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.

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.

SummaryFrancisella tularensis , the causative agent of tularaemia, is a highly infectious and virulent intracellular pathogen. There are two main human pathogenic subspecies, Francisella tularensis ssp. tularensis (type A), and Francisella tularensis ssp. holarctica (type B). So far, knowledge regarding key virulence determinants is limited but it is clear that intracellular survival and multiplication is one major virulence strategy of Francisella . In addition, genome sequencing has revealed the presence of genes encoding type IV pili (Tfp). One genomic region encoding three proteins with signatures typical for type IV pilins contained two 120 bp direct repeats. Here we establish that repeat-mediated loss of one of the putative pilin genes in a type B strain results in severe virulence attenuation in mice infected by subcutaneous route. Complementation of the mutant by introduction of the pilin gene in cis resulted in complete restoration of virulence. The level of attenuation was similar to that of the live vaccine strain and this strain was also found to lack the pilin gene as result of a similar deletion event mediated by the direct repeats. Presence of the pilin had no major effect on the ability to interact, survive and multiply inside macrophage-like cell lines. Importantly, the pilinnegative strain was impaired in its ability to spread from the initial site of infection to the spleen. Our findings indicate that this putative pilin is critical for Francisella infections that occur via peripheral routes.

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.