PorZ, an Essential Component of the Type IX Secretion System of Porphyromonas gingivalis, Delivers Anionic Lipopolysaccharide to the PorU Sortase for Transpeptidase Processing of T9SS Cargo Proteins

© 2021 Madej et al.
Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two β-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro. Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the “attachment complex” composed of PorU, PorZ, and the integral OM β-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface.
This study was supported in part by grants from Spanish and Catalan public and private bodies (PID2019-107725RG-I00, 2017SGR3, and Fundació “La Marató de TV3” 201815 to F.X.G.-R.); National Science Centre, Poland grants UMO-2015/19/N/NZ1/ 00322 to M.M., UMO-2012/04/A/NZ1/00051 to J.P., and UMO-2016/23/N/NZ1/01513 to Z.N.; Polish Ministry of Science and Higher Education grant 1306/MOB/IV/2015/0 to M. K.; U.S. National Institutes of Health, NIDCR, grants DE09761 and DE026280 to J.P.; and Medical Research Council grant MR/P012175/2 to M.A.C.