Hemagglutinin/Adhesin domains of Porphyromonas gingivalis play key roles in coaggregation with Treponema denticola

Porphyromonas gingivalis and Treponema denticola are major pathogens of periodontal disease. Coaggregation between microorganisms plays a key role in the colonization of the gingival crevice and the organization of periodontopathic biofilms. We investigated the involvement of surface ligands of P. gingivalis in coaggregation. Two triple mutants of P. gingivalis lacking Arg-gingipain A (RgpA), Lys-gingipain (Kgp) and Hemagglutinin A (HagA) or RgpA, Arg-gingipain B (RgpB) and Kgp showed significantly decreased coaggregation with T. denticola, whereas coaggregation with a major fimbriae (FimA)-deficient mutant was the same as that with the P. gingivalis wild-type parent strain. rgpA, kgp and hagA code for proteins that contain 44 kDa Hgp44 adhesin domains. The coaggregation activity of an rgpA kgp mutant was significantly higher than that of the rgpA kgp hagA mutant. Furthermore, anti-Hgp44 immunoglobulin G reduced coaggregation between P. gingivalis wild type and T. denticola. Treponema denticola sonicates adhered to recombinant Rgp domains. Coaggregation following co-culture of the rgpA kgp hagA mutant expressing the RgpB protease with the rgpA rgpB kgp mutant expressing the unprocessed HagA protein was enhanced compared with that of each triple mutant with T. denticola. These results indicate that the processed P. gingivalis surface Hgp44 domains are key adhesion factors for coaggregation with T. denticola.     

Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin

Treponema denticola has been reported to coaggregate with Porphyromonas gingivalis and localize closely together in matured subgingival plaque. In this study of the interaction of T. denticola with P. gingivalis, the P. gingivalis fimbria-binding protein of T. denticola was identified by two-dimensional electrophoresis followed by a ligand overlay assay with P. gingivalis fimbriae, and was determined to be dentilisin, a chymotrypsin-like proteinase of T. denticola. The binding was further demonstrated with a ligand overlay assay using an isolated GST fusion dentilisin construct. Our results suggest that P. gingivalis fimbriae and T. denticola dentilisin are implicated in the coaggregation of these bacteria.     

Differential proteomic analysis of a polymicrobial biofilm

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis. The aim of this study was to culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions. In a flow cell all three species attached and grew as a biofilm; however, after 90 h of culture P. gingivalis and T. denticola were closely associated and dominated the polymicrobial biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H(2)(16)O/H(2)(18)O labeling. From two replicates, 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to large increases in the abundance of HusA and HusB in the polymicrobial biofilm while HmuY and other iron/haem transport systems decreased. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These data indicate an intimate association between P. gingivalis and T. denticola in a biofilm that may play a role in disease pathogenesis.     

Fimbria-mediated coaggregation between human oral anaerobes Treponema medium and Porphyromonas gingivalis.

Bacterial binding phenomena among different bacterial genera or species play an important role in bacterial colonization in a mixed microbiota such as in the human oral cavity. The coaggregation reaction between two gram-negative anaerobes, Treponema medium and Porphyromonas gingivalis, was characterized using fimbria-deficient mutants of P. gingivalis and specific antisera against purified fimbriae and bacterial whole cells. T. medium ATCC 700273 strongly coaggregated with fimbriate P. gingivalis strains ATCC 33277 and 381, but not with afimbriate strains including transposon-induced fimbria-deficient mutants and KDP98 as a fimA-disrupted mutant of P. gingivalis ATCC 33277. In the P. gingivalis-T. medium coaggregation assay, the presence of rabbit antiserum against the purified fimbriae or the whole cells of P. gingivalis ATCC 33277 produced different "aggregates" consisting predominantly of P. gingivalis cells with few spirochetes, but both preimmune serum and the antiserum against the afimbriate KDP98 cells did not inhibit the coaggregation reaction. Heated P. gingivalis cells lost their ability to bind both heated and unheated T. medium cells. This T. medium-P. gingivalis coaggregation reaction was inhibited by a cysteine proteinase inhibitor, leupeptin, and also by arginine and lysine, but not by EDTA or sugars including lactose. A binding assay on nitrocellulose membranes and immunoelectron microscopy demonstrated that a heat-stable 37 kDa surface protein on the T. medium cell attached to the P. gingivalis fimbriae.     

Coaggregation of Porphyromonas gingivalis and Prevotella intermedia.

Porphyromonas gingivalis cells coaggregated with Prevotella intermedia cells. The coaggregation was inhibited with L-arginine, L-lysine, Nalpha-p-tosyl-L-lysine chloromethyl ketone, trypsin inhibitor, and leupeptin. Heat- and proteinase K-treated P. gingivalis cells showed no coaggregation with P. intermedia cells, whereas heat and proteinase K treatments of P. intermedia cells did not affect the coaggregation. The vesicles from P. gingivalis culture supernatant aggregated with P. intermedia cells, and this aggregation was also inhibited by addition of L-arginine or L-lysine and by heat treatment of the vesicles. The rgpA rgpB, rgpA kgp, rgpA rgpB kgp, and rgpA kgp hagA mutants of P. gingivalis did not coaggregate with P. intermedia. On the other hand, the fimA mutant lacking the FimA fimbriae showed coaggregation with P. intermedia as well as the wild type parent. These results strongly imply that a heat-labile and proteinous factor on the cell surface of P gingivalis, most likely the gingipain-adhesin complex, is involved in coaggregation of P. gingivalis and P. intermedia.     

Localization of the Fusobacterium nucleatum T18 adhesin activity mediating coaggregation with Porphyromonas gingivalis T22.

Adherence of pathogenic bacteria is often an essential first step in the infectious process. The ability of bacteria to adhere to one another, or to coaggregate, may be an important factor in their ability to colonize and function as pathogens in the periodontal pocket. Previously, a strong and specific coaggregation was demonstrated between two putative periodontal pathogens, Fusobacterium nucleatum and Porphyromonas gingivalis. The interaction appeared to be mediated by a protein adhesin on the F. nucleatum cells and a carbohydrate receptor on the P. gingivalis cells. In this investigation, we have localized the adhesin activity of F. nucleatum T18 to the outer membrane on the basis of the ability of F. nucleatum T18 vesicles to coaggregate with whole cells of P. gingivalis T22 and the ability of the outer membrane fraction of F. nucleatum T18 to inhibit coaggregation between whole cells of F. nucleatum T18 and P. gingivalis T22. Proteolytic pretreatment of the F. nucleatum T18 outer membrane fraction resulted in a loss of coaggregation inhibition, confirming the proteinaceous nature of the adhesin. The F. nucleatum T18 outer membrane fraction was found to be enriched for several proteins, including a 42-kDa major outer membrane protein which appeared to be exposed on the bacterial cell surface. Fab fragments prepared from antiserum raised to the 42-kDa outer membrane protein were found to partially but specifically block coaggregation. These data support the conclusion that the 42-kDa major outer membrane protein of F. nucleatum T18 plays a role in mediating coaggregation with P. gingivalis T22.     

Mixed infections with Porphyromonas gingivalis and Treponema denticola cause excessive inflammatory responses in a mouse pneumonia model compared with monoinfections

Periodontopathic anaerobes such as Porphyromonas gingivalis are frequently found in aspiration pneumonia and lung abscesses. However, defense mechanisms and responses to these bacterial infections in the lung in vivo remain poorly understood. The coexistence of P. gingivalis with Treponema denticola has been found at higher levels and proportions in periodontally diseased sites. We hypothesized that mixed infections with P. gingivalis and T. denticola can cause severe respiratory disease. In the present study, inflammatory responses to mono- and mixed inoculations with P. gingivalis and T. denticola in the bronchoalveolar lavage (BAL) fluid were investigated. Acute pneumonia and lung abscesses in mice with the mixed infection resulted in a 40% mortality rate within 72 h, compared with only 10% mortality for the respective monoinfections. Pulmonary clearance of P. gingivalis was delayed in the mice with mixed infections with P. gingivalis and T. denticola. Tumor necrosis factor alpha (TNFalpha) interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) levels from BAL fluid of mice with mixed infections at 24 h after inoculation were significantly higher than those after P. gingivalis monoinfection (TNFalpha: P < 0.05, Il-1beta: P < 0.001, IL-6: P < 0.05). The chemokine KC level from BAL fluid of mice at 48 h (P < 0.05) and 72 h after mixed infection was also significantly increased when compared with that after P. gingivalis monoinfection (P < 0.001). The present study demonstrates that a mixed infection of P. gingivalis with T. denticola in mouse causes a marked bronchopneumonia and lung abscess in the mouse model.     

A universal stress protein of Porphyromonas gingivalis is involved in stress responses and biofilm formation

Porphyromonas gingivalis is recognized as one of the major periodontal pathogens in subgingival plaque, which is implicated in the progression of chronic periodontal disease. We analyzed the role of upsA in P. gingivalis 381 and its uspA-deficient mutant CW301 under various stress conditions. In general, the uspA mutant was less tolerant to a variety of environmental stresses relative to the parental strain. In addition, gene expression of uspA is upregulated during biofilm formation. Biofilm formation of the uspA mutant was also less than that of strain 381. In conclusion, the uspA gene affecting the stress responses of P. gingivalis is required for optimal biofilm formation.     

Synergy in biofilm formation between Fusobacterium nucleatum and Prevotella species

The formation of biofilm by anaerobic, Gram-negative bacteria in the subgingival crevice plays an important role in the development of chronic periodontitis. The aim of this study was to characterize the role of coaggregation between Fusobacterium nucleatum and Prevotella species in biofilm formation. Coaggregation between F. nucleatum and Prevotella species was determined by visual assay. Effect of co-culture of the species on biofilm formation was assessed by crystal violet staining. Effect of soluble factor on biofilm formation was also examined using culture supernatant and two-compartment co-culture separated by a porous membrane. Production of autoinducer-2 (AI-2) by the organisms was evaluated using Vibrio harveyi BB170. Cells of all F. nucleatum strains coaggregated with Prevotella intermedia or Prevotella nigrescens with a score of 1-4. Addition of ethylenediamine tetraacetic acid or l-lysine inhibited coaggregation. Coaggregation disappeared after heating of P. intermedia or P. nigrescens cells, or Proteinase K treatment of P. nigrescens cells. Co-culture of F. nucleatum ATCC 25586 with P. intermedia or P. nigrescens strains increased biofilm formation compared with single culture (p < 0.01); co-culture with culture supernatant of these strains, however, did not enhance biofilm formation by F. nucleatum. Production of AI-2 in Prevotella species was not related to enhancement of biofilm formation by F. nucleatum. These findings indicate that physical contact by coaggregation of F. nucleatum strains with P. intermedia or P. nigrescens plays a key role in the formation of biofilm by these strains.     

Synergistic effect on biofilm formation between Fusobacterium nucleatum and Capnocytophaga ochracea

The formation of dental plaque biofilm by specific Gram-negative rods and spirochetes plays an important role in the development of periodontal disease. The aim of this study was to characterize biofilm formation by Fusobacterium nucleatum and Capnocytophaga ochracea. Coaggregation between F. nucleatum and Capnocytophaga species was determined by visual assay. Biofilm formation was assessed by crystal violet staining. Enhancement of biofilm formation by F. nucleatum via soluble factor of C. ochracea was evaluated by addition of culture supernatant and a two-compartment separated co-culture system. Production of autoinducer-2 by the tested organisms was evaluated using Vibrio harveyi BB170. F. nucleatum strains coaggregated with C. ochracea ATCC 33596 or ONO-26 strains. Ethylenediamine tetraacetic acid, N-acetyl-d-galactosamine or lysine inhibited coaggregation. Heating or proteinase K treatment of F. nucleatum cells affected coaggregation, whereas the same treatment of C. ochracea cells did not. Co-culture of F. nucleatum with C. ochracea in the same well resulted in a statistically significant increase in biofilm formation. Enhancement of F. nucleatum biofilm formation by a soluble component of C. ochracea was observed using the two-compartment co-culture system (P < 0.05) and confirmed by addition of culture supernatant of C. ochracea (P < 0.01). The present findings indicate that induction of coaggregation and intracellular interaction by release of a diffusible molecule by C. ochracea play a significant role in the formation of biofilm by F. nucleatum and C. ochracea.     

Porphyromonas gingivalis and Treponema denticola Synergistic Polymicrobial Biofilm Development

Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola.     

LuxS-based signaling affects Streptococcus mutans biofilm formation

Streptococcus mutans is implicated as a major etiological agent in human dental caries, and one of the important virulence properties of this organism is its ability to form biofilms (dental plaque) on tooth surfaces. We examined the role of autoinducer-2 (AI-2) on S. mutans biofilm formation by constructing a GS-5 luxS-null mutant. Biofilm formation by the luxS mutant in 0.5% sucrose defined medium was found to be markedly attenuated compared to the wild type. Scanning electron microscopy also revealed that biofilms of the luxS mutant formed larger clumps in sucrose medium compared to the parental strain. Therefore, the expression of glucosyltransferase genes was examined and the gtfB and gtfC genes, but not the gtfD gene, in the luxS mutant were upregulated in the mid-log growth phase. Furthermore, we developed a novel two-compartment system to monitor AI-2 production by oral streptococci and periodontopathic bacteria. The biofilm defect of the luxS mutant was complemented by strains of S. gordonii, S. sobrinus, and S. anginosus; however, it was not complemented by S. oralis, S. salivarius, or S. sanguinis. Biofilm formation by the luxS mutant was also complemented by Porphyromonas gingivalis 381 and Actinobacillus actinomycetemcomitans Y4 but not by a P. gingivalis luxS mutant. These results suggest that the regulation of the glucosyltransferase genes required for sucrose-dependent biofilm formation is regulated by AI-2. Furthermore, these results provide further confirmation of previous proposals that quorum sensing via AI-2 may play a significant role in oral biofilm formation.     

Coaggregation of Porphyromonas gingivalis and Fusobacterium nucleatum PK 1594 is mediated by capsular polysaccharide and lipopolysaccharide

Previous reports have shown that coaggregation between Porphyromonas gingivalis and Fusobacterium nucleatum, two important periodontopathogens, is mediated by a galactoside on the surface of P. gingivalis and a lectin on F. nucleatum. In the present study, purified capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of P. gingivalis PK 1924 (serotype K5) were found to be able to bind to F. nucleatum cells and to inhibit binding of F. nucleatum to P. gingivalis serotype K5. Sugar binding studies showed that the requirements for binding of P. gingivalis serotype K5 CPS and LPS to the F. nucleatum lectin are: the presence of a metal divalent ion, an axial free hydroxyl group at position 4 and free equatorial hydroxyl groups at position 3 and 6 of d-galactose. These data suggest that P. gingivalis serotype K5- CPS and LPS act as receptors mediating coaggregation between P. gingivalis and fusobacteria.     

Enhanced biofilm formation and loss of capsule synthesis: deletion of a putative glycosyltransferase in Porphyromonas gingivalis

Periodontitis is a biofilm-mediated disease. Porphyromonas gingivalis is an obligate anaerobe consistently associated with severe manifestations of this disease. As an opportunistic pathogen, the ability to proliferate within and disseminate from subgingival biofilm (plaque) is central to its virulence. Here, we report the isolation of a P. gingivalis transposon insertion mutant altered in biofilm development and the reconstruction and characterization of this mutation in three different wild-type strains. The mutation responsible for the altered biofilm phenotype was in a gene with high sequence similarity ( approximately 61%) to a glycosyltransferase gene. The gene is located in a region of the chromosome that includes up to 16 genes predicted to be involved in the synthesis and transport of capsular polysaccharide. The phenotype of the reconstructed mutation in all three wild-type backgrounds is that of enhanced biofilm formation. In addition, in strain W83, a strain that is encapsulated, the glycosyltransferase mutation resulted in a loss of capsule. Further experiments showed that the W83 mutant strain was more hydrophobic and exhibited increased auto-aggregation. Our results indicate that we have identified a gene involved in capsular-polysaccharide synthesis in P. gingivalis and that the production of capsule prevented attachment and the initiation of in vitro biofilm formation on polystyrene microtiter plates.     

Inflammatory responses of a macrophage/epithelial cell co-culture model to mono and mixed infections with Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia

Accumulated evidence points to Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia as three major etiologic agents of chronic periodontitis. Epithelial cells and macrophages play a major role in the host response to periodontopathogens, and the secretion of inflammatory mediators and matrix metalloproteinases (MMPs) by these host cells is believed to contribute to periodontal tissue destruction. The aim of this study was to investigate the inflammatory response of a macrophage/epithelial cell co-culture model following mono or mixed infections with the above three periodontopathogens. An in vitro co-culture model composed of epithelial-like transformed cells (HeLa cell line) and macrophage-like cells (phorbol myristic acid-differentiated U937 monocytic cell line) was challenged with whole cells or lipopolysaccharides (LPS) of P. gingivalis, T. denticola, and T. forsythia, individually and in combination. Following stimulation, the production of interleukin-1 beta (IL-1beta), IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), regulated on activation normal T cell expressed and secreted (RANTES), prostaglandin E2 (PGE2), and MMP-9 were quantified by enzyme-linked immunoassays. We observed that mono or mixed infections of the co-culture model induced the secretion of IL-1beta, IL-6, IL-8, PGE2, and MMP-9. P. gingivalis and T. forsythia induced an increase in RANTES secretion, whereas T. denticola alone or in combination resulted in a significant decrease in RANTES levels. All LPS challenges induced an increase in chemokine, MMP-9, and PGE2 production. No synergistic effect on the production of cytokines, chemokines, PGE2, and MMP-9 was observed for any of the bacterial or LPS mixtures tested. This study supports the view that P. gingivalis, T. denticola, and T. forsythia may induce high levels of pro-inflammatory mediators and MMP-9 in periodontal lesions, thus contributing to the progression of periodontitis.     

Salivary detection of periodontopathic bacteria in periodontally healthy children

BACKGROUND: Salivary occurrence of periodontopathic bacteria is of interest especially in children as a risk indicator for the transmission, development and control of periodontal disease. We assessed the prevalence of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, Prevotella nigrescens and Treponema denticola as microbial complexes in the saliva of children with mixed dentition and healthy gingiva. MATERIALS AND METHODS: Paraffin-stimulated saliva samples were collected from 41 children (22 boys and 19 girls), aged 6-13 years old. Gingival health was determined during the initial screening exam. The test bacteria were identified using a 16S rRNA-based PCR analysis. RESULTS: P. nigrescens was the most frequent species (80%), followed by T. denticola (32%), A. actinomycetemcomitans (24%) and P. gingivalis (12%). P. intermedia and T. forsythia were not detected. P. nigrescens was also common species in combinations. Paired and triple bacterial combinations were found in 24% and 20% of all children, respectively. There was no positive association between bacterial combinations in colonization and subject's gender (P>0.05, Fisher exact test). CONCLUSION: The salivary presence of P. nigrescens, T. denticola, A. actinomycetemcomitans and P. gingivalis but not P. intermedia and T. forsythia can occur in childhood without clinical signs of gingival disease. Thus, the possible risk of bacterial transmissions through saliva and, the need to screen for periodontal pathogens should be considered before mixed dentition.     

Molecular approaches to the identification and treatment monitoring of periodontal pathogens

Two different PCR-based molecular approaches, a commercial kit for detection of A. actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus and T. denticola (Amplimedical "Paradonthosis") and a home-made multiplex PCR for A. actinomycetemcomitans, P. gingivalis and B. forsythus were compared for monitoring the efficacy of different dental treatments on localized persistent periodontal pockets. 44 sites were randomized in two treatment groups: mechanical treatment (22 control sites) and in conjunction with the application of tetracycline fibres (22 experimental sites). 40/44 sites were found positive with both tests for A. actinomycetemcomitans, P. gingivalis and B. forsythus pretheraphy. P. intermedia was detected alone in only three sites during the follow-up, while T. denticola. was always associated with the other pathogens. 20 sites were positive in conventional cultures for one to three of the pathogens. PCR-based approaches provided a sensitive and reliable method for identification and monitoring treatment of periodontal pathogens.     

Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae

Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD+ to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization.     

LuxS-based signaling in Streptococcus gordonii: autoinducer 2 controls carbohydrate metabolism and biofilm formation with Porphyromonas gingivalis

Communication based on autoinducer 2 (AI-2) is widespread among gram-negative and gram-positive bacteria, and the AI-2 pathway can control the expression of genes involved in a variety of metabolic pathways and pathogenic mechanisms. In the present study, we identified luxS, a gene responsible for the synthesis of AI-2, in Streptococcus gordonii, a major component of the dental plaque biofilm. S. gordonii conditioned medium induced bioluminescence in an AI-2 reporter strain of Vibrio harveyi. An isogenic mutant of S. gordonii, generated by insertional inactivation of the luxS gene, was unaffected in growth and in its ability to form biofilms on polystyrene surfaces. In contrast, the mutant strain failed to induce bioluminescence in V. harveyi and was unable to form a mixed species biofilm with a LuxS-null strain of the periodontal pathogen Porphyromonas gingivalis. Complementation of the luxS mutation in S. gordonii restored normal biofilm formation with the luxS-deficient P. gingivalis. Differential display PCR demonstrated that the inactivation of S. gordonii luxS downregulated the expression of a number of genes, including gtfG, encoding glucosyltransferase; fruA, encoding extracellular exo-beta-D-fructosidase; and lacD encoding tagatose 1,6-diphosphate aldolase. However, S. gordonii cell surface expression of SspA and SspB proteins, previously implicated in mediating adhesion between S. gordonii and P. gingivalis, was unaffected by inactivation of luxS. The results suggest that S. gordonii produces an AI-2-like signaling molecule that regulates aspects of carbohydrate metabolism in the organism. Furthermore, LuxS-dependent intercellular communication is essential for biofilm formation between nongrowing cells of P. gingivalis and S. gordonii.     

Tobacco smoke augments Porphyromonas gingivalis-Streptococcus gordonii biofilm formation

Smoking is responsible for the majority of periodontitis cases in the US and smokers are more susceptible than non-smokers to infection by the periodontal pathogen Porphyromonas gingivalis. P. gingivalis colonization of the oral cavity is dependent upon its interaction with other plaque bacteria, including Streptococcus gordonii. Microarray analysis suggested that exposure of P. gingivalis to cigarette smoke extract (CSE) increased the expression of the major fimbrial antigen (FimA), but not the minor fimbrial antigen (Mfa1). Therefore, we hypothesized that CSE promotes P. gingivalis-S. gordonii biofilm formation in a FimA-dependent manner. FimA total protein and cell surface expression were increased upon exposure to CSE whereas Mfa1 was unaffected. CSE exposure did not induce P. gingivalis auto-aggregation but did promote dual species biofilm formation, monitored by microcolony numbers and depth (both, p<0.05). Interestingly, P. gingivalis biofilms grown in the presence of CSE exhibited a lower pro-inflammatory capacity (TNF-α, IL-6) than control biofilms (both, p<0.01). CSE-exposed P. gingivalis bound more strongly to immobilized rGAPDH, the cognate FimA ligand on S. gordonii, than control biofilms (p<0.001) and did so in a dose-dependent manner. Nevertheless, a peptide representing the Mfa1 binding site on S. gordonii, SspB, completely inhibited dual species biofilm formation. Thus, CSE likely augments P. gingivalis biofilm formation by increasing FimA avidity which, in turn, supports initial interspecies interactions and promotes subsequent high affinity Mfa1-SspB interactions driving biofilm growth. CSE induction of P. gingivalis biofilms of limited pro-inflammatory potential may explain the increased persistence of this pathogen in smokers. These findings may also be relevant to other biofilm-induced infectious diseases and conditions.