Modulation of interleukin-8 activity by gingipains from Porphyromonas gingivalis: implications for pathogenicity of periodontal disease.

Gingipains are the major cysteine proteinases synthesized by Porphyromonas gingivalis which, in soluble form, are able to initially convert IL-8 (77 amino acid residues) to a more potent species truncated at the amino terminus, followed by slow degradation and destruction of chemokine biological activity. In contrast, the same enzymes when associated with bacterial outer-membrane blebs (vesicles), instantly degrade this chemokine. This division of enhancing and inactivating activity between soluble and membrane-bound gingipains can cause the compartmentalization of pro- and anti-inflammatory reactions to distal and proximal positions from bacterial plaque, respectively, which may explain why, despite the massive neutrophil accumulation at periodontitis sites, there is no elimination of infection.

Role of gingipains R in the pathogenesis of Porphyromonas gingivalis-mediated periodontal disease.

It has been demonstrated that the Porphyromonas gingivalis cysteine proteinases (gingipains) activate and/or degrade a broad range of host proteins. Inactivation of gingipains R prior to infection of mice results in a decrease in the virulence of P. gingivalis. Analysis of mouse, rabbit, and chicken antisera raised to gingipain R1 demonstrated that the hemagglutinin domains of gingipains are very immunogenic; however, immunization of mice with a peptide derived from the hemagglutinin domain did not protect mice from P. gingivalis infection. Our recent studies indicate that immunization of mice with a peptide corresponding to the N-terminus of the catalytic domain of gingipains R results in the generation of an immune response that affords protection of mice from P. gingivalis infection. It is postulated that the protection observed results from the inactivation of the enzymatic activity of gingipains R as a result of antibody recognition of a processing site on the gingipain R precursor.

A peptide domain on gingipain R which confers immunity against Porphyromonas gingivalis infection in mice.

The cysteine proteinases referred to as gingipains R (gingipain R1 and gingipain R2) and gingipain K produced by Porphyromonas gingivalis are virulence factors of this periodontal pathogen which likely act by interrupting host defense mechanisms and by participating in the penetration and destruction of host connective tissue. To examine the effect of immunization with gingipains R on the ability of P. gingivalis to colonize and invade in the mouse chamber model, BALB/c mice were immunized intraperitoneally with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or multiple antigenic peptide (MAP)-conjugated gingipain R-derived peptides and then challenged with P. gingivalis. Immunization of mice with the 95-kDa gingipain R1, the 50-kDa gingipain R2, or a peptide derived from the N-terminal sequence of the catalytic domain of gingipains R (peptide A) followed by challenge with P. gingivalis A7436 resulted in protection from P. gingivalis invasion. In contrast, immunization with peptides corresponding to either a sequence encompassing the catalytic cysteine residue of gingipains R (peptide B) or an identical sequence within the catalytic domains of gingipain R1 and gingipain K (peptide C), followed by challenge with P. gingivalis, did not protect animals, nor did immunization with a peptide corresponding to sequences within the adhesion/hemagglutinin domain of gingipain R1 (peptide D) which have been shown to be directly involved in the hemagglutinin activity of gingipain R1. However, the immunoglobulin G (IgG) titer obtained following immunization with peptide D was comparable to that obtained following immunization with the N-terminal peptide (peptide A). Competitive enzyme-linked immunosorbent assays, using either the 95-kDa gingipain R1 or gingipain K as the competing soluble antigen, indicated that 42 and 53% of the antibodies induced by immunization with heat-killed bacteria recognize gingipain R1 and gingipain K, respectively; however, even at very high concentrations, the 50-kDa gingipain R2 did not hinder IgG binding to P. gingivalis. These results indicate that antibodies directed to the amino-terminal region of the catalytic domain of gingipains R are capable of inducing a protective immune response against P. gingivalis infection in the mouse chamber model.

Transposition of the endogenous insertion sequence element IS1126 modulates gingipain expression in Porphyromonas gingivalis.

We have previously reported on a Tn4351-generated mutant of Porphyromonas gingivalis (MSM-3) which expresses enhanced arginine-specific proteinase activity and does not utilize hemin or hemoglobin for growth (C. A. Genco et al., Infect. Immun. 63:2459-2466, 1995). In the process of characterizing the genetic lesion in P. gingivalis MSM-3, we have determined that the endogenous P. gingivalis insertion sequence element IS1126 is capable of transposition within P. gingivalis. We have also determined that IS1126 transposition modulates the transcription of the genes encoding the lysine-specific proteinase, gingipain K (kgp) and the arginine-specific proteinase, gingipain R2 (rgpB). Sequence analysis of P. gingivalis MSM-3 revealed that Tn4351 had inserted 60 bp upstream of the P. gingivalis endogenous IS element IS1126. Furthermore, P. gingivalis MSM-3 exhibited two additional copies of IS1126 compared to the parental strain A7436. Examination of the first additional IS1126 element, IS1126(1), indicated that it has inserted into the putative promoter region of the P. gingivalis kgp gene. Analysis of total RNA extracted from P. gingivalis MSM-3 demonstrated no detectable kgp transcript; likewise, P. gingivalis MSM-3 was devoid of lysine-specific proteinase activity. The increased arginine-specific proteinase activity exhibited by P. gingivalis MSM-3 was demonstrated to correlate with an increase in the rgpA and rgpB transcripts. The second additional IS1126 element, IS1126(2), was found to have inserted upstream of a newly identified gene, hmuR, which exhibits homology to a number of TonB-dependent genes involved in hemin and iron acquisition. Analysis of total RNA from P. gingivalis MSM-3 demonstrated that hmuR is transcribed, indicating that the insertion of IS1126 had not produced a polar effect on hmuR transcription. The hemin-hemoglobin defect in P. gingivalis MSM-3 is proposed to result from the inactivation of Kgp, which has recently been demonstrated to function in hemoglobin binding. Taken together, the results presented here demonstrate that the introduction of Tn4351 into the P. gingivalis chromosome has resulted in two previously undocumented phenomena in P. gingivalis: (i) the transposition of the endogenous insertion sequence element IS1126 and (ii) the modulation of gingipain transcription and translation as a result of IS1126 transposition.

Comparative properties of two cysteine proteinases (gingipains R), the products of two related but individual genes of Porphyromonas gingivalis.

Proteolytic enzymes produced by Porphyromonas gingivalis are important virulence factors of this periodontopathogen. Two of these enzymes, referred to as arginine-specific cysteine proteinases (gingipains R), are the product of two related genes. Here, we describe the purification of an enzyme translated from the rgpB/rgp-2 gene (gingipain R2, RGP-2) and secreted as a single chain protein of 422 residues. The enzyme occurs in several isoforms differing in pI, molecular mass, mobility in gelatin zymography gels, and affinity to arginine-Sepharose. In comparison to the 95-kDa gingipain R1, a complex of catalytic and hemagglutinin/adhesin domains, RGP-2 showed five times lower proteolytic activity, although its activity on various P1-arginine p-nitroanilide substrates was generally higher. Gingipains R amidolytic activity, but not general proteolytic activity, was stimulated by glycyl-glycine. However, in cases of limited proteolysis, such as the inactivation of alpha-1-antichymotrypsin, glycyl-glycine potentiated inhibitor cleavage. In contrast, alpha-1-proteinase inhibitor was not inactivated by gingipains R and only underwent proteolytic degradation during boiling in reducing SDS-polyacrylamide gel electrophoresis treatment buffer. Similarly, native type I collagen was completely resistant to cleavage by gingipains but readily degraded after denaturation. Together, these data explain much of the controversy regarding gingipains structure and substrate specificity and indicate that these enzymes function as P. gingivalis virulence factors by proteolysis of selected target proteins rather than random degradation of host connective tissue components.

Genetic variation of Porphyromonas gingivalis genes encoding gingipains, cysteine proteinases with arginine or lysine specificity.

Porphyromonas gingivalis hemagglutinating cysteine proteinases (gingipains) are considered as important virulence factors in the development of adult periodontitis. Using Southern blot analysis it was determined that genes of three distinct but related gingipains (gingipain-R1, gingipain-R2 and gingipain-K) were present in all tested strains including HG66, ATCC 33277, W50, E-20-1, EM-3, 381, A7436, and IKG5, with a region encoding a part of the hemagglutinin domain of gingipain-R1 and gingipain-K showing considerable variability. In contrast, the loci encoding gingipain-R1 (rgp1) and gingipain-R2 (rgp2) were strongly conserved excluding the concurrent occurrence of other gingipain-R-like genes such as cpgR and agp. Significantly, no evidence could be found to support the expression of a gene coding for the putative proteinase porphypain, an enzyme suggested to have both gingipain-R and gingipain-K activity.