During infection of cultured epithelial cells, surface‐located Yersinia pseudotuberculosis deliver Yop (Yersinia outer protein) virulence factors into the cytoplasm of the target cell. A non‐polar yopB mutant strain displays a wild‐type phenotype with respect to in vitro Yop regulation and secretion but fails to elicit a cytotoxic response in cultured HeLa cells and is unable to inhibit phagocytosis by macrophage‐like J774 cells. Additionally, the yopB mutant strain was avirulent in the mouse model. No YopE or YopH protein were observed within HeLa cells infected with the yopB mutant strain, suggesting that the loss of virulence of the mutant strain was due to its inability to translocate Yop effector proteins through the target cell plasma membrane. Expression of YopB is necessary for Yersinia‐induced lysis of sheep erythrocytes. Purified YopB was shown to have membrane disruptive activity in vitro. YopB‐dependent haemolytic activity required cell contact between the bacteria and the erythrocytes and could be inhibited by high, but not low, molecular weight carbohydrates. Similarly, expression of YopE reduced haemolytic activity. Therefore, we propose that YopB is essential for the formation of a pore in the target cell membrane that is required for the cell‐to‐cell transfer of Yop effector proteins.
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.
Upon exposure to bacteria, eukaryotic cells activate signalling pathways that result in the increased expression of several defence‐related genes. Here, we report that the yopJ locus of the enteropathogen Yersinia pseudotuberculosis encodes a protein that inhibits the activation of NF‐κB transcription factors by a mechanism(s), which prevents the phosphorylation and subsequent degradation of the inhibitor protein IκB. Consequently, eukaryotic cells infected with YopJ‐expressing Yersinia become impaired in NF‐κB‐dependent cytokine expression. In addition, the blockage of inducible cytokine production coincides with yopJ‐dependent induction of apoptosis. Interestingly, the YopJ protein contains a region that resembles a src homology domain 2 (SH2), and we show that a wild‐type version of this motif is required for YopJ activity in suppressing cytokine expression and inducing apoptosis. As SH2 domains are found in several eukaryotic signalling proteins, we propose that YopJ, which we show is delivered into the cytoplasm of infected cells, interacts directly with signalling proteins involved in inductive cytokine expression. The repressive activity of YopJ on the expression of inflammatory mediators may account for the lack of an inflammatory host response observed in experimental yersiniosis. YopJ‐like activity may also be a common feature of commensal bacteria that, like Yersinia, do not provoke a host inflammatory response.
Both low temperatures and encounters with host phagocytes are two stresses that have been relatively well studied in many species of bacteria. Previous work has shown that the exoribonuclease polynucleotide phosphorylase (PNPase) is required for Yersiniae to grow at low temperatures. Here, we show that PNPase also enhances the ability of Yersinia pseudotuberculosis and Yersinia pestis to withstand the killing activities of murine macrophages. PNPase is required for the optimal functioning of the Yersinia type three secretion system (TTSS), an organelle that injects effector proteins directly into host cells. Unexpectedly, the effect of PNPase on the TTSS is independent of its ribonuclease activity and instead requires its S1 RNA binding domain. In contrast, catalytically inactive enzyme does not enhance the low temperature growth effect of PNPase. Surprisingly, wild-type-like TTSS functioning was restored to the pnp mutant strain by expressing just the ϳ70 amino acid S1 domains from either PNPase, RNase R, RNase II, or RpsA. Our findings suggest that PNPase plays multifaceted roles in enhancing Yersinia survival in response to stressful conditions.
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