A Gatekeeper Chaperone Complex Directs Translocator Secretion during Type Three Secretion

Abstract

Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

Hierarchical protein targeting and secretion is controlled by an affinity switch in the type III secretion system of enteropathogenic Escherichia coli

“…We anticipate that the novel receptor role revealed here and analyzed in detail for CesAB might be universal for translocator chaperones, even those structurally distinct from CesAB [e.g., the CesD and CesD2 class III chaperones of EspB and D (Wainwright & Kaper, ; Neves et al , )]. This would be in agreement with, structures of gatekeepers co‐complexed with chaperone homologues (Archuleta & Spiller, ), pull‐down assays (Kubori & Galan, ; Silva‐Herzog et al , ), and the abolished secretion of all translocators in SepL, SepD, and CesL mutants (Fig EV5H; Wang et al , ; Younis et al , ; Deng et al , ). In this capacity, SepL may act indeed as an early pseudo‐effector (Younis et al , ), that occupies EscV, switches it to preferentially accepting translocators and blocks binding of all other effectors.…”

Structural analysis of SepL, an enteropathogenicEscherichia colitype III secretion-system gatekeeper protein

“…In contrast, deletion of the Yersinia homologue YopN results in constitutive secretion of effector and translocon proteins, suggesting some variability in phenotype between species (Day et al, 2003;Ferracci et al, 2005). The mechanism by which the gatekeeper regulates secretion of the translocon and effectors is not well understood; however, the recently characterized interaction between the Chlamydia gatekeeper CopN and the translocon chaperone Scc3 have led to a model in which a complex formed by the gatekeeper, translocon chaperone and translocon is required to form prior to the secretion of the translocon (Archuleta & Spiller, 2014).…”

Regulation of Type III Secretion of Translocon and Effector Proteins by the EsaB/EsaL/EsaM Complex in Edwardsiella tarda

“…In Salmonella, the SPI-2 senses the neutral pH in the host cytosol and triggers the dissociation of a gatekeeper-containing protein complex (SsaL, SpiC, and SsaM), leading to the translocation of effectors into the host cytosol (18). Similarly, in Chlamydia pneumonia, the gatekeeper protein CopN forms a complex with a chaperone, Scc3, and translocon protein, CopB, ensuring the ordered secretion of translocon proteins and effectors (19). Yersinia sp.…”