A bacterial secretion system caught in the act

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 (Sec) pathway to cross the inner membrane before being translocated across the outer membrane (OM) 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 IM motor complex powered by the proton motive force (PMF). 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 different T9SS components demonstrate that partner proteins control access to the lateral opening and to the periplasmic end of the pore, 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 using both in vitro and in vivo approaches. 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 a TPR repeat-containing lipoprotein SprE, a homologue of the periplasmic chaperone Skp, and the protein Fjoh_3466. The cryoEM structure of the ~ 700kDa substrate-bound ETC shows that the substrate protein CTD binds to the extracellular loops of the translocon partner protein PorV within the SprA pore and that the extended N-terminus of SprA is bound within the cavity of the Skp trimer. Biochemical and structural data show that SprE and Skp play important roles in T9SS transport and the stability of the ETC but are not required for substrate binding to the translocon. In vitro reconstitution experiments confirm that substrate proteins can bind to the translocon independent of the PMF, SprE, or Skp. Our data show that the function of the PMF, SprE, and Skp in Type 9 transport is to extract the substrate from the translocon and thus complete the transport process across the outer membrane