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.

Figure 1. Crystal structure of Scc3-CopN_Δ84.

CopN is colored green, with the YopN homology region in dark green and the TyeA homology region in light green. A. A ribbon diagram of the Scc3-CopN_Δ84 structure. Approximate domain boundaries are indicated. Scc3, salmon, binds across the domain 2-domain 3 domain interface. B. A close-up of the Scc3-CopN_Δ84 interface, oriented as in A, with Scc3 shown as a molecular surface. Scc3 forms a relatively flat surface that bridges domains 2 and 3 of CopN_Δ84. C., D. Comparisons of CopN and homologs. C. Comparison between MxiC and CopN. MxiC is colored tan and shown with thin helices. D. Comparison between CopN and the YopN/TyeA complex. YopN is tan and TyeA is brown. YopN and TyeA are and shown with thin helices. E. Overlay of TyeA in orientation shown in D. and when aligned as a rigid body to the carboxy terminal 91 residues of CopN (rmsd = 0.4 Å).

Figure 2. Sequence conservation in CopN and Scc3.

A., B. Sequence conservation displayed on the CopN_Δ84 structure. Residues are colored by conservation (pink is conserved, blue is variable). A. Scc3 interacts with two conserved regions, site 1 and site 2, on CopN. B. An expanded view of the CopN-Scc3 interface. For clarity only the GBR region is shown. The interface is primarily composed of hydrophobic residues from Scc3 that surround a conserved arginine (R365) on CopN. C. Sequence conservation within GBR's is minimal. Scc3 homologs from multiple species are aligned based on conservation throughout their sequences, revealing that the GBR region is present, but not highly conserved in homologs. Sequences used are from C. pneumonia, S flexneri, S. enterica, B. pertussis, Y. enterocolitica, P. aeruginosa, V. parahaemolyticus. Multiple sequences from each genera used in C. were used in A. and B. (see methods).

Figure 3. The Scc3-CopN_Δ84 complex binds directly to translocators.

A. An overlay of class II T3S chaperones. All structures except the Scc3 structure were determined bound to translocator peptides. For clarity, only the IpaB peptide from Shigella is shown. Scc3 is shown in salmon, IpgC (Shigella) in teal, PcrH (Pseudomonas) in green, and SycD (Yersinia) in blue. The peptide-binding site is conserved and open in Scc3. B. CopN-Scc3 complex directly binds to the CopB translocator. Top: gel filtration traces reveal that the ternary complex is tight enough to survive gel filtration. Bottom: SDS PAGE confirming complex formation.

Figure 4. The gatekeeper-chaperone complex is needed for efficient translocator secretion.

A. T3S was induced from wild type and mxiC mutant Shigella strains. M90T is a wild type strain. ΔmxiC is M90T derived, with mxiC deleted . EV: empty vector. Strains were complemeted with mxiC, mxiC mutant (RDR), or empty vector (EV). Proteins were visualized by silver staining and identified by MS. IpaA, OspCs, and IpgB are effectors. IpaB, IpaC, and IpaD are translocators. Bottom: anti-MxiC blot of the same samples indicates that MxiC and MxiC-RDR are secreted normally. B. A secretion timecourse. The experiment shown in A. was repeated and samples taken a 10, 15, 30, and 60 minutes post induction. IpaA, an effector, is secreted at early time points in the absence of functional Mxic, but in the presence of MxiC it is not efficiently secreted until later time points.