Porphyromonas gingivalis virulence in a Drosophila melanogaster model

Porphyromonas gingivalis has been implicated in the etiology of adult periodontitis. In this study, we examined the viability of Drosophila melanogaster as a new model for examining P. gingivalis-host interactions. P. gingivalis (W83) infection of Drosophila resulted in a systemic infection that killed in a dose-dependent manner. Differences in the virulence of several clinically prevalent P. gingivalis strains were observed in the Drosophila killing model, and the results correlated well with studies in mammalian infection models and human epidemiologic studies. P. gingivalis pathobiology in Drosophila did not result from uncontrolled growth of the bacterium in the Drosophila hemolymph (blood) or overt damage to Drosophila tissues. P. gingivalis killing of Drosophila was multifactorial, involving several bacterial factors that are also involved in virulence in mammals. The results from this study suggest that many aspects of P. gingivalis pathogenesis in mammals are conserved in Drosophila, and thus the Drosophila killing model should be useful for characterizing P. gingivalis-host interactions and, potentially, polymicrobe-host interactions.     

Humoral immunity of older adults with periodontal disease to Porphyromonas gingivalis.

The effect of age on the humoral response to Porphyromonas gingivalis was assessed in groups of adults (25 to 54 years and 55 to 74 years) with periodontal disease and compared with that in age-matched healthy controls. To determine whether there was an antibody response against P. gingivalis, we measured serum antibodies against whole cells of P. gingivalis 381, A7A1-28, and W50. In addition, antibody levels against purified P. gingivalis outer membrane proteins (i.e., the 43-kDa fimbrial protein and a 75-kDa protein) were also evaluated. Elderly subjects showed the same response to P. gingivalis as younger subjects. Immunoglobulin G (IgG) antibodies to both purified proteins were also elevated in both diseased groups as compared with the normal groups. Total serum IgG, IgA, and IgM levels were also determined by an enzyme-linked immunosorbent assay for all four groups. Total serum IgG levels were elevated in older adults with periodontitis and total IgA levels were elevated in both groups of older adults compared with the younger groups of similar disease status. Total serum IgM levels were comparable for the four groups. Antinuclear antibody titers were assessed in the two groups of older adults and were also found to be higher for the group with periodontitis. These studies show that older adults as well as younger adults have markedly elevated specific antibodies of the IgG and IgA classes to antigens of P. gingivalis, a putative pathogen in both groups. Furthermore, older adults with periodontitis have significantly elevated levels of total serum IgG which may possibly be related to higher levels of autoantibodies.     

Sialidase and sialoglycoproteases can modulate virulence in Porphyromonas gingivalis

The Porphyromonas gingivalis recombinant VimA can interact with the gingipains and several other proteins, including a sialidase. Sialylation can be involved in protein maturation; however, its role in virulence regulation in P. gingivalis is unknown. The three sialidase-related proteins in P. gingivalis showed the characteristic sialidase Asp signature motif (SXDXGXTW) and other unique domains. To evaluate the roles of the associated genes, randomly chosen P. gingivalis isogenic mutants created by allelic exchange and designated FLL401 (PG0778::ermF), FLL402 (PG1724::ermF), and FLL403 (PG0352::ermF-ermAM) were characterized. Similar to the wild-type strain, FLL402 and FLL403 displayed a black-pigmented phenotype in contrast to FLL401, which was not black pigmented. Sialidase activity in P. gingivalis FLL401 was reduced by approximately 70% in comparison to those in FLL402 and FLL403, which were reduced by approximately 42% and 5%, respectively. Although there were no changes in the expression of the gingipain genes, their activities were reduced by 60 to 90% in all the isogenic mutants compared to that for the wild type. Immunoreactive bands representing the catalytic domains for RgpA, RgpB, and Kgp were present in FLL402 and FLL403 but were missing in FLL401. While adhesion was decreased, the capacity for invasion of epithelial cells by the isogenic mutants was increased by 11 to 16% over that of the wild-type strain. Isogenic mutants defective in PG0778 and PG0352 were more sensitive to hydrogen peroxide than the wild type. Taken together, these results suggest that the P. gingivalis sialidase activity may be involved in regulating gingipain activity and other virulence factors and may be important in the pathogenesis of this organism.     

In vivo induction of apoptosis and immune responses in mice by administration of lipopolysaccharide from Porphyromonas gingivalis.

In vivo administration of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) to mice induced apoptosis before a specific immune response. Apoptosis was associated with the expression of immunoglobulin and Ia on B cells and of CD5 and several markers on T cells. Apoptosis peaked in the spleen and lymph nodes on day 2, and the second peak occurred in the thymus on day 9. Tumor necrosis factor alpha (TNF-alpha) could mediate apoptosis, because the serum TNF-alpha levels were significantly higher than those of controls at 1 day before apoptosis and recombinant murine TNF-alpha induced apoptosis. The apoptosis induced by Pg-LPS was similar to that induced by Escherichia coli LPS in its basic manner, but it was unique in the response of thymus T cells. It was suggested that Pg-LPS could induce apoptosis for the elimination of early nonspecific activated lymphocytes.     

The effects of exogenous interleukin-12 on the induction of immune response to Porphyromonas gingivalis in vivo in mice

The effects of treatment with exogenous interleukin-12 (IL-12) on the induction of immune response to Porphyromonas gingivalis, a black pigmented periodontopathic oral bacterium in mice, were determined in the present study. An increased footpad swelling representing a delayed type hypersensitivity (DTH) response to P. gingivalis in IL-12-treated mice could be observed, although increasing doses of IL-12 did not produce cumulative effects on this cellular Immune response. Multiple injections with IL-12 also resulted in elevated serum IFN-gamma levels. Treatment with this cytokine the day before, on and after immunization with heat-killed P. gingivalis augmented the levels of serum antigen-specific IgG2a and IgG3 antibodies, but had obviously little or no effects on those of serum antigen-specific IgG1 and IgG2b antibodies. The results of this study suggest that treatment with exogenous IL-12 In P. gingivalis-immunized mice may enhance DTH response and Th1 cell-associated antibody production.     

成人唾液对牙龈卟啉菌及其提取物毒理学作用的影响

用羟基磷灰石层析法分离成人混合唾液成分，并分别与牙龈卟啉菌及其膜泡和外膜成分混合，观察细菌及其成分对Ｌ９２９细胞的毒性作用以及唾液成分对细菌的影响。结果：分离出三个唾液组分（Ｓ１，Ｓ２，Ｓ３），Ｓ３可刺激细菌产生粘附结构，Ｓ２中可能主要含杀菌成分，Ｓ１影响最小；毒性检测结果显示：细菌的膜泡和外膜成分对细胞都具有较明显的毒性作用，而Ｓ２唾液组分在１００μｇ时就具有明显地抑制细菌的细胞毒作用，说明唾液的Ｓ２成分具有较强的中和能力，Ｓ３作用次之，Ｓ１影响最小。     

Induction of B7-H1 receptor by bacterial cells fractions of Porphyromonas gingivalis on human oral epithelial cells: B7-H1 induction by Porphyromonas gingivalis fractions

The immune-regulatory B7-H1 receptor, also known as programmed death-ligand 1 (PD-L1), plays an important role in cell-mediated immune response. It is a co-signaling molecule that mediates regulation of T cell activation and tolerance and is able to negatively regulate activated T cell functions and survival. High expression of B7-H1 in host cells may contribute to the chronicity of inflammatory disorders and represents a possible mechanism of immune evasion. Porphyromonas gingivalis is regarded as a keystone pathogen in periodontitis and is able to invade host cells and disposes a variety of virulence factors including lipopolysaccharide (LPS), fimbriae and proteases such as gingipains. Based on previous studies that demonstrated the capability of P. gingivalis to induce up-regulation of PD-L1 in malignant and non-malignant oral epithelial cells, the aim of the present work was to analyse the potential of various cellular components of P. gingivalis to induce the PD-L1 receptor. Human squamous carcinoma cells and primary gingival keratinocytes were stimulated with total, inner and outer membrane fractions, cytosolic proteins, as well as LPS and peptidoglycans. PD-L1 protein expression was investigated by Western blot analysis and RT-PCR. It was demonstrated that the total membrane fraction induced the highest up-regulation in B7-H1 expression, followed by the outer and inner membrane, whereas cytosolic proteins and LPS did not. In conclusion, we provide evidence that the membrane fraction of P. gingivalis is responsible for up-regulation of the immune-regulatory receptor PD-L1 in squamous carcinoma cells and gingival keratinocytes, and thus may support immune evasion of oral carcinomas.     

Specific antibodies to Porphyromonas gingivalis Lys-gingipain by DNA vaccination inhibit bacterial binding to hemoglobin and protect mice from infection

Lys-gingipain (KGP), a lysine-specific cysteine proteinase, is one of the major virulence factors of Porphyromonas gingivalis. Here we examined the involvement of the catalytic domain of KGP (KGP(cd)) in hemoglobin binding by P. gingivalis, using a specific immunoglobulin G (IgG) elicited by the administration of plasmid DNA encoding KGP(cd) or the catalytic domain of Arg-gingipain (RGP(cd)). The pSeq2A/kgp(cd) and pSeq2B/rgp(cd) plasmids were constructed by the ligation of kgp(cd) and rgp(cd) DNA fragments, respectively. Female BALB/c mice were immunized with each of these plasmids. pSeq2A/kgp(cd) elicited a strong response to recombinant KGP(cd) (rKGP(cd)), as well as to comparably produced rRGP(cd)-reactive antibodies. The serum antibodies elicited by pSecTag2B/rgp(cd) also cross-reacted with rKGP(cd) as well as rRGP(cd). Anti-KGP(cd) IgG significantly inhibited hemoglobin binding by P. gingivalis. Furthermore, the inhibition of hemoglobin binding was markedly enhanced by a combination of anti-KGP(cd) and anti-fimbriae. Anti-RGP(cd) IgG showed a negligible inhibitory effect, while both anti-KGP(cd) and anti-RGP(cd) IgGs showed significant inhibitory effects on Lys- and Arg-specific proteolytic activities and on the growth of P. gingivalis under iron-restricted conditions where supplemented hemoglobin was the sole iron source. Immunized mice were challenged by intraperitoneal inoculation with P. gingivalis. All nonimmunized mice died within 72 h; however, vaccination with pSeq2A/kgp(cd) and pSeq2B/rgp(cd) prevented inflammatory responses and prolonged the survival rate of immunized mice by 43 and 27%, respectively. These results suggest that KGP(cd) acts as a hemoglobin-binding protein and can also be useful as an immunogen inducing a protective response to P. gingivalis infection.     

Experimental Porphyromonas gingivalis infection in nonimmune athymic BALB/c mice.

The purpose of this report was to study the role of T lymphocytes following injection of Porphyromonas gingivalis in a mouse abscess model. Three invasive P. gingivalis isolates (ATCC 53977, W83, and AJW4) were injected into athymic BALB/c mice and their heterozygous (nu/+) littermates. The athymic BALB/c (nu/nu) mice were able to localize the invasive P. gingivalis isolates at the injection site. By comparison, the heterozygous BALB/c (nu/+) littermates developed hemorrhagic secondary lesions within 24 h after subcutaneous injection of the same invasive P. gingivalis isolates. These results suggest that naive T lymphocytes may contribute to the pathology associated with P. gingivalis infection.     

Evidence of recombination in Porphyromonas gingivalis and random distribution of putative virulence markers

The association of Porphyromonas gingivalis to periodontal disease is not clearly understood. Similar proportions of P. gingivalis may be cultivated from both inactive and actively degrading periodontal pockets. Differences in virulence among strains of P. gingivalis exist, but the molecular reason for this remains unknown. We examined the population structure of P. gingivalis to obtain a framework in which to study pathogenicity in relation to evolution. Phylogenetic trees derived from the sequencing of fragments of four housekeeping genes, ahp, thy, rmlB, and infB, in 57 strains were completely different with no correlation between clustering of strains in the four dendrograms. Combining the various alleles of the four gene fragments sequenced resulted in 41 different sequence types. The index of association, I(A), based on a single representative of each sequence type was 0.143 +/- 0.202, indicating a population at linkage equilibrium. Inclusion of all isolates for the calculation of I(A) resulted in a value of 0.206 +/- 0.171. This suggests an epidemic population structure supported by the finding of genetically identical strains in different parts of the world. We observed a random distribution of two virulence-associated mobile genetic elements, the ragB locus and the insertion sequence IS1598, among 132 strains tested. In conclusion, P. gingivalis has a nonclonal population structure characterized by frequent recombination. Our study suggests that particular genotypes, possibly with increased pathogenic potential, may spread successfully in the human population.     

A novel mouse model to study the virulence of and host response to Porphyromonas (Bacteroides) gingivalis.

We describe here the development of a mouse subcutaneous chamber model that allows for the examination of host-parasite interactions as well as the determination of gross pathology with Porphyromonas (Bacteroides) gingivalis challenge. When inoculated into stainless-steel chambers implanted subcutaneously in female BALB/c mice, P. gingivalis W83, W50, and A7436 (10(8) to 10(10) CFU) caused cachexia, ruffling, general erythema and phlegmonous, ulcerated, necrotic lesions, and death. P. gingivalis W50/BEI, HG405, and 33277 (10(10) CFU) produced localized abscesses in the mouse chamber model with rejection of chambers at the injection site. Analysis of chamber fluid from 33277-, HG405-, and W50/BEI-infected mice by cytocentrifugation revealed inflammatory cell debris, polymorphonuclear leukocytes, and high numbers of dead bacteria. In contrast, fluid from A7436-, W50-, and W83-infected mice revealed infiltration predominantly of polymorphonuclear leukocytes and live bacteria. Bacteria were found primarily associated with polymorphonuclear leukocytes in the fluid from W50-, HG405-, and W83-infected mice but not from A7436-infected mice. Viable isolates were recoverable from the chamber fluid through day 3 for W50/BEI, day 5 for 33277, day 6 for HG405, day 7 for W50, day 14 for W83, and day 26 for A7436. All strains induced a systemic immunoglobulin G response in serum and chamber fluid samples. The use of this model will allow us to examine the virulence of P. gingivalis as defined by the interaction of host response to localized infection with P. gingivalis.     

Immune response gene regulation of the humoral immune response to Porphyromonas gingivalis fimbriae in mice.

Among various strains of mice immunized orally with Porphyromonas gingivalis fimbriae and adjuvant GM-53 in liposomes, BALB/c and DBA/2 mice (H-2d) were found to be high responders to the fimbriae, CBA/J and C3H mice (H-2k) were intermediate, while C57BL/6 mice were low responders in terms of serum IgG and salivary IgA responses. Furthermore, humoral immune responses were examined using congeneic mice of B10 background showing different H-2 haplotypes, and it was revealed that B10.D2 mice (H-2d), followed by B10.BR (H-2k), responded well to antigenic stimulation of the fimbriae, while C57BL/10 mice (B10, H-2b) were low responders to the fimbriae. Hybrids between BALB/c and C57BL/6 mice were found to reflect a phenotype of low responders. Thus, the humoral immune responses to P. gingivalis in mice are restricted by H-2 haplotype.     

The rag locus of Porphyromonas gingivalis contributes to virulence in a murine model of soft tissue destruction

The rag locus of Porphyromonas gingivalis encodes a putative TonB-dependent outer membrane receptor, RagA, and a 55-kDa immunodominant antigen, RagB. Inactivation of either ragA or ragB prevented expression of both RagA and RagB. Both the ragA and ragB mutants were significantly less virulent than wild-type strains in a murine model of infection.     

Concordance of Porphyromonas gingivalis colonization in families.

Periodontitis is a widespread disease that appears to be due to a specific bacterial infection. Several species of bacteria have been investigated as potential pathogens, and particularly strong evidence links the presence of Porphyromonas gingivalis with indicators of periodontitis. Information concerning the transmission of P. gingivalis between human contacts may be important in determining risk factors for disease and developing preventive strategies. A few small studies have provided some evidence of transmission between related individuals, but no large-scale study of families that would reflect the typical transmission of this pathogen in the population has been reported. The purpose of this study was to investigate the transmission of P. gingivalis within randomly selected, extended families. The colonization status of 564 members of multigeneration families was determined, and the degree of concordance observed among members of these families was then compared to that expected to occur based on the prevalence of colonization in the population studied. A PCR assay was used for detection of P. gingivalis. Concordance in colonization was more frequently observed within entire families (P = 0.0000) and for spouses (P < 0.001), children and their mothers (P < 0.001), children and their fathers (P < 0.01), adults and their mothers (P < 0.005), and siblings (P < 0.05) than would be expected if P. gingivalis were randomly distributed in the population studied. Results showed that contact with an infected family member substantially increased the relative risk of colonization in these intrafamilial pairs. This indicates that P. gingivalis is commonly transmitted by contact with an infected family member.     

Functional properties of nonhuman primate antibody to Porphyromonas gingivalis.

The nonhuman primate (NHP) serves as a useful model for examining the host-parasite interactions in Porphyromonas gingivalis-associated periodontal disease. This study determined the influence of NHP sera on (i) the direct killing of P. gingivalis, (ii) P. gingivalis-induced superoxide anion (O2-) release from human polymorphonuclear leukocytes (PMNs), and (iii) the ability of PMNs to bind and phagocytize P. gingivalis. Three types of NHP sera were utilized: (i) normal or baseline sera; (ii) sera obtained after ligature-induced periodontitis; and (iii) sera obtained following active immunization with formalinized P. gingivalis. All assays were performed with or without the addition of human complement. Significantly more (P < 0.01) direct killing of P. gingivalis occurred with immunized sera and complement than with any of the other treatments. The sera from ligature-induced periodontitis NHPs had significantly less (P < 0.03) killing capacity than the baseline sera, which contained natural antibody produced to P. gingivalis colonization. Sera from immunized NHPs were used to opsonize P. gingivalis and caused significantly greater (P < 0.01) levels of O2- release from PMNs. Finally, the sera from immunized NHPs significantly enhanced (P < 0.009) the uptake of P. gingivalis by PMNs, although binding of the bacteria to PMNs was similar among all three serum types. Active immunization of NHPs with P. gingivalis elicited a functional antibody that enhanced direct killing, positively influenced the activation of PMNs, and enhanced the ability of PMNs to phagocytize P. gingivalis. Moreover, antibody produced as a sequela of progressing periodontitis appeared to lack these functions. A wide variability in functional capacity of the sera from individual NHPs, which may contribute to an individual's susceptibility to P. gingivalis-induced disease, was noted. This variability suggested that results from functional tests of serum antibody may aid in predicting host susceptibility to disease and response to therapy.     

Effect of aging on BCG immunostimulation of Porphyromonas gingivalis infection in mice

The need for population care increases with the age of the population. Pneumonia is the fourth leading cause of death in Japan, yet the risk of pneumonia could be reduced by eliminating opportunistic infection sources such as oral bacteria (e.g. Porphyromonas gingivalis). Previously, we reported removal of P. gingivalis by macrophages during the early stages of cellular immunity, although neither neutrophils nor antibodies participated in the antimicrobial activity. BCG is a live vaccine against tuberculosis, and is thought to maintain cellular immunity as the antigen remains in vivo for longer periods. In this experiment, we attempted to clarify the relationship between aging and the elimination of P. gingivalis by examining the protective capacity of BCG against P. gingivalis infection in mice of various ages. In young mice, the reduction in numbers of P. gingivalis was accompanied by increased IFN-γ and IL-12 levels, and nitric oxide was continuously produced. The augmentation of bactericidal activity, namely the effects of the vaccine, was clear in young mice, but weaker in older mice. Activation of cellular immunity was not observed in older mice, even when boosters were administered.     

Porphyromonas gingivalis infection exacerbates the onset of rheumatoid arthritis in SKG mice

Epidemiological studies have linked periodontitis to rheumatoid arthritis (RA). Porphyromonas gingivalis (Pg) was reported recently to produce citrullinated protein (CP) and increase anti‐cyclic CP antibody (ACPA), both of which have been identified as causative factors of RA. In the present study, we determined the effects of Pg infection on the exacerbation of RA in a mouse model. RA model mice (SKG mice) were established by an intraperitoneal (i.p.) injection of laminarin (LA). Mice were divided into six groups, Ctrl (PBS injection), LA (LA injection), Pg/LA (Pg + LA injection), Pg (Pg injection), Ec/LA (Escherichia coli and LA injection) and Ec (E. coli injection). In order to evaluate RA, joint swelling by the arthritis score, bone morphology by microcomputed tomography (microCT), haematoxylin and eosin staining, ACPA, matrix metalloproteinase‐3 (MMP‐3) and cytokine level in serum by enzyme‐linked immunosorbent assay were determined. Osteoclast differentiation from bone marrow mononuclear cells (BMCs) was examined to clarify the underlying mechanisms of RA. The presence of Pg and CP in joint tissue was also investigated. The arthritis score was threefold higher in the Pg/LA group than in the LA group. Severe bone destruction was observed in joint tissue of the Pg/LA group. A microCT analysis of the Pg/LA group revealed a decrease in bone density. ACPA, MMP‐3, interleukin (IL)‐2, IL‐6, CXCL1 and macrophage inflammatory protein (MIP)‐1α levels from the Pg/LA group were the highest. The osteoclastogenesis of BMCs was enhanced in the Pg/LA group. Furthermore, large amounts of Pg components and CP were detected in the Pg/LA group. In conclusion, Pg infection has the potential to exacerbate RA.     

Age and prevalence of Porphyromonas gingivalis in children.

The acquisition of Porphyromonas gingivalis was examined in a cross-sectional study of 198 subjects from 0 to 18 years of age using a PCR-based assay. P. gingivalis was detected in the oral cavities of 37% of subjects and at similar frequencies among subjects of all ages. These data indicate that P. gingivalis may be acquired in the first days of life.     

Interactions of Porphyromonas gingivalis with epithelial cells.

The invasion of gingival epithelial cells by certain pathogenic periodontal bacteria may account for their presence within diseased gingival tissue. To dissect the initial steps of a potential invasion pathway for the periodontal pathogen Porphyromonas gingivalis, laboratory and clinical bacterial isolates were tested for their interactions with a human oral epithelial cell line (KB). Several P. gingivalis strains immobilized on filters could bind oral epithelial cells. Quantitative adherence assays supported these results. The invasion of epithelial cells by P. gingivalis 33277 was measured by assay and confirmed by transmission electron microscopy. These preliminary results demonstrate that certain P. gingivalis strains are capable of internalization by human oral epithelial cells in vitro.