to express YopE are avirulent after oral or intraperitoneal infection but virulent following intravenous injection. On the basis of these results, we propose a role for YopE in the virulence process of Yersinia.

Phosphorylation of proteins catalysed by protein kinases is associated with central functions in growth and proliferation of the eukaryotic cell, and kinases are particularly important in the signal transduction pathways. Enterobacterial protein kinases are structurally and functionally different from eukaryotic protein kinases, and no prokaryotic kinase has so far been described implicating a direct role for this activity in virulence. Virulent Yersinia possess a common virulence plasmid that encodes a number of secreted proteins (Yops), of which YopH has protein-tyrosine phosphatase activity with a key function in the block of phagocytosis by the pathogen. Here we report that the virulence plasmid of Yersinia pseudotuberculosis encodes a secreted protein kinase (YpkA) with extensive homology to eukaryotic Ser/Thr protein kinases. Specific mutants of ypkA resulted in avirulent strains. Thus, YpkA is, to our knowledge, the first reported prokaryotic secreted protein kinase involved in pathogenicity, presumably by interfering with the signal transduction pathways of the target cell.

SummaryExoenzyme S (ExoS) is an ADP-ribosyltransferase secreted by the opportunistic pathogen Pseudomonas aeruginosa. The amino-terminal half of ExoS exhibits homology to the YopE cytotoxin of pathogenic Yersinia. Recently, YopE was found to be translocated into the host cell by a bacteria-cell contact-dependent mechanism involving the ysc-encoded type III secretion system. By using an approach in which exoS was expressed in different strains of Yersinia, including secretion and translocation mutants, we could demonstrate that ExoS was secreted and translocated into HeLa cells by a similar mechanism to that described previously for YopE. Similarly to YopE, the presence of ExoS in the host cell elicited a cytotoxic response, correlating with disruption of the actin microfilament structure. A similar cytotoxic response was also induced by a mutated form of ExoS with a more than 2000-fold reduced ADP-ribosyltransferase activity. However, the enzymatically active ExoS elicited a more definite rounding up of the HeLa cells, which also correlated with decreased viability of the cells after prolonged infection compared with cells infected with strains expressing mutated ExoS or YopE. This suggests that ExoS can act through two different mechanisms on the host cell. The expression of ExoS by Yersinia also mediated an anti-phagocytic effect on macrophages. In addition, we present evidence that extracellularly located P. aeruginosa is able to target ExoS into eukaryotic cells. Taken together, our data suggest that P. aeruginosa, by analogy with Yersinia, targets virulence proteins into the eukaryotic cytosol via a type III secretion-dependent mechanism as part of an anti-phagocytic strategy.

Type III‐mediated translocation of Yop effectors is an essential virulence mechanism of pathogenic YersiniaLcrV is the only protein secreted by the type III secretion system that induces protective immunity. LcrV also plays a significant role in the regulation of Yop expression and secretion. The role of LcrV in the virulence process has, however, remained elusive on account of its pleiotropic effects. Here, we show that anti‐LcrV antibodies can block the delivery of Yop effectors into the target cell cytosol. This argues strongly for a critical role of LcrV in the Yop translocation process. Additional evidence supporting this role was obtained by genetic analysis. LcrV was found to be present on the bacterial surface before the establishment of bacteria target cell contact. These findings suggest that LcrV serves an important role in the initiation of the translocation process and provides one possible explanation for the mechanism of LcrV‐induced protective immunity.

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