A bacterial secretion system caught in the act

Abstract

The T9SS is a novel secretion system exclusively found in Gram-negative bacteria of the phylum Bacteroidetes. It is most notably associated with the human pathogen Porphyromonas gingivalis, the etiologic agent of chronic periodontitis, in which it is responsible for the secretion of the bacterium’s main virulence factors, so-called gingipains. T9SS substrates are secreted in a two-step process, using the general secretory pathway to cross the inner membrane before being translocated across the outer membrane via the T9SS. This second step is mediated by a specific, folded, recognition signal located in the C-terminus of T9SS substrates (the CTD) and requires an inner membrane motor complex powered by the proton motive force. We recently showed that the T9SS OM translocon is formed from the 36-stranded β-barrel protein SprA1. The barrel pore is capped on the extracellular end, but has a lateral opening to the external membrane surface. Structures of SprA bound to the T9SS components PorV and Plug demonstrate that these proteins control access to the lateral opening and to the periplasmic end of the pore, respectively, suggesting an alternating access mechanism in which the two ends of the protein conducting channel are open at different times. This model also suggests that only one conformation of SprA is able to interact with its substrates. We now report our progress in probing this mechanistic model by capturing transport intermediates of the T9SS translocon. We show that removing the T9SS energy source traps substrate proteins during passage through the translocon and allows the isolation of an extended translocon complex (ETC) that contains as additional components the proteins SprE, Fjoh_3466, and a homologue (SkpA) of the Escherichia coli periplasmic chaperone Skp. A structure of the extended translocon shows that the additional translocon components form a disc-shaped structure to one side of the SprA pore at the periplasmic side of the complex. Structural analysis of substrate-translocon complexes isolated by in vivo trapping or in vitro reconstitution reveal that the substrate CTD binds to the extracellular loops of PorV located within the SprA pore. Live cell single molecule tracking experiments imply that the extended translocon is the physiologically relevant form of the transporter. Strikingly, deletion of SprE alone also leads to substrate trapping on the translocon. Our data show that SprE and energization of the T9SS are required for the substrate release step of transport. We propose a model for Type 9 transport in which PorV is used to pull substrate proteins through the translocon