Proteases of an early colonizer can hinder Streptococcus mutans colonization in vitro.

Streptococcus mutans is the primary cariogen that produces several virulence factors that are modulated by a competence-stimulating peptide (CSP) signaling system. In this study, we sought to determine if proteases produced by early dental plaque colonizers such as Streptococcus gordonii interfere with the subsequent colonization of S. mutans BM71 on the existing streptococcal biofilms. We demonstrated that S. mutans BM71 colonized much less efficiently in vitro on streptococcal biofilms than on Actinomyces naeslundii biofilms. Several oral streptococci, relative to A. naeslundii, produced proteases that inactivated the S. mutans CSP. We further demonstrated that cell protein extracts from S. gordonii, but not from A. naeslundii, interfered with S. mutans BM71 colonization. In addition, S. mutans BM71 colonized more efficiently on the sgc protease knockout mutant of S. gordonii than on the parent biofilms. In conclusion, proteases of early colonizers can interfere with subsequent colonization by S. mutans in vitro.

Periodontal pathogens interfere with quorum-sensing-dependent virulence properties in Streptococcus mutans.

BACKGROUND AND OBJECTIVE: The mechanism by which periodontal pathogens dominate at disease sites is not yet understood. One possibility is that these late colonizers antagonize the quorum-sensing systems of early colonizers and render those early colonizers less resistant to environmental factors. In this study, we utilized Streptococcus mutans, a well-documented oral Streptococcus with many quorum-sensing-dependent properties, as an example of an earlier colonizer antagonized by periodontal pathogens. MATERIAL AND METHODS: In this study, S. mutans NG8 and S. mutans LT11 were used in experiments assessing transformation, and S. mutans BM71 was used in experiments investigating bacteriocin production. The effects of the periodontal pathogens Porphyromonas gingivalis and Treponema denticola on these competence-stimulating peptide-dependent properties were evaluated in mixed-broth assays. RESULTS: Both P. gingivalis (either live bacteria or membrane vesicles) and T. denticola antagonized transformation in S. mutans NG8 and LT11. The production of bacteriocin by S. mutans BM71 was also inhibited by P. gingivalis and T. denticola. Boiling of these late colonizers before addition to the broth cultures abolished their ability to inhibit S. mutans transformation and bacteriocin production. P. gingivalis and T. denticola inactivated S. mutans exogenous competence-stimulating peptide, whereas the boiled bacteria did not. CONCLUSIONS: This study demonstrated that periodontal pathogens antagonize S. mutans quorum-sensing properties. This may render S. mutans less virulent and less resistant to environmental antibacterial factors.

Stimulation of Fusobacterium nucleatum biofilm formation by Porphyromonas gingivalis.

BACKGROUND/AIMS: Bacterial infection is a major cause of periapical periodontitis. Eradication of these microorganisms from apical lesions is essential to the success of endodontic treatment. The aim of this study was to clarify the molecular interaction between Fusobacterium nucleatum, Porphyromonas gingivalis and other microorganisms associated with periapical periodontitis. METHODS: Microorganisms isolated from periapical lesions were inoculated into type-I collagen-coated polystyrene microtiter plates and maintained at 37 degrees C under anaerobic conditions for 2 days, after which, the quantity of organized biofilm on the plates was evaluated by crystal violet staining. Growth enhancement via soluble factor was evaluated by separated coculture using a 0.4-mum membrane filter. RESULTS: F. nucleatum exhibited strong adherence to type-I collagen-coated polystyrene microplates. Biofilm formation by F. nucleatum was significantly enhanced by P. gingivalis. It was complemented by compartmentalized coculture with P. gingivalis. Enhancement of biofilm formation by P. gingivalis was only slightly reduced by inactivation of its autoinducer-2-producing gene luxS. CONCLUSION: The results suggest that P. gingivalis enhances biofilm formation by F. nucleatum by releasing diffusible signaling molecules other than autoinducer-2.

Immunization by Arg-gingipain A DNA vaccine protects mice against an invasive Porphyromonas gingivalis infection through regulation of interferon-gamma production.

We previously demonstrated that a Porphyromonas gingivalis rgpA DNA vaccine induced protective immune responses against P. gingivalis infection in mice. In the present study, reduction in lethality against infection by lethal doses of P. gingivalis was observed in the rgpA DNA vaccine-immunized mice. Cytokine levels in the mouse model with nonlethal doses of infection by P. gingivalis were evaluated to analyze the mechanism of protection by immunization with the rgpA DNA vaccine. After nonlethal challenge with invasive P. gingivalis W50, production of interleukin (IL)-2, IL-4, IL-5 and IL-12 was elevated; however, interferon (IFN)-gamma was lower in the serum of the DNA vaccine-immunized mice than in the serum of nonimmunized mice. The regulation of IFN-gamma production elicited by immunization with the rgpA DNA vaccine may play a significant role in protection against P. gingivalis infection in mice.

Synergy between Tannerella forsythia and Fusobacterium nucleatum in biofilm formation.

During dental plaque formation, the interaction of different organisms is important in the development of complex communities. Fusobacterium nucleatum is considered a 'bridge-organism' that facilitates colonization of other bacteria by coaggregation-mediated mechanisms and possibly by making the environment conducive for oxygen intolerant anaerobes. These studies were carried out to determine whether coaggregation between F. nucleatum and Tannerella forsythia is important in the formation of mixed species biofilms. Further, the role of BspA protein, a surface adhesin of T. forsythia, in coaggregation and biofilm formation was investigated. The results showed the development of synergistic mixed biofilms of F. nucleatum and T. forsythia when these bacteria were cocultured. The BspA protein was not involved in biofilm formation. Though BspA plays a role in coaggregation with F. nucleatum, presumably other adhesins are also involved. The synergistic biofilm formation between the two species was dependent on cell-cell contact and soluble components of the bacteria were not required. This study demonstrates that there is a positive synergy between F. nucleatum and T. forsythia in the development of mixed biofilms and that the cell-cell interaction is essential for this phenomenon.

Genetic exchange between Treponema denticola and Streptococcus gordonii in biofilms.

Some gram-positive oral bacteria such as Streptococcus gordonii are naturally transformable. We investigated the possibility of genetic exchange among oral bacteria using an erythromycin-resistant (Erm(r)) shuttle plasmid, pKMR4PE, as an indicator. S. gordonii Challis cells were co-cultivated with purified pKMR4PE or with Trepomena denticola harboring pKMR4PE either in broth or in artificial biofilms. Transformation of S. gordonii occurred both in broth and in biofilms with pKMR4PE or T. denticola harboring pKMR4PE as donor sources. These results provide direct experimental evidence that gene transfer can occur from T. denticola to S. gordonii.

Virulence properties of oral bacteria: impact of molecular biology.

Dental caries and periodontitis, although generally not life threatening, are nevertheless of significant importance. An understanding of the molecular nature of these diseases could aid the development of novel methods of prevention and control, and increase our knowledge of their etiology. The identification of virulence factors in oral bacteria could lead to the development of vaccines directed against these organisms, the design of inhibitors of biofilm formation, and the development of replacement therapy strategies.

Development of a gene inactivation system for Bacteroides forsythus: construction and characterization of a BspA mutant.

Bacteroides forsythus is a gram-negative anaerobic bacterium associated with periodontitis. The bspA gene encoding a cell surface associated leucine-rich repeat protein (BspA) involved in adhesion to fibronectin and fibrinogen was recently cloned from this bacterium in our laboratory. We now describe the construction of a BspA-defective mutant of B. forsythus. This is the first report describing the generation of a specific gene knockout mutant of B. forsythus, and this procedure should be useful in establishing the identity of virulence-associated factors in these organisms.

Effects of the oral spirochete Treponema denticola on interleukin-8 expression from epithelial cells.

This communication demonstrates that the interaction of the oral spirochete Treponema denticola 35405 with KB epithelial cells does not lead to the induction of interleukin-8 production as occurs with a variety of other bacteria. Utilizing the dentilisin protease mutant K1 of T. denticola, this property was demonstrated to be primarily a function of the expression of the protease by strain 35405.

Interleukin-8 and intercellular adhesion molecule 1 regulation in oral epithelial cells by selected periodontal bacteria: multiple effects of Porphyromonas gingivalis via antagonistic mechanisms.

Interaction of bacteria with mucosal surfaces can modulate the production of proinflammatory cytokines and adhesion molecules produced by epithelial cells. Previously, we showed that expression of interleukin-8 (IL-8) and intercellular adhesion molecule 1 (ICAM-1) by gingival epithelial cells increases following interaction with several putative periodontal pathogens. In contrast, expression of IL-8 and ICAM-1 is reduced after Porphyromonas gingivalis ATCC 33277 challenge. In the present study, we investigated the mechanisms that govern the regulation of these two molecules in bacterially infected gingival epithelial cells. Experimental approaches included bacterial stimulation of gingival epithelial cells by either a brief challenge (1.5 to 2 h) or a continuous coculture throughout the incubation period. The kinetics of IL-8 and ICAM-1 expression following brief challenge were such that (i) secretion of IL-8 by gingival epithelial cells reached its peak 2 h following Fusobacterium nucleatum infection whereas it rapidly decreased within 2 h after P. gingivalis infection and remained decreased up to 30 h and (ii) IL-8 and ICAM-1 mRNA levels were up-regulated rapidly 2 to 4 h postinfection and then decreased to basal levels 8 to 20 h after infection with either Actinobacillus actinomycetemcomitans, F. nucleatum, or P. gingivalis. Attenuation of IL-8 secretion was facilitated by adherent P. gingivalis strains. The IL-8 secreted from epithelial cells after F. nucleatum stimulation could be down-regulated by subsequent infection with P. gingivalis or its culture supernatant. Although these results suggested that IL-8 attenuation at the protein level might be associated with P. gingivalis proteases, the Arg- and Lys-gingipain proteases did not appear to be solely responsible for IL-8 attenuation. In addition, while P. gingivalis up-regulated IL-8 mRNA expression, this effect was overridden when the bacteria were continuously cocultured with the epithelial cells. The IL-8 mRNA levels in epithelial cells following sequential challenge with P. gingivalis and F. nucleatum and vice versa were approximately identical and were lower than those following F. nucleatum challenge alone and higher than control levels or those following P. gingivalis challenge alone. Thus, together with the protease effect, P. gingivalis possesses a powerful strategy to ensure the down-regulation of IL-8 and ICAM-1.

Role for periodontal bacteria in cardiovascular diseases.

BACKGROUND: Several epidemiological studies as well as a recent animal model approach have suggested a role for periodontal diseases in the development of cardiovascular disease (CVD). This relationship could be mediated by inflammatory responses induced by periodontal pathogens as well as direct interaction of these organisms with cardiac tissue. METHODS: In order to explore these possibilities, the effects of the periodontal pathogen Porphyromonas gingivalis on cellular events proposed to play a role in CVD were investigated. RESULTS: P. gingivalis, as well as its outer membrane vesicles (OMV), was able to induce foam cell formation (an important characteristic of CVD) in the murine macrophage cell line J774 A.1. This property appears to be mediated by the lipopolysaccharide (LPS) fraction of the cells. Several other oral bacteria were also able to induce foam cell formation. Furthermore, since the rupture of the fibrous cap of plaque appears to be an important factor in acute coronary syndrome, it was demonstrated that P. gingivalis 381 degraded fibrous caps isolated from autopsy samples. In addition, it was observed that strain 381 strongly induced matrix metalloproteinase (MMP)-9 protease activity, implicated in plaque rupture, from the J774 A.1 macrophages. Finally, strain 381 was able to enhance monocyte chemoattractant protein-1 (MCP-1) and NADH oxidase expression from endothelial cells. CONCLUSIONS: Therefore, P. gingivalis exhibits several properties which could play a role in CVD as mediators of LDL oxidation, foam cell formation, and rupture of atherosclerotic plaque.

Polymerase chain reaction for the detection of flaA-1 genes of oral spirochaetes in human advanced periodontal pockets.

A novel group of oral spirochaetes that were specifically labeled by H9-2, a monoclonal antibody against the 37-kDa endoflagellar sheath protein of Treponema pallidum, have earlier been demonstrated in periodontitis-associated plaque samples; two cultivable oral spirochaetes T. vincentii and T. medium displayed cross-reactivity with that monoclonal, but the molecular basis for this cross-reactivity is not yet defined. Here, the flaA-1 genes which encode the 37-kDa sheath protein from T. vincentii and the oral spirochaetes present in samples from advanced periodontitis were examined. A 856 bp fragment of the flaA-1 gene was amplified in T. pallidum and T. vincentii. The same-size polymerase chain reaction fragments were also amplified in two clinical samples from patients with advanced periodontitis but not from samples of healthy plaque. The sequences of the flaA-1 genes of the oral spirochaetes detected in human periodontal plaque were closely similar to those of T. pallidum and T. vincentii, but neither of these two organisms could be detected in these samples using rRNA-specific primers. The identity of the flaA-1 positive spirochaetes associated with periodontitis remains to be determined.

A novel antibacterial agent derived from the C-terminal domain of streptococcus mutans GTP-binding protein.

A decapeptide, P1, homologous to part of the Streptococcus mutans G-protein (SGP) and the Escherichia coli Era protein, was synthesized and examined for growth-inhibitory effects. When P1 10 mg/L was added to E. coli DH5, the viability of the cells was reduced by 13%. Addition of lauric acid enhanced the bactericidal effects of P1 (68% killing in the presence of P1 plus lauric acid). Similar enhancements were observed for mono lauroyl-rac-glycerol (MLG) and sodium dodecyl sulphate (SDS). In cultures treated with both P1 and MLG, there were more elongated cells than in cultures treated with detergent or peptide alone. As with E. coli, the bactericidal effects of P1 on S. mutans were significantly enhanced in the presence of the detergent lauric acid. The combination of the two effectors produced >90% killing of S. mutans. Likewise, the combined action of P1 plus lauric acid reduced the viability of Listeria monocytogenes. P1 did not appear to be toxic to human gingival epithelial cells when added at concentrations < or = 1000 mg/L. Therefore, P1 has properties which could allow it to be used as an antibacterial agent.

Characterization of the relA gene of Porphyromonas gingivalis.

Based upon the nucleotide sequence of the relA gene from Escherichia coli, a gene fragment corresponding to the homologous gene from the pathogenic oral bacterium Porphyromonas gingivalis 381 was isolated by PCR and utilized to construct a relA mutant. The mutant, KS7, was defective in ribosome-mediated ppGpp formation and also in the stringent response.

Protein interactions of SGP, an essential Streptococcus mutans GTPase, revealed by biochemical and yeast two-hybrid system analyses.

SGP, a Streptococcus mutans essential GTPase, plays a role in the stress response of the organism. Recently, we proposed that one of the physiological functions of the SGP is the modulation of the GTP/GDP ratio under different growth conditions. In order to further determine the functions of SGP and its possible interactions with other molecules, we carried out immunoprecipitation, SGP binding, and the yeast two-hybrid system analyses. These approaches suggest that SGP may oligomerize and such interactions could be important for the function of this regulatory protein.

Porphyromonas gingivalis platelet aggregation activity: outer membrane vesicles are potent activators of murine platelets.

Recent evidence has established an association between chronic periodontitis and cardiovascular disease. Periodontitis is a chronic inflammatory disease caused by a small group of gram-negative bacteria, of which Porphyromonas gingivalis is considered an important causative agent. It has been proposed that dental plaque bacteria and their products can disseminate into the bloodstream from the site of infection and promote thromboembolic events associated with atherosclerosis and myocardial infarction. In this regard, Streptococcus sanguis and P. gingivalis have been shown to induce platelet aggregation in vitro. Here we report that P. gingivalis was able to induce platelet aggregation, and that oral strains of Actinobaillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia and Trepenoma denticola failed to aggregate platelets when tested for platelet aggregation activity under similar conditions. Additionally, we show that vesicles (outer membrane evaginations that are shed into the environment by the bacteria) of P. gingivalis are potent inducers of mouse platelet aggregation in vitro. In summary, our data show that i) initial adherence of the bacterium to platelet may be facilitated by P. gingivalis fimbriae and ii) P. gingivalis vesicles possess platelet aggregation-inducing activity.

Stress-induced membrane association of the Streptococcus mutans GTP-binding protein, an essential G protein, and investigation of its physiological role by utilizing an antisense RNA strategy.

SGP (for Streptococcus GTP-binding protein) is a Streptococcus mutans essential GTPase which has significant sequence identity to the previously identified Escherichia coli Era protein and to numerous other prokaryotic GTPase proteins of unknown function. Recent studies in our laboratory have addressed the possible role of SGP in the stress response of the oral pathogen S. mutans. Here we report that during growth in the early stationary phase, and in response to elevated temperatures or acidic pH, the distribution of SGP between the cytoplasm and the membranes of S. mutans cells varies. Immunoblot analysis of soluble and membrane protein fractions collected from the mid-log and early stationary growth phases of bacterial populations grown at normal temperature (37 degrees C) and at the elevated temperature of 43 degrees C, or at acidic pH, demonstrated that the total amount of SGP increased with the age of the bacterial culture, elevated temperature, or acidic pH. Furthermore, it was established that a substantial amount of SGP is associated with the membrane fraction under stress conditions. In order to investigate the physiological role of SGP, we constructed an S. mutans strain capable of chromosomal sgp antisense RNA expression, which interferes with the normal information processing of the sgp gene. Utilizing this strain, we determined conditions whereby the streptococcal cells can be depleted of SGP, thus avoiding the problem of constructing a conditional lethal system. From the results of measurements of the nucleotide pools extracted from the antisense strain and its isogenic counterpart, we propose that one of the physiological roles of SGP is regulation and modulation of the GTP/GDP ratio under different growth conditions. Moreover, we observed that in SGP-depleted cells the levels of glucan-binding protein A (GbpA) substantially increased, suggesting that GbpA may have stress response-related physiological functions. Finally, the potential applications of the antisense RNA approach that we employed are discussed.

Molecular mechanism for the spontaneous generation of pigmentless Porphyromonas gingivalis mutants.

Porphyromonas gingivalis is one of the pathogens associated with periodontal diseases, and its protease activity has been implicated as an important virulence factor. Kgp is the major Lys-gingipain protease of P. gingivalis and appears to be involved not only in enzyme activity but also in hemagglutination and the pigmented phenotype due to heme accumulation and/or hemoglobin binding. However, little information concerning the molecular mechanism for the spontaneous generation of pigmentless P. gingivalis mutants is currently available. In this study, several spontaneous pigmentless mutants of P. gingivalis were isolated and characterized. The results revealed that a portion of the kgp gene had been deleted from the chromosomes of the pigmentless mutants. This deletion appears to result from recombination between the highly homologous DNA sequences encoding the adhesin domains of the tandemly arranged hagA and kgp genes on the chromosomes of P. gingivalis strains.

Role of superoxide dismutase activity in the physiology of Porphyromonas gingivalis.

Porphyromonas gingivalis is a gram-negative, obligate anaerobe strongly associated with chronic adult periodontitis. A previous study has demonstrated that this organism requires superoxide dismutase (SOD) for its modest aerotolerance. In this study, we have constructed a mutant deficient in SOD activity by insertional inactivation as well as a sod::lacZ reporter translational fusion construct to study the regulation of expression of this gene. We have confirmed that SOD is essential for tolerance to atmospheric oxygen but does not appear to be protective against hydrogen peroxide or exogenously generated reactive oxygen species. Furthermore, the sod mutant appeared to be no more sensitive to killing by neutrophils than the parental strain 381. SOD appears to be protective against oxygen-dependent DNA damage as measured by increased mutation to rifampin resistance by the sod mutant. Use of the sod::lacZ construct confirmed that SOD expression is maximal at mid-log phase and is influenced by oxygen, temperature, and pH. However, expression does not appear to be significantly affected by iron depletion, osmolarity, or nutrient depletion. The transcription start site of the sod gene was determined to be 315 bp upstream of the sod start codon and to be within an upstream open reading frame. Our studies demonstrate the essential role that SOD plays in aerotolerance of this organism as well as the selective induction of this enzyme by environmental stimuli.