Structural basis for effector transmembrane domain recognition by type VI secretion system chaperones

Abstract

Type VI secretion systems (T6SSs) deliver antibacterial effector proteins between neighboring bacteria. Many effectors harbor N-terminal transmembrane domains (TMDs) implicated in effector translocation across target cell membranes. However, the distribution of these TMD-containing effectors remains unknown. Here, we discover prePAAR, a conserved motif found in over 6000 putative TMD-containing effectors encoded predominantly by 15 genera of Proteobacteria. Based on differing numbers of TMDs, effectors group into two distinct classes that both require a member of the Eag family of T6SS chaperones for export. Co-crystal structures of class I and class II effector TMD-chaperone complexes from Salmonella Typhimurium and Pseudomonas aeruginosa, respectively, reveals that Eag chaperones mimic transmembrane helical packing to stabilize effector TMDs. In addition to participating in the chaperone-TMD interface, we find that prePAAR residues mediate effector-VgrG spike interactions. Taken together, our findings reveal mechanisms of chaperone-mediated stabilization and secretion of two distinct families of T6SS membrane protein effectors.

Structure of a bacterial Rhs effector exported by the type VI secretion system

“…RhsA forms a cocoon-like structure that undergoes a double autocleavage We previously demonstrated that a fragment of RhsA lacking its N-terminal prePAAR motif and two predicted transmembrane helices of the TMD (Figure 1C, residues 74-1486), RhsA∆NT, is stable in the absence of its EagR1 chaperone and can be purified to homogeneity as a soluble protein when over-expressed in Escherichia coli 14 . We therefore expressed and purified RhsA∆NT (Supplementary Figure 1A) and used it for cryo-EM and single particle analysis.…”

“…To investigate how RhsA is mounted onto VgrG1 we set out to determine the structure of the secretion competent pre-firing complex (PFC) comprising VgrG1 in complex with fulllength RhsA and EagR1. We purified the complex as described previously 14 and examined it by single particle cryo-EM. The 2D class averages enabled us to unequivocally characterize the arrangement of the subunits of the complex (Figure 5A).…”

“…Previously, our group showed that the T6SS of the soil bacterium Pseudomonas protegens secretes the prePAAR and PAAR-containing effector RhsA 14 . The rhsA operon contains genes encoding its cognate VgrG spike and Eag chaperone proteins (Figure 1A), both of which are required for the delivery of this effector into susceptible competitors 14 . Previous negative stain electron microscopy experiments suggest that EagR1, RhsA and VgrG assemble to form a complex necessary for T6SS function (Figure 1B) 14 .…”

“…The rhsA operon contains genes encoding its cognate VgrG spike and Eag chaperone proteins (Figure 1A), both of which are required for the delivery of this effector into susceptible competitors 14 . Previous negative stain electron microscopy experiments suggest that EagR1, RhsA and VgrG assemble to form a complex necessary for T6SS function (Figure 1B) 14 . The N-terminal domain of RhsA consists of a prePAAR motif as well as two predicted transmembrane helices.…”

Systematic Discovery of Antibacterial and Antifungal Bacterial Toxins

“…Alternatively, these short toxins might be loaded into other bacterial secretion systems using adaptor proteins, a phenomenon which is known in many secretion systems 5,[53][54][55][56][57][58][59] .…”