Mechanisms of evasion of complement by Porphyromonas gingivalis.

The complement system is an important host response to invading bacteria. Activation leads to deposition on the bacterial surface of C3b and its' inactivation products and phagocytosis of the opsonised bacteria by host cells. Alternatively the entire complement pathway including terminal components C5b-9 may be activated on the cell surface which gives rise to generation and insertion of the membrane attack complex into the bacterial membrane and cell lysis. Bacterial resistance to complement may be by enzyme digestion of complement components or by the generation or acquisition from the host of cell surface molecules which allow the organism to adopt host complement control proteins. The involvement of surface polysaccharides can be deduced from the very strong association of resistance with the presence of capsule and extended or modified LPS O-antigens in several species. However, in many cases the mechanism is unclear. The proteases of Porphyromonas gingivalis breakdown C3 and C5 and prevent the deposition of C3b on the bacterial cell surface. Greater deposition of both C3b and C5b-9 occurs in protease deficient mutants but mutants do not show loss of resistance to complement mediated lysis. Instead, complement resistance in P. gingivalis is associated with the presence on the cell surface of an anionic branched mannan and appears independent of capsule serotype.

Mechanisms of resistance of Porphyromonas gingivalis to killing by serum complement.

The complement system plays an important role in the host defense against infection, and the formation of the terminal complement complex on the bacterial surface has been shown to be particularly important in killing of gram-negative bacteria. The gram-negative periodontal pathogen Porphyromonas gingivalis is resistant to complement killing, and possible mechanisms suggested for this resistance include protease production and capsule formation. In this study, P. gingivalis Arg- and Lys-gingipain deletion mutants and polysaccharide synthesis deletion mutants have been used to investigate these hypotheses. When Arg- and Lys-gingipain protease mutants were incubated in 20% normal human serum, deposition of complement components on the cell surface was significantly increased compared to that for the wild-type organism. However, despite the increased deposition, the protease mutants maintained resistance to killing and their viability was equal to that seen with heat-inactivated serum. Similar data were obtained when the wild-type organism was treated with gingipain protease inhibitors. K-antigen expression mutants were also resistant to killing. However, mutants which no longer synthesized a surface anionic polysaccharide (APS) (a phosphorylated branched mannan) were extremely sensitive to serum killing. These mutants lack the organized dense glycan surface layer present on the parent strain on the basis of electron microscopy. We conclude that the production of APS at the surface of P. gingivalis rather than Arg- and Lys-gingipain synthesis is the principal mechanism of serum resistance in P. gingivalis.

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.

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.

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. The Arg-gingipains of Porphyromonas gingivalis are recent additions to this list. In this review we present a summary of our investigations on the structure of the glycan additions to these proteolytic enzymes, the genetics of the glycosylation process and some of the effects on enzyme function and recognition. These findings are placed in the context of the current status of understanding of glycoconjugate structure and synthesis in other bacteria. Given the importance of glycosylation of eucaryotic proteins to their stability, structure, resistance to proteolysis and recognition, the modifications to the proteases described in the present report are likely to have a functional role in the properties of these enzymes in periodontal disease.

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. Within 1 h, the protease traversed the plasma membrane and was localized around the nucleus before becoming concentrated in the cytoplasm after 24 to 48 h. In contrast, the purified arginine-specific heterodimeric protease (HRgpA) rapidly entered the nucleus within 15 to 30 min. This nuclear targeting (i) was seen with active and Nalpha-p-tosyl-L-lysine chloromethyl ketone (TLCK)-inactivated HRgpA, indicating it was independent of the proteolytic activity; (ii) occurred at both 4 and 37 degrees C; and (iii) failed to occur with the monomeric protease (RgpA(cat)), indicating the importance of the adhesin chain of the HRgpA protease to this process. Rapid cell entry was also observed with recombinant catalytic (alpha) and adhesin (beta) chains, with the latter again targeting the nuclear area. After 48 h of incubation with HRgpA, significant dose-dependent stimulation of metabolic activity was observed (measured by reduction of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide), and a doubling of mitotic activity combined with the presence of apoptotic cells indicated that HRgpA may interfere with cell cycle control mechanisms. These effects were seen with both active and TLCK-inactivated protease, confirming that they were not dependent on proteolytic activity, and thus provide new insights into the functioning of this P. gingivalis protease.

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. gingivalis lipopolysaccharide (LPS) (Curtis et al., Infect Immun 1999;62:3816-3823). In the present study, we examined the role of these glycan additions in the immune recognition of RgpAcat, by sera from adult periodontal patients (n = 25). Serum IgG antibody levels to P. gingivalis W50, RgpAcat and LPS and to recombinant RgpA were determined by enzyme-linked immunosorbant assay (ELISA). No correlation was observed between the antibody levels to RgpAcat from P. gingivalis and the recombinant form of this enzyme expressed in Escherichia coli. However, a strong association was found between the recognition of LPS and the wild-type enzyme (R = 0.8926; p = 0.0005). Incorporation of LPS into the ELISA led to a significant reduction (mean 25%; range 0.8-43%, SD = 15; p < 0.05) in the recognition of RgpAcat, but had no effect on the recognition of control antigens. Deglycosylation of RgpAcat led to the abolition of immune recognition by patient serum IgG, which suggests that the glycan additions to this molecule are the principal targets of the immune response. Therefore, glycosylation of the RgpAcat protease may play an important role in immune evasion by shielding the primary structure from immune recognition.

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.