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The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence.

The Gram-negative anaerobic bacteriumPorphyromonas gingivalisis a major pathogen in periodontal disease, one of the biofilm-caused infectious diseases. The bacterium possesses potential virulence factors, including fimbriae, proteinases, hemagglutinin, lipopolysaccharide (LPS), and outer membrane vesicles, and some of these factors are associated with biofilm formation; however, the precise mechanism of biofilm formation is still unknown. Colonial pigmentation of the bacterium on blood agar plates is related to its virulence. In this study, we isolated a nonpigmented mutant that had an insertion mutation within the new gene PGN_1251 (gtfB) by screening a transposon insertion library. The gene shares homology with genes encoding glycosyltransferase 1 of several bacteria. ThegtfBmutant was defective in biosynthesis of both LPSs containing O side chain polysaccharide (O-LPS) and anionic polysaccharide (A-LPS). The defect in the gene resulted in a complete loss of surface-associated gingipain proteinases, strong autoaggregation, and a marked increase in biofilm formation, suggesting that polysaccharide portions of LPSs influence attachment of gingipain proteinases to the cell surface, autoaggregation, and biofilm formation ofP. gingivalis.

Porphyromonas gingivalis is one of the most etiologically important microorganisms in periodontal disease. We found in a previous study that PG1385 (TprA) protein, a tetratricopeptide repeat (TPR) protein, was upregulated in P. gingivalis wild-type cells placed in a mouse subcutaneous chamber and that a tprA mutant was clearly less virulent in the mouse subcutaneous abscess model (M. Yoshimura et al., Oral Microbiol. Immunol. 23:413-418, 2008). In the present study, we investigated the gene expression profile of tprA mutant cells placed in a mouse subcutaneous chamber and found that 9 genes, including PG2102 (tapA), PG2101 (tapB), and PG2100 (tapC) genes, were downregulated in the tprA mutant compared with those in the wild type. Expression of a cluster of tapA, tapB, and tapC genes of the mutant was also downregulated in an in vitro culture with enriched brain heart infusion medium. The TprA protein has three TPR motifs known as a protein-protein interaction module. Yeast two-hybrid system analysis and in vitro protein binding assays with immunoprecipitation and surface plasmon resonance detection revealed that the TprA protein could bind to TapA and TapB proteins. TprA and TapB proteins were located in the periplasmic space, whereas TapA, which appeared to be one of the C-terminal domain family proteins, was located at the outer membrane. We constructed tapA, tapB, and tapC single mutants and a tapA-tapB-tapC deletion mutant. In the mouse subcutaneous infection experiment, all of the mutants were less virulent than the wild type. These results suggest that TprA, TapA, TapB, and TapC are cooperatively involved in P. gingivalis virulence., Infection and immunity, 78(6), pp.2846-2856; 2010

Porphyromonas gingivalis , a gram-negative anaerobic bacterium associated with active lesions of chronic periodontitis, produces several proteinases which are presumably involved in host colonization, perturbation of the immune system, and tissue destruction. The aims of this study were to investigate the degradation of human transferrin by gingipain cysteine proteinases of P. gingivalis and to demonstrate the production of toxic hydroxyl radicals (HO · ) catalyzed by the iron-containing transferrin fragments generated or by release of iron itself. Analysis by polyacrylamide gel electrophoresis and Western immunoblotting showed that preparations of Arg- and Lys-gingipains of P. gingivalis cleave transferrin (iron-free and iron-saturated forms) into fragments of various sizes. Interestingly, gingival crevicular fluid samples from diseased periodontal sites but not samples from healthy periodontal sites contained fragments of transferrin. By using 55 Fe-transferrin, it was found that degradation by P. gingivalis gingipains resulted in the production of free iron, as well as iron bound to lower-molecular-mass fragments. Subsequent to the degradation of transferrin, bacterial cells assimilated intracellularly the radiolabeled iron. Growth of P. gingivalis ATCC 33277, but not growth of an Arg-gingipain- and Lys-gingipain-deficient mutant, was possible in a chemically defined medium containing 30% iron-saturated transferrin as the only source of iron and peptides, suggesting that gingipains play a critical role in the acquisition of essential growth nutrients. Finally, the transferrin degradation products generated by Arg-gingipains A and B were capable of catalyzing the formation of HO · , as determined by a hypoxanthine/xanthine oxidase system and spin trapping-electron paramagnetic resonance spectrometry. Our study indicates that P. gingivalis gingipains degrade human transferrin, providing sources of iron and peptides. The iron-containing transferrin fragments or the release of iron itself may contribute to tissue destruction by catalyzing the formation of toxic HO · .

Porphyromonas gingivalis, an important periodontal pathogen, is closely associated with inflammatory alveolar bone resorption, and several components of the organism such as lipopolysaccharides have been reported to stimulate production of cytokines that promote inflammatory bone destruction. We investigated the effect of infection with viableP. gingivalison cytokine production by osteoblasts. Reverse transcription-PCR and real-time PCR analyses revealed that infection withP. gingivalisinduced receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) mRNA expression in mouse primary osteoblasts. Production of interleukin-6 was also stimulated; however, osteoprotegerin was not. SB20350 (an inhibitor of p38 mitogen-activated protein kinase), PD98059 (an inhibitor of classic mitogen-activated protein kinase kinase, MEK1/2), wortmannin (an inhibitor of phosphatidylinositol 3 kinase), and carbobenzoxyl-leucinyl-leucinyl-leucinal (an inhibitor of NF-κB) did not prevent the RANKL expression induced byP. gingivalis. Degradation of inhibitor of NF-κB-alpha was not detectable; however, curcumin, an inhibitor of activator protein 1 (AP-1), prevented the RANKL production induced byP. gingivalisinfection. Western blot analysis revealed that phosphorylation of c-Jun, a component of AP-1, occurred in the infected cells, and an analysis of c-Fos binding to an oligonucleotide containing an AP-1 consensus site also demonstrated AP-1 activation in infected osteoblasts. Infection withP. gingivalisKDP136, an isogenic deficient mutant of arginine- and lysine-specific cysteine proteinases, did not stimulate RANKL production. These results suggest thatP. gingivalisinfection induces RANKL expression in osteoblasts through AP-1 signaling pathways and cysteine proteases of the organism are involved in RANKL production.

Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis . Our results showed that Arg- and Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis , inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipain-deficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis , including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects.

The periodontopathogen Porphyromonas gingivalis is an obligate anaerobe that is devoid of catalase but exhibits a relatively high degree of resistance to peroxide stress. In the present study, we demonstrate that P. gingivalis contains a Dps homologue that plays an important role in the protection of cells from peroxide stress. The Dps protein isolated from P. gingivalis displayed a ferritin-like spherical polymer consisting of 19-kDa subunits. Molecular cloning and sequencing of the gene encoding this protein revealed that it had a high similarity in nucleotide and amino acid sequences to Dps proteins from other species. The expression of Dps was significantly increased by exposure of P. gingivalis to atmospheric oxygen in an OxyR-dependent manner, indicating that it is regulated by the reactive oxygen species-regulating gene oxyR . The Dps-deficient mutants, including the dps single mutant and the ftn dps double mutant, showed no viability loss upon exposure to atmospheric oxygen for 6 h. In contrast to the wild type, however, these mutants exhibited the high susceptibility to hydrogen peroxide, thereby disrupting the viability. On the other hand, no significant difference in sensitivity to mitomycin C and metronidazole was observed between the wild type and the mutants. Furthermore, the dps single mutant, compared with the wild type, showed a lower viability in infected human umbilical vein endothelial cells.

Porphyromonas gingivalis , a bacterium associated with active chronic periodontitis lesions, produces several proteolytic enzymes that are thought to be involved in host colonization, perturbation of the immune system, and tissue destruction. The aim of the present study was to investigate the contribution of Arg- and Lys-gingipains produced by P. gingivalis to its growth. Although all of the proteins studied were degraded by P. gingivalis , only human serum albumin and transferrin supported growth during serial transfers in a chemically defined medium (CDM). Growth studies with site-directed gingipain-deficient mutants of P. gingivalis revealed that inactivation of both gingipains prevents growth, whereas inactivation of either Arg- or Lys-gingipain activity extended the doubling times to 33 or 13 h, respectively, compared to 9 h for the parent strain. Growth of the mutants and the parent strain was similar when the CDM was supplemented with a protein hydrolysate instead of human serum albumin. Incubation of resting P. gingivalis ATCC 33277 cells with fluorophore-labeled albumin indicated that the proteolytic fragments generated by the gingipains were internalized by the bacterial cells. Internalization of fluorophore-labeled albumin fragments was reduced or completely inhibited in the proteinase-deficient mutants. Interestingly, gingival crevicular fluid samples from diseased periodontal sites contained low-molecular-mass albumin fragments, whereas samples from healthy sites did not. The critical role of proteinases in the growth of P. gingivalis was further investigated using specific Arg- and Lys-gingipain inhibitors. Adding the inhibitors to CDM containing albumin revealed that leupeptin (Arg-gingipain A and B inhibitor) was more efficient at inhibiting growth than cathepsin B inhibitor II (Lys-gingipain inhibitor). Our study suggests that Arg-gingipains and, to a lesser extent, Lys-gingipain play an important role in the growth of P. gingivalis in a defined medium containing a human protein as the sole carbon and nitrogen source.

Porphyromonas gingivalis is a major periodontal pathogen that has the pathogenic proteinases Arg-specific gingipain and Lys-specific gingipain. We previously found that a cell surface component on P. gingivalis is able to induce Toll-like receptor 2 (TLR2)- and TLR4-independent signaling in 7.19 cells and that this component can be degraded by gingipains. In this study, we purified this component from the P. gingivalis gingipain-null mutant KDP136 and obtained two candidate proteins. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis showed that the proteins, with molecular masses of 123 and 43 kDa, were encoded by PGN_0748 and PGN_0728 (pgm6), respectively, in the P. gingivalis ATCC 33277 genome sequence. The PGN_0748-encoded protein, which we refer to as gingipain-sensitive ligand A (GslA), reacted with antiserum that could effectively inhibit the activity of KDP136 to induce NF-kappaB activation in 7.19 cells, but Pgm6 did not. To further determine what protein is responsible for the NF-kappaB activation, we constructed gslA, pgm6, and pgm6 pgm7 deletion mutants from KDP136. When 7.19 cells were exposed to those mutants, the gslA deletion mutant did not induce NF-kappaB activation, whereas the pgm6 and pgm6 pgm7 deletion mutants did. Furthermore, NF-kappaB activation in 7.19 cells induced by KDP136 was partially inhibited by antiserum against a recombinant protein expressed from the 5'-terminal third of gslA. These results indicate that GslA is one of the factors that induce NF-kappaB activation in 7.19 cells. Interestingly, the gslA gene was present in four of seven P. gingivalis strains tested. This restricted distribution might be associated with the virulence potential of each strain., Infection and immunity, 77(10), pp.4414-4420; 2009

Porphyromonas gingivalis is one of the principal organisms associated with adult periodontitis. Bacterial surface proteins such as fimbriae and gingipain hemagglutinin domains have been implicated as adhesins that actuate colonization of epithelium lining the gingival sulcus. We investigated the genetics of P. gingivalis adhesion to monolayers of epithelial cells using wild-type and gingipain mutant strains. These experiments suggested that arginine-specific gingipain (Rgp) catalytic activity modulated adhesion. From the data obtained with rgp mutants, we constructed a working hypothesis predicting that attachment and detachment of P. gingivalis to epithelial cells were mediated by gingipain adhesin and Rgp catalytic domains, respectively. A membrane-based epithelial cell binding assay, used to locate adhesins in extracellular fractions of wild-type and mutant strains, recognized gingipain peptides as adhesins rather than fimbriae. We developed a capture assay that demonstrated the binding of gingipain adhesin peptides to oral epithelial cells. The adherence of fimbrillin to epithelial cells was detected after heat denaturation of cell fractions. The prediction that Rgp catalytic activities mediated detachment was substantiated when the high level of attachment of an rgp mutant was reduced in the presence of wild-type cell fractions that contained gingipain catalytic activities.