The Yersinia protein kinase A is a host factor inducible RhoA/Rac-binding virulence factor.

The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoA-binding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA(GDP) affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

The Salmonella YopJ-homologue AvrA does not possess YopJ-like activity.

The YopJ protein of Yersinia pseudotuberculosis inhibits several eukaryotic signalling pathways that are normally activated in cells following their contact with bacteria. Salmonella encodes a protein, AvrA, that is secreted by the typeIII inv/spa secretion system which is clearly homologous to YopJ (56% identical, 87% similarity). Since AvrA and YopJs similarity also encompassed a region of YopJ that had previously been shown to be critical for its biological activity, we were interested whether AvrA and YopJ provoked similar responses in eukaryotic cells. Two different approaches were used to determine whether AvrA possesses YopJ-like activity in modulating cytokine expression or killing macrophages. An avrA strain of Salmonella dublin was constructed and its activity was compared to an isogenic wildtype counterpart in cellular response assays. In a complementary approach, AvrA was expressed in and delivered into eukaryotic cells by a yopJ strain of Yersinia pseudotuberculosis. We show here that AvrA affects neither cytokine expression or plays a role in macrophage killing when expressed by either Salmonella or Yersinia. Additionally, AvrA does not possess SopB/D-like activity in promoting fluid secretion into infected calf ileal loops. These data indicate that Salmonella and Yersinia trigger and/or modulate eukaryotic cell responses by different typeIII-secreted proteins and suggests that despite their close evolutionary relatedness, AvrA and YopJ perform different functions for Salmonella and Yersinia, respectively.

The yopJ locus is required for Yersinia-mediated inhibition of NF-kappaB activation and cytokine expression: YopJ contains a eukaryotic SH2-like domain that is essential for its repressive 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-kappaB transcription factors by a mechanism(s), which prevents the phosphorylation and subsequent degradation of the inhibitor protein IkappaB. Consequently, eukaryotic cells infected with YopJ-expressing Yersinia become impaired in NF-kappaB-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.