Crystal structure of the Yersinia enterocolitica type III secretion chaperone SycT.

Several Gram-negative pathogens deploy type III secretion systems (TTSSs) as molecular syringes to inject effector proteins into host cells. Prior to secretion, some of these effectors are accompanied by specific type III secretion chaperones. The Yersinia enterocolitica TTSS chaperone SycT escorts the effector YopT, a cysteine protease that inactivates the small GTPase RhoA of targeted host cells. We solved the crystal structure of SycT at 2.5 angstroms resolution. Despite limited sequence similarity among TTSS chaperones, the SycT structure revealed a global fold similar to that exhibited by other structurally solved TTSS chaperones. The dimerization domain of SycT, however, differed from that of all other known TTSS chaperone structures. Thus, the dimerization domain of TTSS chaperones does not likely serve as a general recognition pattern for downstream processing of effector/chaperone complexes. Yersinia Yop effectors are bound to their specific Syc chaperones close to the Yop N termini, distinct from their catalytic domains. Here, we showed that the catalytically inactive YopT(C139S) is reduced in its ability to bind SycT, suggesting an ancillary interaction between YopT and SycT. This interaction could maintain the protease inactive prior to secretion or could influence the secretion competence and folding of YopT.

Yersinia enterocolitica type III secretion depends on the proton motive force but not on the flagellar motor components MotA and MotB.

The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

Characterization of YopT effects on Rho GTPases in Yersinia enterocolitica-infected cells.

Pathogenic yersiniae employ a type III secretion system for translocating up to six effector proteins (Yersinia outer proteins (Yops)) into eukaryotic target cells. YopT is a cysteine protease that was shown to remove the C-terminal isoprenoid group of RhoA, Rac, and CDC42Hs. Here we characterized the cell biological and biochemical activities of YopT in cells infected with pathogenic Yersinia enterocolitica. Bacterially injected YopT located to cell membranes from which it released RhoA but not Rac or CDC42Hs. In the infected cells RhoA was dissociated from guanine nucleotide dissociation inhibitor-1 (GDI-1) and accumulated as a monomeric protein in the cytosol, whereas Rac and CDC42Hs remained GDI-bound. Direct transfer of isoprenylated RhoA to YopT and RhoA modification could be reconstituted in vitro by guanosine 5'-3-O-(thio)triphosphate loading of a recombinant RhoA.GDI-1 complex. Finally, in macrophages infected with a Yersinia strain selectively translocating YopT podosomal adhesion structures required for chemotaxis as well as phagocytic cups mediating uptake of yersiniae were disrupted. These findings indicate that bacterially translocated YopT acts on membrane-bound and GDI-complexed RhoA but not Rac or CDC42, and this is sufficient for disruption of macrophage immune functions.