The K1K2 region of Lys-gingipain of Porphyromonas gingivalis blocks induction of HLA expression by gamma interferon.

In the context of periodontal disease, cysteine proteinases or gingipains from Porphyromonas gingivalis have been implicated in the hydrolysis of cytokines, including gamma interferon (IFN-γ). This cytokine plays a crucial role in host defenses, in part, by regulating expression of major histocompatibility complex molecules. Our recent analysis has identified three structurally defined modules, K1, K2, and K3, of the hemagglutinin region of the lysine gingipain Kgp. These three structurally homologous domains have a common ß-sandwich topology that is similar to that found in a superfamily of adhesins and carbohydrate binding domains. The three Kgp hemagglutinin modules are distinguished by variation in some of the loops projecting from the ß-sandwich core. Recombinant products corresponding to both single and multidomain regions as well as native Kgp bound IFN-γ with similar affinities. Among the adhesin domain constructs, only the K1K2 polypeptide inhibited the upregulation of HLA-1 expression in a human erythroleukemia (K562) line induced by both recombinant and native IFN-γ. The K1K2 polypeptide also inhibited HLA-DR expression induced by IFN-γ in human umbilical vein endothelial cells. These effects were competitively inhibited by coincubation with sodium or potassium chloride solution. The N-terminal residues of IFN-γ were implicated in mediating the effect of K1K2, while antibody binding to loop 1 of K2 blocked the action of K1K2. The findings indicate the potential significance of structurally defined Kgp adhesin modules in the inactivation of IFN-γ but also the potential of K1K2 in locating the target for the catalytic domain of Kgp.

The modular structure of haemagglutinin/adhesin regions in gingipains of Porphyromonas gingivalis.

High-molecular-weight arginine- and lysine-specific (Kgp) gingipains are essential virulence factors expressed by the oral pathogen Porphyromonas gingivalis. Haemagglutinin/adhesin (HA) regions of these proteases have been implicated in targeting catalytic domains to biological substrates and in other adhesive functions. We now report the crystal structure of the K3 adhesin domain/module of Kgp, which folds into the distinct ß-jelly roll sandwich topology previously observed for K2. A conserved structural feature of K3, previously observed in the Kgp K2 module, is the half-way point anchoring of the surface exposed loops via an arginine residue found in otherwise highly variable sequences. Small-angle X-ray scattering data for the recombinant construct K1K2K3 confirmed a structure comprising a tandem repeat of three homologous modules, K1, K2 and K3 while also indicating an unusual 'y'-shape arrangement of the modules connected by variable linker sequences. Only the K2 and K3 modules and a K1K2 construct were observed to be potently haemolytic. K2, K3 and the K1K2 construct showed preferential recognition of haem-albumin over albumin whereas only low affinity binding was detected for K1 and the K1K2K3 construct. The data indicate replication of some biological functions over the three adhesin domains of Kgp while other functions are restricted.

Structure determination and analysis of a haemolytic gingipain adhesin domain from Porphyromonas gingivalis.

Porphyromonas gingivalis is an obligately anaerobic bacterium recognized as an aetiological agent of adult periodontitis. P. gingivalis produces cysteine proteinases, the gingipains. The crystal structure of a domain within the haemagglutinin region of the lysine gingipain (Kgp) is reported here. The domain was named K2 as it is the second of three homologous structural modules in Kgp. The K2 domain structure is a 'jelly-roll' fold with two anti-parallel beta-sheets. This fold topology is shared with adhesive domains from functionally diverse receptors such as MAM domains, ephrin receptor ligand binding domains and a number of carbohydrate binding modules. Possible functions of K2 were investigated. K2 induced haemolysis of erythrocytes in a dose-dependent manner that was augmented by the blocking of anion transport. Further, cysteine-activated arginine gingipain RgpB, which degrades glycophorin A, sensitized erythrocytes to the haemolytic effect of K2. Cleaved K2, similar to that found in extracted Kgp, lacks the haemolytic activity indicating that autolysis of Kgp may be a staged process which is artificially enhanced by extraction of the protein. The data indicate a functional role for K2 in the integrated capacity conferred by Kgp to enable the porphyrin auxotroph P. gingivalis to capture essential haem from erythrocytes.

Gingipains of Porphyromonas gingivalis modulate leukocyte adhesion molecule expression induced in human endothelial cells by ligation of CD99.

Porphyromonas gingivalis has been implicated as a key etiologic agent in the pathogenesis of destructive chronic periodontitis. Among virulence factors of this organism are cysteine proteinases, or gingipains, that have the capacity to modulate host inflammatory defenses. Intercellular adhesion molecule expression by vascular endothelium represents a crucial process for leukocyte transendothelial migration into inflamed tissue. Ligation of CD99 on endothelial cells was shown to induce expression of endothelial leukocyte adhesion molecule 1, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and major histocompatibility complex class II molecules and to increase adhesion of leukocytes. CD99 ligation was also found to induce nuclear translocation of NF-kappaB. These results indicate that endothelial cell activation by CD99 ligation may lead to the up-regulation of adhesion molecule expression via NF-kappaB activation. However, pretreatment of endothelial cells with gingipains caused a dose-dependent reduction of adhesion molecule expression and leukocyte adhesion induced by ligation of CD99 on endothelial cells. The data provide evidence that the gingipains can reduce the functional expression of CD99 on endothelial cells, leading indirectly to the disruption of adhesion molecule expression and of leukocyte recruitment to inflammatory foci.

Functional implication of the hydrolysis of platelet endothelial cell adhesion molecule 1 (CD31) by gingipains of Porphyromonas gingivalis for the pathology of periodontal disease.

Periodontitis is a response of highly vascularized tissues to the adjacent microflora of dental plaque. Progressive disease has been related to consortia of anaerobic bacteria, with the gram-negative organism Porphyromonas gingivalis particularly implicated. The gingipains, comprising a group of cysteine proteinases and associated hemagglutinin domains, are major virulence determinants of this organism. As vascular expression of leukocyte adhesion molecules is a critical determinant of tissue response to microbial challenge, the objective of this study was to determine the capacity of gingipains to modulate the expression and function of these receptors. Given the potential multifunctional role of platelet endothelial cell adhesion molecule 1 (PECAM-1) in the vasculature, the effect of gingipains on PECAM-1 expression by endothelial cells was examined. Activated gingipains preferentially down-regulated PECAM-1 expression on endothelial cells compared with vascular cell adhesion molecule 1 and endothelial-leukocyte adhesion molecule 1, but the reduction in PECAM-1 expression was completely inhibited in the presence of the cysteine proteinase inhibitor TLCK (Nalpha-p-tosyl-l-lysine chloromethyl ketone). Endothelial monolayers treated with activated gingipains demonstrated progressive intercellular gap formation that correlated with reduced intercellular junctional PECAM-1 expression as determined by Western blotting and immunofluorescence microscopy. This was accompanied by enhanced transfer of both albumin and neutrophils across the monolayer. The results suggest that degradation of PECAM-1 by gingipains contributes to increased vascular permeability and neutrophil flux at disease sites.

Binding of Porphyromonas gingivalis gingipains to human CD4(+) T cells preferentially down-regulates surface CD2 and CD4 with little affect on co-stimulatory molecule expression.

The role of Porphyromonas gingivalis cysteine proteinases (gingipains) in the evasion of host cell-mediated immunity has not been fully determined. In this study, modulation by gingipains of accessory and co-stimulatory molecule expression on human CD4(+) T cells was evaluated. Arg-gingipain rather than Lys-gingipain binds to resting CD4(+) T cells in the presence of serum. The constitutive expression of CD28 on T cells was slightly up-regulated following challenge with gingipains, whereas CD45 and CD3 were not affected. Binding of anti-CD2 and anti-CD4 monoclonal antibodies (mAbs) was reduced after challenge of T cells with gingipains, but restored to 50 and 100%, respectively, of control levels, after 48h of incubation in medium depleted of gingipains. The induced expression, by anti-CD3 mAb, of CTLA-4, CD25, and CD40 ligand (CD40L) was decreased following incubation of T cells with gingipains which also led to decreased response to anti-CD3 and anti-CD28 mAbs as shown by reduction of interleukin-2 (IL-2) production. Cumulatively, these results indicate that activated gingipains attach to T cells and preferentially cleave CD2 and CD4 molecules, with potential to impair T cell responses at periodontal sites.

Modulation of an interleukin-12 and gamma interferon synergistic feedback regulatory cycle of T-cell and monocyte cocultures by Porphyromonas gingivalis lipopolysaccharide in the absence or presence of cysteine proteinases.

Interleukin 12 (IL-12) is an efficient inducer and enhancer of gamma interferon (IFN-gamma) production by both resting and activated T cells. There is evidence that human monocytes exposed to IFN-gamma have enhanced ability to produce IL-12 when stimulated with lipopolysaccharide (LPS). In this study, it was demonstrated that LPS from the oral periodontal pathogen Porphyromonas gingivalis stimulated monocytes primed with IFN-gamma to release IL-12, thereby enhancing IFN-gamma accumulation in T-cell populations. P. gingivalis LPS was shown to enhance IL-12 induction of IFN-gamma in T cells in a manner independent from TNF-alpha contribution. The levels of T-cell IL-12 receptors were not affected by P. gingivalis LPS and played only a minor role in the magnitude of the IFN-gamma response. These data suggest that LPS from P. gingivalis establishes an activation loop with IL-12 and IFN-gamma with potential to augment the production of inflammatory cytokines in relation to the immunopathology of periodontitis. We previously reported that the major cysteine proteinases (gingipains) copurifying with LPS in this organism were responsible for reduced IFN-gamma accumulation in the presence of IL-12. However, the addition of the gingipains in the presence of LPS resulted in partial restoration of the IFN-gamma levels. In the destructive periodontitis lesion, release of gingipains from the outer membrane (OM) of P. gingivalis could lead to the downregulation of Th1 responses, while gingipain associated with LPS in the OM or in OM vesicles released from the organism could have net stimulatory effects.

Serum antibodies against the hemoglobin-binding domain (HA2) of Porphyromonas gingivalis.

BACKGROUND: The hemoglobin-binding domain (HA2) of the Porphyromonasgingivalis gingipains and hemagglutinins strongly binds hemoglobin and hemin and is thought to play a key role in acquisition of this essential metabolite by the microorganism. METHODS: In this report, we partially characterized human anti-HA2 humoral antibodies and their relationship to periodontal disease in an analysis of titer and function. RESULTS: Overall, serum anti-HA2 antibodies were relatively low and dominated by the immunoglobulin M (IgM) isotype. Pre-therapy titers had a direct association with periodontal health. Levels of P. gingivalis in the plaque were directly related to pre-therapy anti-HA2 IgG levels, and were an important covariant in a significant direct relationship between pre- and post-therapy anti-HA2 titers. Post-therapy anti-HA2 IgG antibody titers were directly related to the capacity of serum IgG fractions to neutralize hemoglobin binding by Lys-gingipain (Kgp). Further, lower levels of neutralizing activity post-therapy were directly related to severe periodontitis within the patient cohort. CONCLUSIONS: These data suggest that anti-HA2 IgG antibodies correspond directly with periodontal health, possibly through their ability to neutralize P. gingivalis hemoglobin capture. The data also suggest that inadvertent or therapeutic inoculation of P. gingivalis in the plaque may contribute to generation of neutralizing anti-HA2 IgG and improvement of periodontal prognosis.