Hans Wolf-Watz

Research articleTargeting Bacterial Virulence: Inhibitors of Type III Secretion in Yersinia

Agents that target bacterial virulence without detrimental effect on bacterial growth are useful chemical probes for studies of virulence and potential candidates for drug development. Several gram-negative pathogens employ type III secretion to evade the innate immune response of the host. Screening of a chemical library with a luciferase reporter gene assay in viable Yersinia pseudotuberculosis furnished several compounds that inhibit the reporter gene signal expressed from the yopE promoter and effector protein secretion at concentrations with no or modest effect on bacterial growth. The selectivity patterns observed for inhibition of various reporter gene strains indicate that the compounds target the type III secretion machinery at different levels. Identification of this set of inhibitors illustrates the approach of utilizing cell-based assays to identify compounds that affect complex bacterial virulence systems.

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 Yersiniaprotein 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 RhoAGDP 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.

Signal transduction in Yersinia pseudotuberculosis

Virulent Yersinia possess a common 70kb virulence plasmid which encodes a number of indispensable Yops virulence determinants. Yops proteins are regulated by external stimuli temperature and calcium concentration. At 37° yop transcription is induced and the rate of transcription is regulated by the Ca2+ concentration of the growth medium. In parallel to this transcriptional regulation, Yops are secreted into the growth medium by a specific Ca2+ regulated plasmid-encoded secretion system. One mutant (yopN) has been isolated and studies using this mutant suggest that the surface-located YopN protein senses the Ca2+ concentration and transmits this signal accordingly. The final step of the regulatory hierarchy involves a yop transcriptional repressor. LcrH is suggested to be this repressor since overproduction of LcrH leads to repression of transcription of yop genes. Several Yop proteins have been shown to be essential virulence determinants. Two of these, YopH and YopE, act in concert to block phagocytosis by macrophages. YopH exhibits a protein tyrosine phosphatase activity suggesting that YopH dephosphorylates host proteins. YopE is a cytotoxin. Recent studies have shown that interaction between the target cell surfaces and the pathogen triggers YopE expression and its polarized transfer through the plasma membrane of the target cell. The previous findings with respect to Yop regulation and Yop secretion is in agreement with the polarized transfer of Yop proteins into the target cell.