LptO (PG0027) Is Required for Lipid A 1-Phosphatase Activity in Porphyromonas gingivalis W50.

produces outer membrane vesicles (OMVs) rich in virulence factors, including cysteine proteases and A-LPS, one of the two lipopolysaccharides (LPSs) produced by this organism. Previous studies had suggested that A-LPS and PG0027, an outer membrane (OM) protein, may be involved in OMV formation. Their roles in this process were examined by using W50 parent and the Δ mutant strains.

Identification of the linkage between A-polysaccharide and the core in the A-lipopolysaccharide of Porphyromonas gingivalis W50.

Unlabelled: Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. The structure of the core oligosaccharide (OS) of O-LPS and the attachment site of the O-polysaccharide (O-PS) repeating unit [ → 3)-α-D-Galp-(1 → 6)-α-D-Glcp-(1 → 4)-α-L-Rhap-(1 → 3)-β-D-GalNAcp-(1 → ] to the core have been elucidated using the ΔPG1051 (WaaL, O-antigen ligase) and ΔPG1142 (Wzy, O-antigen polymerase) mutant strains, respectively. The core OS occurs as an "uncapped" glycoform devoid of O-PS and a "capped" glycoform that contains the attachment site of O-PS via β-d-GalNAc at position O-3 of the terminal α-(1 → 3)-linked mannose (Man) residue.

Characterization of the α- and β-mannosidases of Porphyromonas gingivalis.

Mannose is an important sugar in the biology of the Gram-negative bacterium Porphyromonas gingivalis. It is a major component of the oligosaccharides attached to the Arg-gingipain cysteine proteases, the repeating units of an acidic lipopolysaccharide (A-LPS), and the core regions of both types of LPS produced by the organism (O-LPS and A-LPS) and a reported extracellular polysaccharide (EPS) isolated from spent culture medium. The organism occurs at inflamed sites in periodontal tissues, where it is exposed to host glycoproteins rich in mannose, which may be substrates for the acquisition of mannose by P.

Pseudomonas aeruginosa possesses two putative type I signal peptidases, LepB and PA1303, each with distinct roles in physiology and virulence.

Type I signal peptidases (SPases) cleave signal peptides from proteins during translocation across biological membranes and hence play a vital role in cellular physiology. SPase activity is also of fundamental importance to the pathogenesis of infection for many bacteria, including Pseudomonas aeruginosa, which utilizes a variety of secreted virulence factors, such as proteases and toxins. P.

Temperature-dependent modulation of Porphyromonas gingivalis lipid A structure and interaction with the innate host defenses.

Lipid A structure is a critical determinant of the interaction between pathogens and the innate immune system. Previously, we demonstrated the presence of non- and monophosphorylated tetra-acylated lipid A structures in the outer membrane of Porphyromonas gingivalis, an agent of human periodontal disease. These modifications to lipid A structure lead to evasion and suppression of innate defenses mediated by Toll-like receptor 4 (TLR4) and cationic antimicrobial peptides.

Selective sorting of cargo proteins into bacterial membrane vesicles.

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo.

Structural analysis of the core region of O-lipopolysaccharide of Porphyromonas gingivalis from mutants defective in O-antigen ligase and O-antigen polymerase.

Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. Here, we elucidate the structure of the core oligosaccharide (OS) of O-LPS from two mutants of P. gingivalis W50, Delta PG1051 (WaaL, O-antigen ligase) and Delta PG1142 (O-antigen polymerase), which synthesize R-type LPS (core devoid of O antigen) and SR-type LPS (core plus one repeating unit of O antigen), respectively.

Identification of a second lipopolysaccharide in Porphyromonas gingivalis W50.

We previously described a cell surface anionic polysaccharide (APS) in Porphyromonas gingivalis that is required for cell integrity and serum resistance. APS is a phosphorylated branched mannan that shares a common epitope with posttranslational additions to some of the Arg-gingipains. This study aimed to determine the mechanism of anchoring of APS to the surface of P.

Protease-activated receptor 2 mediates human beta-defensin 2 and CC chemokine ligand 20 mRNA expression in response to proteases secreted by Porphyromonas gingivalis.

The oral pathogen Porphyromonas gingivalis secretes proteases such as Arg-gingipain B (RgpB) that activate protease-activated receptors (PARs). Human beta-defensins (hBDs) and the macrophage inflammatory protein 3alpha/CC chemokine ligand 20 (CCL20) produced by epithelial cells are antimicrobial peptides that provide cytokine function and play an important role in innate immunity. The aim of the present study was to determine whether specific members of the PAR family mediate the expression of these innate immunity markers in gingival epithelial cells (GECs) when exposed to P.

Identification and characterization of the capsular polysaccharide (K-antigen) locus of Porphyromonas gingivalis.

Capsular polysaccharides of gram-negative bacteria play an important role in maintaining the structural integrity of the cell in hostile environments and, because of their diversity within a given species, can act as useful taxonomic aids. In order to characterize the genetic locus for capsule biosynthesis in the oral gram-negative bacterium Porphyromonas gingivalis, we analyzed the genome of P. gingivalis W83 which revealed two candidate loci at PG0106-PG0120 and PG1135-PG1142 with sufficient coding capacity and appropriate gene functions based on comparisons with capsule-coding loci in other bacteria.

Structural analysis of a novel anionic polysaccharide from Porphyromonas gingivalis strain W50 related to Arg-gingipain glycans.

The Arg-gingipains (RgpsA and B) of Porphyromonas gingivalis are a family of extracellular cysteine proteases and are important virulence determinants of this periodontal bacterium. A monoclonal antibody, MAb1B5, which recognizes an epitope on glycosylated monomeric RgpAs also cross-reacts with a cell-surface polysaccharide of P. gingivalis W50 suggesting that the maturation pathway of the Arg-gingipains may be linked to the biosynthesis of a surface carbohydrate.

Expression of Arg-Gingipain RgpB is required for correct glycosylation and stability of monomeric Arg-gingipain RgpA from Porphyromonas gingivalis W50.

Arg-gingipains are extracellular cysteine proteases produced by the gram-negative periodontal pathogen Porphyromonas gingivalis and are encoded by rgpA and rgpB. Three Arg-gingipains, heterodimeric high-molecular-mass Arg-gingipain HRgpA comprising the alpha-catalytic chain and the beta-adhesin chain, the monomeric soluble Arg-gingipain comprising only the alpha-catalytic chain (RgpA(cat)), and the monomeric membrane-type heavily glycosylated Arg-gingipain comprising the alpha-catalytic chain (mt-RgPA(cat)), are derived from rgpA. The monomeric enzymes contain between 14 and 30% carbohydrate by weight.

Glycosylation of the Arg-gingipains of Porphyromonas gingivalis and comparison with glycoconjugate structure and synthesis in other bacteria.

Post-translational modification of proteins by covalent attachment of sugars to the protein backbone (protein glycosylation) is the most common post-translational modification in the eucaryotic cell. However, the addition of carbohydrates to proteins of Eubacteria and Archaea has been demonstrated and accepted only recently. There is now a rapidly expanding list of bacterial glycoproteins that have been characterised from a variety of different organisms including many important pathogens.

Nuclear targeting of Porphyromonas gingivalis W50 protease in epithelial cells.

Porphyromonas gingivalis is an important pathogen associated with destructive periodontal disease and is able to invade the epithelial cell barrier. Its cysteine proteases are recognized as major virulence factors, and in this study, we examined the interaction of the arginine-specific protease with epithelial cells in culture. Three cell lines (KB, HeLa, and SCC4) were incubated with strain W50 culture supernatant; stained with monoclonal antibody 1A1, which recognizes an epitope on the adhesin (beta) component of the cysteine protease-adhesin (alpha/beta) heterodimer; and viewed using immunofluorescence microscopy.

Recognition of the carbohydrate modifications to the RgpA protease of Porphyromonas gingivalis by periodontal patient serum IgG.

Periodontal infections by Porphyromonas gingivalis are associated with a sustained systemic IgG antibody response and elevations in local antibody synthesis to this organism. One of the targets of this response is a protease, RgpAcat, which is an important virulence determinant of this organism. Recently, we demonstrated that this molecule is glycosylated and that the glycan chains are immunologically related to P.

Generation of lys-gingipain protease activity in Porphyromonas gingivalis W50 is independent of Arg-gingipain protease activities.

Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, rgpA and rgpB, encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain). The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain with large N- and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of rgpB contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme.

Altered expression and modification of proteases from an avirulent mutant of Porphyromonas gingivalis W50 (W50/BE1).

Proteases of Porphyromonas gingivalis are considered to be important factors in the virulence of this organism. A non-pigmenting mutant of P. gingivalis W50 (W50/BE1) has been shown to be less virulent in animal models and to produce significantly less Arg-specific protease activity than the parent strain.