The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss.

Aging is linked to greater susceptibility to chronic inflammatory diseases, several of which, including periodontitis, involve neutrophil-mediated tissue injury. Here we found that aging-associated periodontitis was accompanied by lower expression of Del-1, an endogenous inhibitor of neutrophil adhesion dependent on the integrin LFA-1, and by reciprocal higher expression of interleukin 17 (IL-17). Consistent with that, IL-17 inhibited gingival endothelial cell expression of Del-1, thereby promoting LFA-1-dependent recruitment of neutrophils. Young Del-1-deficient mice developed spontaneous periodontitis that featured excessive neutrophil infiltration and IL-17 expression; disease was prevented in mice doubly deficient in Del-1 and LFA-1 or in Del-1 and the IL-17 receptor. Locally administered Del-1 inhibited IL-17 production, neutrophil accumulation and bone loss. Therefore, Del-1 suppressed LFA-1-dependent recruitment of neutrophils and IL-17-triggered inflammatory pathology and may thus be a promising therapeutic agent for inflammatory diseases.

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

Porphyromonas gingivalis 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 ΔPG0027 mutant strains. Inactivation of PG0027 caused a reduction in the yield of OMVs. Lipid A from cells and OMVs of P. gingivalis W50 and the ΔPG0027 mutant strains were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Lipid A from W50 cells contained bis-P-pentaacyl, mono-P-pentaacyl, mono-P-tetraacyl, non-P-pentaacyl, and non-P-tetraacyl species, whereas lipid A from ΔPG0027 mutant cells contained only phosphorylated species; nonphosphorylated species were absent. MALDI-TOF/TOF tandem MS of mono-P-pentaacyl (m/z 1,688) and mono-P-tetraacyl (m/z 1,448) lipid A from ΔPG0027 showed that both contained lipid A 1-phosphate, suggesting that the ΔPG0027 mutant strain lacked lipid A 1-phosphatase activity. The total phosphatase activities in the W50 and the ΔPG0027 mutant strains were similar, whereas the phosphatase activity in the periplasm of the ΔPG0027 mutant was lower than that in W50, supporting a role for PG0027 in lipid A dephosphorylation. W50 OMVs were enriched in A-LPS, and its lipid A did not contain nonphosphorylated species, whereas lipid A from the ΔPG0027 mutant (OMVs and cells) contained similar species. Thus, OMVs in P. gingivalis are apparently formed in regions of the OM enriched in A-LPS devoid of nonphosphorylated lipid A. Conversely, dephosphorylation of lipid A through a PG0027-dependent process is required for optimal formation of OMVs. Hence, the relative proportions of nonphosphorylated and phosphorylated lipid A appear to be crucial for OMV formation in this organism.IMPORTANCE Gram-negative bacteria produce outer membrane vesicles (OMVs) by "blebbing" of the outer membrane (OM). OMVs can be used offensively as delivery systems for virulence factors and defensively to aid in the colonization of a host and in the survival of the bacterium in hostile environments. Earlier studies using the oral anaerobe Porphyromonas gingivalis as a model organism to study the mechanism of OMV formation suggested that the OM protein PG0027 and one of the two lipopolysaccharides (LPSs) synthesized by this organism, namely, A-LPS, played important roles in OMV formation. We suggest a novel mechanism of OMV formation in P. gingivalis involving dephosphorylation of lipid A of A-LPS controlled/regulated by PG0027, which causes destabilization of the OM, resulting in blebbing and generation of OMVs.

Periodontitis-associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14(+) monocytes leading to enhanced Th17/IL-17 responses.

The Th17/IL-17 pathway is implicated in the pathogenesis of periodontitis (PD), however the mechanisms are not fully understood. We investigated the mechanism by which the periodontal pathogens Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) promote a Th17/IL-17 response in vitro, and studied IL-17(+) CD4(+) T-cell frequencies in gingival tissue and peripheral blood from patients with PD versus periodontally healthy controls. Addition of Pg or Aa to monocyte/CD4(+) T-cell co-cultures promoted a Th17/IL-17 response in vitro in a dose- and time-dependent manner. Pg or Aa stimulation of monocytes resulted in increased CD40, CD54 and HLA-DR expression, and enhanced TNF-α, IL-1ß, IL-6 and IL-23 production. Mechanistically, IL-17 production in Pg-stimulated co-cultures was partially dependent on IL-1ß, IL-23 and TLR2/TLR4 signalling. Increased frequencies of IL-17(+) cells were observed in gingival tissue from patients with PD compared to healthy subjects. No differences were observed in IL-17(+) CD4(+) T-cell frequencies in peripheral blood. In vitro, Pg induced significantly higher IL-17 production in anti-CD3 mAb-stimulated monocyte/CD4(+) T-cell co-cultures from patients with PD compared to healthy controls. Our data suggest that periodontal pathogens can activate monocytes, resulting in increased IL-17 production by human CD4(+) T cells, a process that appears enhanced in patients with PD.

Naked Polyamidoamine Polymers Intrinsically Inhibit Angiotensin II-Mediated EGFR and ErbB2 Transactivation in a Dendrimer Generation- and Surface Chemistry-Dependent Manner.

The effects of naked polyamidoamine (PAMAM) dendrimers on renin-angiotensin system (RAS) signaling via Angiotensin (Ang) II-mediated transactivation of the epidermal growth factor receptor (EGFR) and the closely related family member ErbB2 (HER2) were investigated. In primary aortic vascular smooth muscle cells, a cationic fifth-generation (G5) PAMAM dendrimer dose- and time-dependently inhibited Ang II/AT1 receptor-mediated transactivation of EGFR and ErbB2 as well as their downstream signaling via extracellular-regulated kinase 1/2 (ERK1/2). Inhibition even occurred at noncytotoxic concentrations at short (1 h) exposure times and was dependent on dendrimer generation (G7 > G6 > G5 > G4) and surface group chemistry (amino > carboxyl > hydroxyl). Mechanistically, the cationic G5 PAMAM dendrimer inhibited Ang II-mediated transactivation of EGFR and ErbB2 via inhibition of the nonreceptor tyrosine kinase Src. This novel, early onset, intrinsic biological action of PAMAM dendrimers as inhibitors of the Ang II/AT1/Src/EGFR-ErbB2/ERK1/2 signaling pathway could have important toxicological and pharmacological implications.

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)-ß-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 ß-d-GalNAc at position O-3 of the terminal α-(1 → 3)-linked mannose (Man) residue. In this study, the attachment site of A-PS to the core OS was determined based on structural analysis of SR-type LPS (O-LPS and A-LPS) isolated from a P. gingivalis ΔPG1142 mutant strain by extraction with aqueous hot phenol to minimize the destruction of A-LPS. Application of one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy in combination with methylation analysis showed that the A-PS repeating unit is linked to a nonterminal α-(1 → 3)-linked Man of the "capped core" glycoform of outer core OS at position O-4 via a → 6)-[α-D-Man-α-(1 → 2)-α-D-Man-1-phosphate → 2]-α-D-Man-(1 → motif. In order to verify that O-PS and A-PS are attached to almost identical core glycoforms, we identified a putative α-mannosyltransferase (PG0129) in P. gingivalis W50 that may be involved in the formation of core OS. Inactivation of PG0129 led to the synthesis of deep-R-type LPS with a truncated core that lacks α-(1 → 3)-linked mannoses and is devoid of either O-PS or A-PS. This indicated that PG0129 is an α-1,3-mannosyltransferase required for synthesis of the outer core regions of both O-LPS and A-LPS in P. gingivalis. IMPORTANCE: Porphyromonas gingivalis, a Gram-negative anaerobe, is considered to be an important etiologic agent in periodontal disease, and among the virulence factors produced by the organism are two lipopolysaccharides (LPSs), O-LPS and A-LPS. The structures of the O-PS and A-PS repeating units, the core oligosaccharide (OS), and the linkage of the O-PS repeating unit to the core OS in O-LPS have been elucidated by our group. It is important to establish whether the attachment site of the A-PS repeating unit to the core OS in A-LPS is similar to or differs from that of the O-PS repeating unit in O-LPS. As part of understanding the biosynthetic pathway of the two LPSs in P. gingivalis, PG0129 was identified as an α-mannosyltransferase that is involved in the synthesis of the outer core regions of both O-LPS and A-LPS.

Commensal bacteria-dependent select expression of CXCL2 contributes to periodontal tissue homeostasis.

The oral and intestinal host tissues both carry a heavy microbial burden. Although commensal bacteria contribute to healthy intestinal tissue structure and function, their contribution to oral health is poorly understood. A crucial component of periodontal health is the recruitment of neutrophils to periodontal tissue. To elucidate this process, gingival tissues of specific-pathogen-free and germ-free wild-type mice and CXCR2KO and MyD88KO mice were examined for quantitative analysis of neutrophils and CXCR2 chemoattractants (CXCL1, CXCL2). We show that the recruitment of neutrophils to the gingival tissue does not require commensal bacterial colonization but is entirely dependent on CXCR2 expression. Strikingly, however, commensal bacteria selectively upregulate the expression of CXCL2, but not CXCL1, in a MyD88-dependent way that correlates with increased neutrophil recruitment as compared with germ-free conditions. This is the first evidence that the selective use of chemokine receptor ligands contributes to neutrophil homing to healthy periodontal tissue.

Characterization of the α- and ß-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. gingivalis. Five potential mannosidases were identified in the P. gingivalis W83 genome that may play a role in mannose acquisition. Four mannosidases were characterized in this study: PG0032 was a ß-mannosidase, whereas PG0902 and PG1712 were capable of hydrolyzing p-nitrophenyl α-d-mannopyranoside. PG1711 and PG1712 were α-1 → 3 and α-1 → 2 mannosidases, respectively. No enzyme function could be assigned to PG0973. α-1 → 6 mannobiose was not hydrolyzed by P. gingivalis W50. EPS present in the culture supernatant was shown to be identical to yeast mannan and a component of the medium used for culturing P. gingivalis and was resistant to hydrolysis by mannosidases. Synthesis of O-LPS and A-LPS and glycosylation of the gingipains appeared to be unaffected in all mutants. Thus, α- and ß-mannosidases of P. gingivalis are not involved in the harnessing of mannan/mannose from the growth medium for these biosynthetic processes. P. gingivalis grown in chemically defined medium devoid of carbohydrate showed reduced α-mannosidase activity (25%), suggesting these enzymes are environmentally regulated.

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. Structural analyses were performed using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy in combination with composition and methylation analysis. The outer core OS of O-LPS occurs in two glycoforms: an "uncapped core," which is devoid of O polysaccharide (O-PS), and a "capped core," which contains the site of O-PS attachment. The inner core region lacks L(D)-glycero-D(l)-manno-heptosyl residues and is linked to the outer core via 3-deoxy-D-manno-octulosonic acid, which is attached to a glycerol residue in the outer core via a monophosphodiester bridge. The outer region of the "uncapped core" is attached to the glycerol and is composed of a linear alpha-(1-->3)-linked d-Man OS containing four or five mannopyranosyl residues, one-half of which are modified by phosphoethanolamine at position 6. An amino sugar, alpha-D-allosamine, is attached to the glycerol at position 3. In the "capped core," there is a three- to five-residue extension of alpha-(1-->3)-linked Man residues glycosylating the outer core at the nonreducing terminal residue. beta-D-GalNAc from the O-PS repeating unit is attached to the nonreducing terminal Man at position 3. The core OS of P. gingivalis O-LPS is therefore a highly unusual structure, and it is the basis for further investigation of the mechanism of assembly of the outer membrane of this important periodontal bacterium.

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. gingivalis. APS was purified on concanavalin A affinity columns to minimize the loss of the anchoring system that occurred during chemical extraction. (1)H nuclear magnetic resonance spectroscopy of the lectin-purified APS confirmed the previous structure but also revealed additional signals that suggested the presence of a lipid A. This was confirmed by fatty acid analysis of the APS and matrix-assisted laser desorption ionization-time of flight mass spectrometry of the lipid A released by treatment with sodium acetate buffer (pH 4.5). Hence, P. gingivalis synthesizes two distinct lipopolysaccharide (LPS) macromolecules containing different glycan repeating units: O-LPS (with O-antigen tetrasaccharide repeating units) and A-LPS (with APS repeating units). Nonphosphorylated penta-acylated and nonphosphorylated tetra-acylated species were detected in lipid A from P. gingivalis total LPS and in lipid A from A-LPS. These lipid A species were unique to lipid A derived from A-LPS. Biological assays demonstrated a reduced proinflammatory activity of A-LPS compared to that of total LPS. Inactivation of a putative O-antigen ligase (waaL) at PG1051, which is required for the final step of LPS biosynthesis, abolished the linkage of both the O antigen and APS to the lipid A core of O-LPS and A-LPS, respectively, suggesting that WaaL in P. gingivalis has dual specificity for both O-antigen and APS repeating units.

Porphyromonas gingivalis regulates the RANKL-OPG system in bone marrow stromal cells.

Porphyromonas gingivalis is a Gram-negative anaerobe implicated in chronic periodontitis, a bacterial-induced inflammatory condition that causes destruction of the periodontal connective tissues and underlying alveolar bone. The receptor activator of nuclear factor-kappaB ligand (RANKL) is a cytokine that directly stimulates osteoclastogenesis and bone resorption, whereas its decoy receptor osteoprotegerin (OPG) blocks this action. This study aimed to investigate the effects of P. gingivalis culture supernatants on RANKL and OPG expression in W20-17 bone marrow stromal cells, and evaluate the involvement of its virulence factors, particularly gingipains and lipopolysaccharide. P. gingivalis up-regulated RANKL and down-regulated OPG mRNA expression and protein production. These effects were blocked by indomethacin, suggesting mediation by prostaglandins. Furthermore, P gingivalis induced the production of prostaglandin E(2). Heat-inactivation, or chemical inhibition of P. gingivalis gingipains did not affect RANKL and OPG regulation. However, lipopolysaccharide depletion by polymyxin B abolished RANKL induction, and partly rescued the suppression of OPG. In conclusion, P. gingivalis regulates the RANKL-OPG system via prostaglandin E(2) in bone marrow stromal cells, in a manner that favours osteoclastogenesis. A non-proteolytic and non-proteinaceous P. gingivalis component is involved in these events, most probably its lipopolysaccharide. This activity may contribute to the bone loss characteristic of periodontitis.

Porphyromonas gingivalis manipulates complement and TLR signaling to uncouple bacterial clearance from inflammation and promote dysbiosis.

Certain low-abundance bacterial species, such as the periodontitis-associated oral bacterium Porphyromonas gingivalis, can subvert host immunity to remodel a normally symbiotic microbiota into a dysbiotic, disease-provoking state. However, such pathogens also exploit inflammation to thrive in dysbiotic conditions. How these bacteria evade immunity while maintaining inflammation is unclear. As previously reported, P. gingivalis remodels the oral microbiota into a dysbiotic state by exploiting complement. Now we show that in neutrophils P. gingivalis disarms a host-protective TLR2-MyD88 pathway via proteasomal degradation of MyD88, whereas it activates an alternate TLR2-Mal-PI3K pathway. This alternate TLR2-Mal-PI3K pathway blocks phagocytosis, provides "bystander" protection to otherwise susceptible bacteria, and promotes dysbiotic inflammation in vivo. This mechanism to disengage bacterial clearance from inflammation required an intimate crosstalk between TLR2 and the complement receptor C5aR and can contribute to the persistence of microbial communities that drive dysbiotic diseases.

Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement.

Porphyromonas gingivalis is a low-abundance oral anaerobic bacterium implicated in periodontitis, a polymicrobial inflammatory disease, and the associated systemic conditions. However, the mechanism by which P. gingivalis contributes to inflammation and disease has remained elusive. Here we show that P. gingivalis, at very low colonization levels, triggers changes to the amount and composition of the oral commensal microbiota leading to inflammatory periodontal bone loss. The commensal microbiota and complement were both required for P. gingivalis-induced bone loss, as germ-free mice or conventionally raised C3a and C5a receptor-deficient mice did not develop bone loss after inoculation with P. gingivalis. These findings demonstrate that a single, low-abundance species can disrupt host-microbial homeostasis to cause inflammatory disease. The identification and targeting of similar low-abundance pathogens with community-wide impact may be important for treating inflammatory diseases of polymicrobial etiology.

Regulation of RANKL and OPG gene expression in human gingival fibroblasts and periodontal ligament cells by Porphyromonas gingivalis: a putative role of the Arg-gingipains.

Porphyromonas gingivalis is highly implicated in the pathogenesis of periodontitis, which is characterized by the destruction of periodontal connective tissues and the supporting alveolar bone. Receptor Activator of NF-kappaB Ligand (RANKL) stimulates bone resorption, whereas osteoprotegerin (OPG) blocks its action, and this bi-molecular system is implicated in periodontitis. The aim of this work was (a) to investigate the regulation of RANKL and OPG gene expression in human periodontal ligament (PDL) cells and gingival fibroblasts (GF), in response to P. gingivalis culture supernatants, by quantitative real-time PCR and (b) to attempt to identify putative virulence factors involved in this process. The results indicated that P. gingivalis induced RANKL and reduced OPG mRNA expression by the studied cells, resulting in an increased RANKL/OPG expression ratio. Heat-inactivation of P. gingivalis resulted in significant reduction of RANKL mRNA expression. A Lys-gingipain mutant strain did not affect, whereas an Arg-gingipain mutant strain further enhanced RANKL mRNA expression, compared to their parental wild-type strain. In conclusion, P. gingivalis up-regulates the RANKL/OPG expression ratio in GF and PDL cells, denoting an enhanced osteoclastogenic potential by the cells. The component mainly responsible for RANKL induction appears to be proteinaceous, and it may be regulated by the Arg-gingipains.

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. Insertion and deletion mutants were prepared at PG0106-PG0120 in P. gingivalis W50-a K1 serotype. Deletion of PG0109-PG0118 and PG0116-PG0120 both yielded mutants which no longer reacted with antisera to K1 serotypes. Restriction fragment length polymorphism analysis of the locus in strains representing all six K-antigen serotypes and K(-) strains demonstrated significant variation between serotypes and limited conservation within serotypes. In contrast, PG1135-PG1142 was highly conserved in this collection of strains. Sequence analysis of the capsule locus in strain 381 (K(-) strain) demonstrated synteny with the W83 locus but also significant differences including replacement of PG0109-PG0110 with three unique open reading frames, deletion of PG0112-PG0114, and an internal termination codon within PG0106, each of which could contribute to the absence of capsule expression in this strain. Analysis of the Arg-gingipains in the capsule mutants of strain W50 revealed no significant changes to the glycan modifications of these enzymes, which indicates that the glycosylation apparatus in P. gingivalis is independent of the capsule biosynthetic machinery.

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. rgpB encodes two monomeric enzymes, RgpB and mt-RgpB. Earlier work indicated that rgpB is involved in the glycosylation process, since inactivation of rgpB results in the loss of not only RgpB and mt-RgpB but also mt-RgpA(cat). This work aims to confirm the role of RgpB in the posttranslational modification of RgpA(cat) and the effect of aberrant glycosylation on the properties of this enzyme. Two-dimensional gel electrophoresis of cellular proteins from W50 and an inactivated rgpB strain (D7) showed few differences, suggesting that loss of RgpB has a specific effect on RgpA maturation. Inactivation of genes immediately upstream and downstream of rgpB had no effect on rgpA-derived enzymes, suggesting that the phenotype of the rgpB mutant is not due to a polar effect on transcription at this locus. Matrix-assisted laser desorption ionization-time of flight analysis of purified RgpA(cat) from W50 and D7 strains gave identical peptide mass fingerprints, suggesting that they have identical polypeptide chains. However, RgpA(cat) from D7 strain had a higher isoelectric point and a dramatic decrease in thermostability and did not cross-react with a monoclonal antibody which recognizes a glycan epitope on the parent strain enzyme. Although it had the same total sugar content as the parent strain enzyme, there were significant differences in the monosaccharide composition and linking sugars. These data suggest that RgpB is required for the normal posttranslational glycosylation of Arg-gingipains derived from rgpA and that this process is required for enzyme stabilization.

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. We report the purification and structural characterization of the cross-reacting anionic polysaccharide (APS), which is distinct from both the lipopolysaccharide and serotype capsule polysaccharide of P. gingivalis W50. The structure of APS was determined by 1D and 2D NMR spectroscopy and methylation analysis, which showed it to be a phosphorylated branched mannan. The backbone is built up of alpha-1,6-linked mannose residues and the side-chains contain alpha-1,2-linked mannose oligosaccharides of different lengths (one to two sugar residues) attached to the backbone via 1,2-linkage. One of the side-chains in the repeating unit contains Manalpha1-2Manalpha1-phosphate linked via phosphorus to a backbone mannose at position 2. De-O-phosphorylation of APS abolished cross-reactivity suggesting that Manalpha1-2Manalpha1-phosphate fragment forms part of the epitope recognized by MAb1B5. This phosphorylated branched mannan represents a novel polysaccharide that is immunologically related to the post-translational additions of Arg-gingipains.

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. An rgpA rgpB double mutant (E8) of P. gingivalis was constructed to study the role of RGPs in the processing of KGP. A kgp mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs. E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) and exhibited the same pigmentation, colony morphology and identical growth rates to the parent W50 strain in the absence of antibiotics, in both complex and chemically defined media. The KGP activity of E8, grown in the absence of tetracycline, in whole cultures and in culture supernatants (up to 6 d) was identical to levels in W50. However, in the presence of tetracycline in the growth medium, the level of KGP was reduced to 50% of levels present in whole cultures of W50. Since tetracycline had no effect on RGP or KGP activity when incorporated into assay buffer, this effect is most likely to be on the synthesis of Kgp polypeptide. K1A was also stable in the absence of antibiotics but was unable to pigment, and remained straw-coloured throughout growth. RGP activity in whole cultures of K1A was identical to levels in W50, but RGP activity in 6 d culture supernatants was reduced to 50% of levels present in W50. Thus, although KGP is not required for generation of RGP activity from RgpA and RgpB polypeptides, its absence affects the release/transport of RGP into culture supernatant.