Mechanism of secretion activation in the Shigella TTSS

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Gram-negative bacteria commonly interact with eukaryotic host cells by using type III secretion systems (TTSSs or secretons). TTSSs serve to inject bacterial proteins directly into the host cell membrane and cytoplasm during infection. Secretons are activated for secretion only upon physical contact with host cells. How this host-pathogen signaling event occurs is not understood. Secretons are composed of cytoplasmic, transmembrane and extracellular domains. The extracellular domain is a hollow needle protruding 45 nm above the bacterial surface. The monomeric unit of the Shigella flexneri needle, MxiH, forms a helical assembly with parameters similar to those defining the architecture of the extracellular, axial components of bacterial flagella (Cordes et al., 2003). The segments of helically arranged single protein types that make up the different parts of the axial flagellum display remarkable kinds of different structural plasticity, for instance supercoiling and strong flexibility/curvature (Samatey et al., 2001 and 2004). The architectural homology between the TTSS needle and bacterial flagella along with other genetic, morphological and functional homologies, led us to propose (Blocker et al., 2003) that signaling of host cell-contact through TTSSs might occur via structural changes in the needle analogous to some controlling the structural plasticity of the flagellar filament (Samatey et al., 2001). To test our hypothesis, we propose to: 1) define the molecular architecture of the external needle that underlies activation of the Shigella TTSS for secretion by determining the helical structure of needles derived from MxiH mutants that lock the TTSS into different secretion states and by similar studies of physiologically activated TTSS, 2) to understand how the external needle communicates the activation signal to the basal body by using biochemical, genetic and EM techniques to study how the other components of the needle (including the probable MxiI periplasmic rod and a putative distal needle cap) interact with the basal body of the apparatus .