PorV is an Outer Membrane Shuttle Protein for the Type IX Secretion System

Michelle D. Glew,Dhana G. Gorasia,Ben Peng,Eric C. Reynolds,Paul D. Veith,Dina Chen

doi:10.1038/s41598-017-09412-w

Michelle D. Glew, Dhana G. Gorasia + Show 4 more

Open Access

https://doi.org/10.1038/s41598-017-09412-w

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Journal: Scientific Reports	Publication Date: Aug 18, 2017
Citations: 46	License type: open-access

Affiliation: University of Melbourne

Abstract

Porphyromonas gingivalis is a keystone pathogen associated with chronic periodontitis. Major virulence factors named gingipains (cysteine proteinases, RgpA, RgpB and Kgp) are secreted via the Type IX Secretion System (T9SS). These, together with approximately 30 other proteins, are secreted to the cell surface and anchored to the outer membrane by covalent modification to anionic lipopolysaccharide (A-LPS) via the novel Gram negative sortase, PorU. PorU is localised on the cell surface and cleaves the C-terminal domain signal (CTD) of T9SS substrates and conjugates their new C-termini to A-LPS. A 440 kDa-attachment complex was identified in the wild-type (WT) comprising of PorU:PorV:PorQ:PorZ. In mutant strains, sub-complexes comprising PorU:PorV or PorQ:PorZ were also identified at smaller native sizes suggesting that PorU and PorZ are anchored to the cell surface via interaction with the PorV and PorQ outer membrane proteins, respectively. Analysis of porU mutants and a CTD cleavage mutant revealed accumulation of immature T9SS substrates in a PorV-bound form. Quantitative label-free proteomics of WT whole cell lysates estimated that the proportion of secretion channels:attachment complexes:free PorV:T9SS substrates was 1:6:110:2000 supporting a role for PorV as a shuttle protein delivering secreted proteins to the attachment complex for CTD signal cleavage and A-LPS modification.

Highlights

Gingipains, together with approximately 30 other proteins, are secreted to the cell surface and anchored to the outer membrane (OM) by covalent attachment to an anionic lipopolysaccharide (A-LPS)[6, 10]
Four spots were found to co-align at a native size of 440 kDa and mass spectrometry (MS) analysis identified them as PorU, PorZ, PorV and PorQ, suggesting all four proteins constitute a complex (Supplementary Fig. S1 and Supplementary Table S1)
The absolute stoichiometry of these various complexes are difficult to estimate since the migration of membrane proteins in BN-PAGE can differ from soluble standards[32]

Summary

Introduction

Gingipains, together with approximately 30 other proteins, are secreted to the cell surface and anchored to the outer membrane (OM) by covalent attachment to an anionic lipopolysaccharide (A-LPS)[6, 10]. This secretion and attachment is performed by the Type IX Secretion System (T9SS) that has been found only in members of the Bacteroidetes/Chlorobi phyla[11,12,13,14]. The cleavage site occurs in a disordered linker region between the Ig-like and CTD domains consistent with it being flexible and exposed for PorU cleavage[29]. Very recently PG1604, named PorZ, was experimentally demonstrated to be a T9SS component and its crystal structure elucidated[23]

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