The outer membrane protein LptO is essential for the O‐deacylation of LPS and the co‐ordinated secretion and attachment of A‐LPS and CTD proteins in Porphyromonas gingivalis

Abstract

SummaryProtein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) essential for secretion and attachment to the cell surface. Inactivation of lptO (PG0027) or porT produced mutants that lacked surface protease activity and an electron-dense surface layer. Both mutants showed co-accumulation of A-LPS and unmodified CTD proteins in the periplasm. Lipid profiling by mass spectrometry showed the presence of both tetra-and pentaacylated forms of mono-phosphorylated lipid A in the wild-type and porT mutant, while only the penta-acylated forms of mono-phosphorylated lipid A were found in the lptO mutant, indicating a specific role of LptO in the O-deacylation of mono-phosphorylated lipid A. Increased levels of non-phosphorylated lipid A and the presence of novel phospholipids in the lptO mutant were also observed that may compensate for the missing monophosphorylated tetra-acylated lipid A in the outer membrane (OM). Molecular modelling predicted LptO to adopt a b-barrel structure characteristic of an OM protein, supported by the enrichment of LptO in OM vesicles. The results suggest that LPS deacylation by LptO is linked to the co-ordinated secretion of A-LPS and CTD proteins by a novel secretion and attachment system to form a structured surface layer.

Porphyromonas gingivalis Lipopolysaccharide Weakly Activates M1 and M2 Polarized Mouse Macrophages but Induces Inflammatory Cytokines

“…No amino acids were detected without acid hydrolysis, indicating that the amino acids were present as proteins/peptides. As A-LPS or a glycolipid reactive with MAb 1B5 is recognized as the covalently attached anchor of a class of secreted proteins referred to as CTD proteins, which include the abundant gingipains (RgpA/B and Kgp) (30,32), we probed the LPS preparation for the presence of RgpA/B using antibodies raised to RgpB (30,32). The presence of the modified form of RgpB as a diffuse band at 60 to 80 kDa was confirmed in the LPS preparation, whereas the discrete (unconjugated) form of the protease, which runs as a sharp band at 50 kDa, was not detected.…”

“…The authors also demonstrated differences in the lipid A structures of the two types of LPS, with the A-LPS containing nonphosphorylated tetra-and penta-acylated isoforms which exhibited reduced proinflammatory activity compared with total LPS, containing both O-LPS and A-LPS (37). Recently, Chen et al (30) suggested that A-LPS or a glycolipid containing the phosphorylated mannan epitope that reacts with MAb 1B5 was the cell surface anchor for a unique range of proteins secreted through the outer membrane and covalently attached to the surface of P. gingivalis. These secreted proteins contain a C-terminal signal sequence (CTD) which is cleaved from the protein at the surface and then is covalently attached to the membrane anchor (30,32).…”

“…Recently, Chen et al (30) suggested that A-LPS or a glycolipid containing the phosphorylated mannan epitope that reacts with MAb 1B5 was the cell surface anchor for a unique range of proteins secreted through the outer membrane and covalently attached to the surface of P. gingivalis. These secreted proteins contain a C-terminal signal sequence (CTD) which is cleaved from the protein at the surface and then is covalently attached to the membrane anchor (30,32). The most abundant CTD proteins are the gingipains (RgpA/B and Kgp), and the RgpB conjugate is displayed on an SDS-PAGE gel as a diffuse 60-to 80-kDa band, whereas the unconjugated form of the protein is a sharp 50-kDa band (30,32).…”

“…LPS separated by SDS-PAGE was transferred to a nitrocellulose membrane using the X-cell II transblot module (Life Technologies) at 30 V for 60 min. The membrane then was blocked with 5% (wt/vol) skim milk powder in PBS for 1 h at room temperature and probed with the detection antibodies anti-A-LPS (30,31) and antiRgpA/B (30,32) in 1% (wt/vol) skim milk powder in PBS overnight at 4°C. The anti-A-LPS antibody (monoclonal antibody [MAb] 1B5) was a gift from M. A. Curtis (31).…”

Flavobacterium johnsoniae Chitinase ChiA Is Required for Chitin Utilization and Is Secreted by the Type IX Secretion System

“…The F. johnsoniae motility proteins SprB and RemA and the P. gingivalis gingipains and adhesins are examples of such proteins (8,18,23,24,(39)(40)(41). Some of these surface- ChiA may undergo multiple processing events during or after secretion from the cell.…”