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One virulence strategy used by the opportunistic pathogen Pseudomonas aeruginosa is to target toxic proteins into eukaryotic cells by a type III secretion mechanism. Two of these proteins, ExoS and ExoT, show 75% homology on amino acid level. However, compared with ExoS, ExoT exhibits highly reduced ADP-ribosylating activity and the role of ExoT in pathogenesis is poorly understood. To study the biological effect of ExoT, we used a strategy by which ExoT was delivered into host cells by the heterologous type III secretion system of Yersinia pseudotuberculosis. ExoT was found to induce a rounded cell morphology and to mediate disruption of actin microfilaments, similar to that induced by an ADP-ribosylation defective ExoS (E381A) and the related cytotoxin YopE of Y. pseudotuberculosis. In contrast to ExoS, ExoT had no major effect on cell viability and did not modify or inactivate Ras by ADP-ribosylation in vivo. However, similar to ExoS and YopE, ExoT exhibited GAP (GTPase activating protein) activity on RhoA GTPase in vitro. Interestingly, ExoT(R149K), deficient for GAP activity, still caused a morphological change of HeLa cells. Based on our findings, we suggest that the ADP-ribosylating activity of ExoT target another, as yet unidentified, host protein that is distinct from Ras.

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Type III-mediated translocation of Yop effectors is an essential virulence mechanism of pathogenic Yersinia. LcrV 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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Virulent Yersinia species cause systemic infections in rodents, and Y. pestis is highly pathogenic for humans. Pseudomonas aeruginosa, on the other hand, is an opportunistic pathogen, which normally infects only compromised individuals. Surprisingly, these pathogens both encode highly related contact-dependent secretion systems for the targeting of toxins into eukaryotic cells. In Yersinia, YopB and YopD direct the translocation of the secreted Yop effectors across the target cell membrane. In this study, we have analysed the function of the YopB and YopD homologues, PopB and PopD, encoded by P. aeruginosa. Expression of the pcrGVHpopBD operon in defined translocation-deficient mutants (yopB/yopD) of Yersinia resulted in complete complementation of the cell contact-dependent, YopE-induced cytotoxicity of Y. pseudotuberculosis on HeLa cells. We demonstrated that the complementation fully restored the ability of Y. pseudotuberculosis to translocate the effector molecules YopE and YopH into the HeLa cells. Similar to YopB, PopB induced a lytic effect on infected erythrocytes. The lytic activity induced by PopB could be prevented if the erythrocytes were infected in the presence of sugars larger than 3 nm in diameter, indicating that PopB induced a pore of similar size compared with that induced by YopB. Our findings show that the contact-dependent toxin-targeting mechanisms of Y. pseudotuberculosis and P. aeruginosa are conserved at the molecular level and that the translocator proteins are functionally interchangeable. Based on these similarities, we suggest that the translocation of toxins such as ExoS, ExoT and ExoU by P. aeruginosa across the eukaryotic cell membrane occurs via a pore induced by PopB.

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Exoenzyme 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.

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Pathogenic yersiniae deliver a number of different effector molecules, which are referred to as Yops, into the cytosol of eukaryotic cells via a type III secretion system. To identify the regions of YopE from Yersinia pseudotuberculosis that are necessary for its translocation across the bacterial and eukaryotic cellular membranes, we constructed a series of hybrid genes which consisted of various amounts of yopE fused to the adenylate cyclase-encoding domain of the cyclolysin gene (cyaA) of Bordetella pertussis. By assaying intact cells for adenylate cyclase activity, we show that a YopE-Cya protein containing just the 11 amino-terminal residues of YopE is efficiently exported to the exterior surface of the bacterial cell. Single amino acid replacements of the first seven YopE residues significantly decreased the amount of reporter protein detected on the cell surface, suggesting that the extreme amino-terminal region of YopE is recognized by the secretion machinery. As has recently been shown for the Y. enterocolitica YopE protein (M.-P. Sory, A. Boland, I. Lambermont, and G. R. Cornelis, Proc. Natl. Acad. Sci. USA 92:11998-12002, 1995), we found that export to the cell surface was not sufficient for YopE-Cya proteins to be delivered into the eukaryotic cytoplasm. For traversing the HeLa cell membrane, at least 49 yopE-encoded residues were required. Replacement of leucine 43 of YopE with glycine severely affected the delivery of the reporter protein into HeLa cells. Surprisingly, export from the bacterial cell was also not sufficient for YopE-Cya proteins to be released from the bacterial cell surface into the culture supernatant. At least 75 residues of YopE were required to detect activity of the corresponding reporter protein in the culture supernatant, suggesting that a release domain exists in this region of YopE. We also show that the chaperone-like protein YerA required at least 75 YopE residues to form a stable complex in vitro with YopE-Cya proteins and, furthermore, that YerA is not required to target YopE-Cya proteins to the secretion complex. Taken together, our results suggest that traversing the bacterial and eukaryotic membranes occurs by separate processes that recognize distinct domains of YopE and that these processes are not dependent on YerA activity.

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The virulence plasmid-encoded YopE cytotoxin of Yersinia pseudotuberculosis is secreted across the bacterial membranes and subsequently translocated into the eukaryotic cell. Translocation of YopE into target cells was recently shown to be polarized and only occurred at the zone of contact between the pathogen and the eukaryotic cell. Immunogold electron microscopy on cryosectioned Y. pseudotuberculosis revealed that YopE is secreted and deposited on the bacterial cell surface when the bacteria are grown in Ca(2+)-depleted media at 37 degrees C. No YopE was detected in the cytoplasm or in the membranes. In yerA mutants which are downregulated for YopE at a post-transcriptional level, the cytotoxin could only be detected in the cytoplasm. The overall recovery of YopE from the yerA mutant strain was, however, considerably lower than from the wild-type strain. yerA had no major effect on the translation of YopE, but was found to stabilize YopE in the cytoplasm. YerA was shown to specifically interact with YopE in the cytoplasm in vivo and this binding also correlated with YopE secretion. Targeting of YopE to the secretion loci as well as translocation of YopE into HeLa cells occurred also in the absence of YerA. Based on our findings, we suggest that YerA by binding to YopE stabilizes and maintains the cytotoxin in a secretion-competent conformation.

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