Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37°C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface.

The pathogenic species of the genus Yersinia (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis) cause infections of highly varied severity in humans. Y. pestis causes plague and is transmitted by flea bites or infectious aerosols, whereas Y. enterocolitica and Y. pseudotuberculosis are enteric pathogens that cause gastroenteritis after the ingestion of contaminated food or water (for reviews, see references 9 and 35). Still, the virulence mechanisms of the different species show a lot of similarities. One such similarity is the ability to inhibit phagocytosis, which enables the pathogens to replicate in lymphoid tissues. This is conferred by an ca. 70-kb plasmid that is required for virulence in all three species. The plasmid encodes a type III secretion system (TTSS) that delivers antihost proteins or virulence effectors called Yops (Yersinia outer proteins) into the cytosol of eukaryotic cells (12, 13). Yop secretion is normally triggered by eukaryotic cell contact (36, 37), but it can also be induced in vitro by growing the bacteria in calcium-depleted medium at 37°C (13).TTSSs are found in several gram-negative animal and plant pathogens (20, 39). The overall mechanism of secretion appears to be conserved in the different systems. Typically, 20 to 25 proteins are required to assemble a functional secretion system. Nine of these proteins are conserved not only in the TTSSs of different pathogens but also in the bacterial flagellar export apparatus (for reviews, see references 1, 20, and 25). For several animal pathogens, the type III secretion organelle, also referred to as the secreton, has been isolated and analyzed (5,6,21,22,40,44). The basal body of this structure possesses two sets of rings resembling the flagellar basal body. The components common to the virulence associated and the flagellar TTSS are believed either to associate with the cytoplasmic face of the basal body-like structure or to form a pore in the inner membrane ring (15,42). A common feature of secretons isolated so far is a needle-like structure that protrudes from the ring structure located in the outer membrane. This needle is required for secretion, suggesting that the combination of the basal portion and needle extension (needle complex) constitutes an intact secretion organelle (5,6,21,22,40,44). In Yersinia spp. the needle-like structure is comprised of the YscF protein and localizes to the bacterial cell surface prior to eukaryotic cell contact (18; P. Edqvist, J. Olsson, M. Lavander, L. Sundberg, Å. Forsberg, H. Wolf-Watz, and S. Lloyd, unpublished data).The proteins forming the actual secretion apparatus are believed to somehow identify the type III secretion substrates to enable their secretion through the basal body-like structure. One key protein in the export of flagellar components is FlhB, a membrane protein with a large cytoplasmic C-terminal domain (29,30). YscU, the corresponding protein of the TTSS of Yersinia spp. has also been shown to localize to the cytoplasmic membrane (3). The flagellum is a tripar...

Three signal transduction pathways, the two-component systems CpxRA and BaeSR and the alternative sigma factor E , respond to extracytoplasmic stress that facilitates bacterial adaptation to changing environments. At least the CpxRA and E pathways control the production of protein-folding and degradation factors that counter the effects of protein misfolding in the periplasm. This function also influences the biogenesis of multicomponent extracellular appendages that span the bacterial envelope, such as various forms of pili. Herein, we investigated whether any of these regulatory pathways in the enteropathogen Yersinia pseudotuberculosis affect the functionality of the Ysc-Yop type III secretion system. This is a multicomponent molecular syringe spanning the bacterial envelope used to inject effector proteins directly into eukaryotic cells. Disruption of individual components revealed that the Cpx and E pathways are important for Y. pseudotuberculosis type III secretion of Yops (Yersinia outer proteins). In particular, a loss of CpxA, a sensor kinase, reduced levels of structural Ysc (Yersinia secretion) components in bacterial membranes, suggesting that these mutant bacteria are less able to assemble a functional secretion apparatus. Moreover, these bacteria were no longer capable of localizing Yops into the eukaryotic cell interior. In addition, a cpxA lcrQ double mutant engineered to overproduce and secrete Yops was still impaired in intoxicating cells. Thus, the Cpx pathway might mediate multiple influences on bacterium-target cell contact that modulate Yersinia type III secretion-dependent host cell cytotoxicity.

To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III translocon to microinject several anti-host Yop effectors into the cytosol of target eukaryotic cells. YopD has been implicated in several key steps during Yop effector translocation, including maintenance of yop regulatory control and pore formation in the target cell membrane through which effectors traverse. These functions are mediated, in part, by an interaction with the cognate chaperone, LcrH. To gain insight into the complex molecular mechanisms of YopD function, we performed a systematic mutagenesis study to search for discrete functional domains. We highlighted amino acids beyond the first three N-terminal residues that are dispensable for YopD secretion and confirmed that an interaction between YopD and LcrH is essential for maintenance of yop regulatory control. In addition, discrete domains within YopD that are essential for both pore formation and translocation of Yop effectors were identified. Significantly, other domains were found to be important for effector microinjection but not for pore formation. Therefore, YopD is clearly essential for several discrete steps during efficient Yop effector translocation. Recognition of this modular YopD domain structure provides important insights into the function of YopD.

SummaryNon-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.