The AtoC family response regulator upregulates an operon encoding putative outer membrane proteins sorted by type IX secretion system in Porphyromonas gingivalis.

OBJECTIVES: Porphyromonas gingivalis, a keystone periodontopathogen, has multiple two-component systems that are thought to modulate virulence. In this study, we focused on PGN_0775 response regulator (RR), an AtoC homolog, and attempted to identify the target gene that it regulates in P. gingivalis. METHODS: Comparative proteomic analyses comprising two-dimensional electrophoresis and peptide mass fingerprinting were applied to total protein samples from parent (WT) and atoC gene knockout (KO) strains to screen for affected protein spots. Fluctuations in the expression of corresponding genes were further confirmed using relative quantitative real-time polymerase chain reaction (RQPCR). RESULTS: Five protein spots with fluctuating expression levels were identified in pgn_0775 KO strains along with their masses and physiological features, which contained two hypothetical proteins with higher expression levels in the WT than in the KO strains. RQPCR analysis confirmed that mRNA levels were consistently decreased in KO and recovered in pgn_0775-complemented KO strains. The two hypothetical proteins appeared to be the products of an operon that comprises four genes encoding three hypothetical but putative type IX secretion system sorting domain-containing proteins and an N-terminal region of the C25 cysteine peptidase. CONCLUSIONS: The AtoC RR homolog in P. gingivalis upregulates the expression of the operon encoding potentially antigenic proteins retained on the cell surface; thus, it could be a promising target for P. gingivalis-specific antivirulence therapy.

Effects of cleaning sports mouthguards with ethylene-vinyl acetate on oral bacteria.

Background: Sports mouthguards, worn in the oral cavity to prevent sports injuries, are constantly exposed to various microorganisms that cause oral infections. Hence, the optimal cleaning methods for sports mouthguards have been thoroughly examined. In this study, we evaluated the efficiency of cleaning effects with a mouthguard cleaner (MC) on microbial biofilm formation in sports mouthguards in vitro and in vivo. Methods: We evaluated the cleaning effects of the discs produced by ethylene-vinyl acetate (EVA) on bacterial biofilms formed by the commensal bacterium Streptococcus oralis, the cariogenic bacterium Streptococcus mutans, and the opportunistic pathogen Staphylococcus aureus in vitro. EVA discs with biofilm were subjected to sterile distilled water (CTRL) and ultrasonic washing (UW), followed by treatment with MC and sodium hypochlorite (NaClO) as positive controls. Thereafter, the viable bacterial cell counts were determined. The bacteria adhering to the sheets before and after the treatment were observed under an electron microscope. The degree of cleanliness and measurement of viable microbial cell counts for total bacteria, Streptococci and Candida, opportunistic fungi, were evaluated on the used experimental sports mouthguards with and without UW and MC treatment in vivo. Results: The number of bacterial cells significantly decreased against all the tested biofilm bacteria upon treatment with MC, compared with CTRL and UW. Electron microscopy analysis revealed the biofilm formation by all bacteria on the EVA discs before cleaning. We observed fewer bacteria on the EVA discs treated with MC than those treated with CTRL and UW. Furthermore, the degree of cleanliness of the used experimental sports mouthguards cleaned using MC was significantly higher than that of the CTRL-treated mouthguards. Moreover, the viable microbial cell counts on the used experimental sports mouthguard were considerably lower than those on the CTRL ones. Conclusion: The cleaning effect of MC against oral bacteria was more effective than that of UW. MC treatment might have a potential future application as a cleaning method for sports mouthguards to protect athletes from oral infection.

Porphyromonas gingivalis Fimbriae Induce Osteoclastogenesis via Toll-like Receptors in RAW264 Cells.

The effect of Mfa1 fimbriae of Porphyromonas gingivalis on the progression of bone resorption remains unclear, especially compared with another fimbriae, FimA. We investigated the effect of Mfa1 on osteoclastogenesis together with FimA. We also investigated the role of Toll-like receptors (TLRs) in Mfa1 recognition during osteoclast differentiation. Receptor activator of nuclear factor κß ligand (RANKL)-prestimulated RAW264 cells were used to examine the effects of purified Mfa1 fimbriae. The number of osteoclasts was examined by tartrate-resistant acid phosphate (TRAP) staining, osteoclast activation was investigated by bone resorption assays, and gene expression of differentiation markers was examined by quantitative real-time PCR. Transfection of Tlr2 and Tlr4 siRNAs into RAW264 cells was also employed and their role in Mfa1 recognition was investigated. Mfa1 effectively induced the formation of TRAP-positive multinucleated cells and activated osteoclasts. Mfa1 also increased gene expression of Acp5, Mmp9, and Ctsk in RANKL-prestimulated RAW264 cells compared with the control. The osteoclastogenesis induced by Mfa1 was significantly decreased in cells transfected with Tlr2 or Tlr4 siRNAs compared with control siRNA. Our results revealed the role of Mfa1 fimbriae in osteoclastogenesis that may contribute to the partial elucidation of the mechanisms of periodontal disease progression and the development of new therapeutic strategies.

Porphyromonas gingivalis Mfa1 fimbria putatively binds to TLR2 and induces both IL-6 and IL-8 production in human bronchial epithelial cells.

Porphyromonas gingivalis (Pg) a major periodontal pathogen involved in periodontal disease development and progression. Moreover, Pg has two fimbriae surface proteins (FimA and Mfa1) that are genetically distinct and make-up the fimbrial shaft which in-turn form crucial attachment to oral bacteria and multiple host cells. However, unlike FimA, Mfa1 attachment to non-periodontal cells has not been fully elucidated. Considering Pg-associated periodontal disease contributes to pulmonary disease development, we investigated whether Mfa1 can functionally interact with human bronchial epithelial cells and, likewise, trigger a functional response. Initially, we simulated molecular docking and performed both luciferase and neutralization assays to confirm Mfa1-related functional interaction. Subsequently, we treated BEAS-2B cells with purified Mfa1 and performed cytokine quantification through real time-PCR and ELISA to establish Mfa1-related functional response. We found that both Mfa1-TLR2 and Mfa1-TLR4 docking is possible, however, only Mfa1-TLR2 showed a functional interaction. Additionally, we observed that both IL-8 and IL-6 gene expression and protein levels were induced confirming Mfa1-related functional response. Taken together, we propose that BEAS-2B human bronchial epithelial cells are able to recognize Pg Mfa1 and induce both IL-8 and IL-6 inflammatory responses.

Clinical classification of tooth position in the alveolar bone housing with periodontal defects.

A new classification of tooth position in the alveolar bone housing, which indicates the width of alveolar bone for buccolingual direction, with bone defects caused by periodontal disease is proposed. This classification highlights the importance of tooth position in the alveolar bone housing in terms of the progression of the regenerative process and the factors that may affect the prognosis of compromised teeth after regenerative surgery. Tooth positions were divided into two groups: (i) The whole tooth is centrally positioned in the existing alveolar bone housing (Grade I) and (ii) A part of the tooth is exposed out of the existing alveolar bone housing (Grade II). Grade II is further divided into two subgroups according to situations encountered in clinical practice. The following subclasses are suggested: Subgroup A, where the alveolar bone housing is broader than the tooth, and Subgroup B, where the alveolar bone housing is narrower than the tooth. These subgroups represent a discrepancy between tooth size and alveolar bone dimensions in the buccolingual orientation. This classification could be useful for planning the correct regenerative treatment for each type of the tooth position in the alveolar bone housing with periodontal defects.

Efficacy of antimicrobial photodynamic therapy with Rose Bengal and blue light against cariogenic bacteria.

OBJECTIVE: We evaluated the effect of antimicrobial photodynamic therapy (a-PDT) with Rose Bengal and blue light LED on bacteria that initiate and promote dental caries. DESIGN: Colony forming units of Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis, and Lactobacillus salivarius under planktonic and biofilm conditions were counted after a-PDT treatment using Rose Bengal and blue light LED. In addition, cariogenic bacteria from saliva and dental plaques from ten volunteers were used for evaluation of a-PDT treatment. RESULTS: We found that a-PDT using Rose Bengal at > 10 µg/mL had antimicrobial effects on oral Gram-positive S. mutans, S. sobrinus, S. sanguinis, and L. salivarius under both planktonic and biofilm conditions. The effect was also observed for cariogenic bacteria that formed biofilms containing water-insoluble glucans, through which the bacteria are firmly attached to the tooth surface. Moreover, a-PDT led to a marked reduction in cariogenic bacteria in saliva and dental plaques. CONCLUSION: a-PDT could be a useful approach for controlling dental caries in dental surgery.

Construction of a Gene-Deletion Mutant in Tannerella forsythia.

Tannerella forsythia, a gram-negative anaerobic bacterium, is one of the most important pathogens in periodontal disease. However, it has been difficult to construct a gene-deletion mutant in this organism, which may serve as a useful tool in microbiological research. We reported a highly efficient method to construct a gene-deletion mutant of T. forsythia in 2007, and it was accomplished by preparing competent cells from a colony grown on an agar medium instead of a broth culture. Here, we describe the same method with some improvements.

Biological and Biochemical Roles of Two Distinct Cyclic Dimeric Adenosine 3',5'-Monophosphate- Associated Phosphodiesterases in Streptococcus mutans.

Cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP), a recently identified secondary messenger in bacteria, plays a role in several bacterial processes, including biofilm formation. It is enzymatically produced by diadenylate cyclase and cleaved by c-di-AMP phosphodiesterase. c-di-AMP is believed to be essential for the viability of bacterial cells that produce it. In the current study, the biochemical and biological roles of GdpP (SMU_2140c) and DhhP (SMU_1297), two distinct Streptococcus mutans phosphodiesterases involved in the pathway producing AMP from c-di-AMP, were investigated. Liquid chromatography-tandem mass spectrometry revealed that c-di-AMP was degraded to phosphoadenylyl adenosine (pApA) by truncated recombinant GdpP, and pApA was cleaved by recombinant DhhP to yield AMP. In-frame deletion mutants lacking the dhhP gene (ΔdhhP) and both the gdpP and dhhP genes (ΔgdpPΔdhhP) displayed significantly more biofilm formation than the wild-type and a mutant strain lacking the gdpP gene (ΔgdpP; p < 0.01). Furthermore, biofilm formation was restored to the level of the wild type strain upon complementation with dhhP. Optical and electron microscopy observations revealed that ΔdhhP and ΔgdpPΔdhhP mutants self-aggregated into large cell clumps, correlated with increased biofilm formation, but cell clumps were not observed in cultures of wild-type, ΔgdpP, or strains complemented with gdpP and dhhP. Thus, deletion of dhhP presumably leads to the formation of bacterial cell aggregates and a subsequent increase in biofilm production.

Maturation of the Mfa1 Fimbriae in the Oral Pathogen Porphyromonas gingivalis.

The Mfa1 fimbriae of the periodontal pathogen Porphyromonas gingivalis are involved in adhesion, including binding to synergistic species in oral biofilms. Mfa1 fimbriae are comprised of 5 proteins: the structural component Mfa1, the anchor Mfa2, and Mfa3-5 which constitute the fimbrial tip complex. Interactions among the Mfa proteins and the polymerization mechanism for Mfa1 are poorly understood. Here we show that Mfa3 can bind to Mfa1, 2, 4, and 5 in vitro, and may function as an adaptor protein interlinking other fimbrial subunits. Polymerization of Mfa1 is independent of Mfa3-5 and requires proteolytic processing mediated by the RgpA/B arginine gingipains of P. gingivalis. Both the N- and C- terminal regions of Mfa1 are necessary for polymerization; however, potential ß-strand disrupting amino acid substitutions in these regions do not impair Mfa1 polymerization. In contrast, substitution of hydrophobic amino acids with charged residues in either the N- or C- terminal domains yielded Mfa1 proteins that failed to polymerize. Collectively, these results indicate that Mfa3 serves as an adaptor protein between Mfa1 and other accessory fimbrial proteins. Mfa1 fimbrial polymerization is dependent on hydrophobicity in both the N- and C-terminal regions, indicative of an assembly mechanism involving the terminal regions forming a hydrophobic binding interface between Mfa1 subunits.

Angiopoietin-like protein 2 regulates Porphyromonas gingivalis lipopolysaccharide-induced inflammatory response in human gingival epithelial cells.

Angiopoietin-like protein 2 (ANGPTL2) maintains tissue homeostasis by inducing inflammation and angiogenesis. It is produced in infiltrating immune cells or resident cells, such as adipocytes, vascular endothelial cells, and tumor cells. We hypothesized that ANGPTL2 might play an important role as a unique mediator in both systemic and periodontal disease. We demonstrated an increased ANGPTL2 concentration in gingival crevicular fluid from chronic periodontitis patients. Porphyromonas gingivalis lipopolysaccharide (LPS) treatment strongly induced ANGPTL2 mRNA and protein levels in Ca9-22 human gingival epithelial cells. Recombinant human ANGPTL2 increased interleukin 1ß (IL-1ß), IL-8, and tumor necrosis factor-α (TNF-α) mRNA and protein levels in Ca9-22 cells. Small-interfering (si)RNA-mediated ANGPTL2 knockdown in Ca9-22 cells reduced IL-1ß, IL-8 and TNF-α mRNA and protein levels compared with control siRNA (p<0.01) in P. gingivalis LPS-stimulated Ca9-22 cells. Antibodies against integrin α5ß1, an ANGPTL receptor, blocked induction of these inflammatory cytokines in P. gingivalis LPS-treated Ca9-22 cells, suggesting that secreted ANGPTL induces inflammatory cytokines in gingival epithelial cells via an autocrine loop. The classic sequential cascade of P. gingivalis LPS → inflammatory cytokine induction is well established. However, in the current study, we reveal a novel cascade comprising sequential P. gingivalis LPS → ANGPTL2 → integrin α5ß1 → inflammatory cytokine induction, which might be responsible for inducing potent periodontal disorganization activity in gingival epithelial cells. Via this pathway, ANGPTL2 functions in the pathogenesis of periodontitis and contributes to prolonging chronic inflammation in patients with systemic disease.

Comparative analysis of motility and other properties of Treponema denticola strains.

The periodontitis-associated pathogen Treponema denticola is a spirochetal bacterium that swims by rotating its cell body like a corkscrew using periplasmic flagella. We compared physiologic and pathogenic properties, including motility, in four strains of T. denticola. Phase-contrast microscopy showed differential motility between the strains; ATCC 35404 showed the highest motility, followed by ATCC 33521, and the remaining two strains (ATCC 35405 and ATCC 33520) showed the lowest motility. Transmission electron microscopy showed that the low motility strains exhibited extracellular flagellar protrusions resulting from elongated flagella. Treponemal flagellar filaments are composed of three flagellins of FlaB1, FlaB2 and FlaB3. FlaB1 expression was comparable between the strains, whereas FlaB2 expression was lowest in ATCC 35404. FlaB3 expression varied among strains, with ATCC 35405, ATCC 33520, ATCC 33521, and ATCC 35404 showing the highest to lowest expression levels, respectively. Additionally, the low motility strains showed faster electrophoretic mobility of FlaB3, suggesting that posttranslational modifications of these proteins may have varied, because the amino acid sequences of FlaB3 were identical between the strains. These results suggest that inappropriate expression of FlaB2 and FlaB3 caused the unusual elongation of flagella that resulted in decreased motility. Furthermore, the low motility strains grew to higher bacterial density, and showed greater chymotrypsin-like protease activity, and more bacterial cells associated with gingival epithelial cells in comparison with the high motility strains. There may be a relationship between motility and these properties, but the genetic factors underlying this association remain unclear.

IL-15 and RANKL Play a Synergistically Important Role in Osteoclastogenesis.

Interleukin-15 (IL-15), a cytokine secreted by several cell types, has important physiological roles in the activity, proliferation, and viability of immune cells. It has both chemoattractant and proinflammatory properties, and may promote bone destruction. A previous study has shown that IL-15 alone exerts no effect on osteoclastogenesis. Therefore, the current study addressed the synergistic effect of IL-15 on osteoclast formation using RAW264.7 (RAW) cells by co-stimulation with receptor activator of nuclear factor (NF)-κB ligand (RANKL) that has a major role in osteoclastogenesis involving the pathogenesis of rheumatoid arthritis and periodontal disease. Co-stimulation of RAW cells by IL-15 and RANKL significantly increased the gene expression of osteoclast differentiation and osteoclastogenesis markers compared with stimulation by RANKL or IL-15 independently as evaluated by tartrate-resistant acid phosphate-positive cell numbers, the fusion index, a pit formation assay with Alizarin red staining (calcification estimation), and quantitative polymerase chain reaction. Phosphorylation of extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase, p38 mitogen-activated protein kinase, and NF-κB was significantly increased by RANKL and IL-15 (P < 0.05) compared with RANKL alone. In addition, these differentiation activities induced by RANKL and IL-15 were comparatively suppressed by inhibition of ERK, suggesting that this synergistic effect on osteoclastogenesis is mainly mediated by ERK. Taken together, our results demonstrate that IL-15 and RANKL induce osteoclastogenesis synergistically, and IL-15 might play a novel and major role in destructive inflammatory bone diseases. J. Cell. Biochem. 118: 739-747, 2017. © 2016 Wiley Periodicals, Inc.

Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients.

We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.

Identification of OmpA-Like Protein of Tannerella forsythia as an O-Linked Glycoprotein and Its Binding Capability to Lectins.

Bacterial glycoproteins are associated with physiological and pathogenic functions of bacteria. It remains unclear whether bacterial glycoproteins can bind to specific classes of lectins expressed on host cells. Tannerella forsythia is a gram-negative oral anaerobe that contributes to the development of periodontitis. In this study, we aimed to find lectin-binding glycoproteins in T. forsythia. We performed affinity chromatography of wheat germ agglutinin, which binds to N-acetylglucosamine (GlcNAc) and sialic acid (Sia), and identified OmpA-like protein as the glycoprotein that has the highest affinity. Mass spectrometry revealed that OmpA-like protein contains O-type N-acetylhexosamine and hexose. Fluorometry quantitatively showed that OmpA-like protein contains Sia. OmpA-like protein was found to bind to lectins including E-selectin, P-selectin, L-selectin, Siglec-5, Siglec-9, Siglec-10, and DC-SIGN. The binding of OmpA-like protein to these lectins, except for the Siglecs, depends on the presence of calcium. N-acetylneuraminic acid (NeuAc), which is the most abundant Sia, inhibited the binding of OmpA-like protein to all of these lectins, whereas GlcNAc and mannose only inhibited the binding to DC-SIGN. We further found that T. forsythia adhered to human oral epithelial cells, which express E-selectin and P-selectin, and that this adhesion was inhibited by addition of NeuAc. Moreover, adhesion of an OmpA-like protein-deficient T. forsythia strain to the cells was reduced compared to that of the wild-type strain. Our findings indicate that OmpA-like protein of T. forsythia contains O-linked sugar chains that can mediate interactions with specific lectins. This interaction is suggested to facilitate adhesion of T. forsythia to the surface of host cells.

Mfa4, an Accessory Protein of Mfa1 Fimbriae, Modulates Fimbrial Biogenesis, Cell Auto-Aggregation, and Biofilm Formation in Porphyromonas gingivalis.

Porphyromonas gingivalis, a gram-negative obligate anaerobic bacterium, is considered to be a key pathogen in periodontal disease. The bacterium expresses Mfa1 fimbriae, which are composed of polymers of Mfa1. The minor accessory components Mfa3, Mfa4, and Mfa5 are incorporated into these fimbriae. In this study, we characterized Mfa4 using genetically modified strains. Deficiency in the mfa4 gene decreased, but did not eliminate, expression of Mfa1 fimbriae. However, Mfa3 and Mfa5 were not incorporated because of defects in posttranslational processing and leakage into the culture supernatant, respectively. Furthermore, the mfa4-deficient mutant had an increased tendency to auto-aggregate and form biofilms, reminiscent of a mutant completely lacking Mfa1. Notably, complementation of mfa4 restored expression of structurally intact and functional Mfa1 fimbriae. Taken together, these results indicate that the accessory proteins Mfa3, Mfa4, and Mfa5 are necessary for assembly of Mfa1 fimbriae and regulation of auto-aggregation and biofilm formation of P. gingivalis. In addition, we found that Mfa3 and Mfa4 are processed to maturity by the same RgpA/B protease that processes Mfa1 subunits prior to polymerization.

Role of Porphyromonas gingivalis SerB in gingival epithelial cell cytoskeletal remodeling and cytokine production.

The SerB protein of Porphyromonas gingivalis is a HAD family serine phosphatase that plays a critical role in entry and survival of the organism in gingival epithelial cells. SerB is secreted by P. gingivalis upon contact with epithelial cells. Here it is shown by microarray analysis that SerB impacts the transcriptional profile of gingival epithelial cells, with pathways involving the actin cytoskeleton and cytokine production among those significantly overpopulated with differentially regulated genes. Consistent with the transcriptional profile, a SerB mutant of P. gingivalis exhibited defective remodeling of actin in epithelial cells. Interaction between gingival epithelial cells and isolated SerB protein resulted in actin rearrangement and an increase in the F/G actin ratio. SerB protein was also required for P. gingivalis to antagonize interleukin-8 accumulation following stimulation of epithelial cells with Fusobacterium nucleatum. SerB is thus capable of modulating host cell signal transduction that impacts the actin cytoskeleton and cytokine production.

P. gingivalis accelerates gingival epithelial cell progression through the cell cycle.

P. gingivalis, an opportunistic pathogen in periodontal disease, can reside within the epithelial cells that line the gingival crevice. A proteomic analysis revealed that infection of gingival epithelial cells with P. gingivalis induces broadly based changes in the level and phosphorylation status of proteins that exert multi-level control on the eukaryotic cell cycle. Pathways that were impacted by P. gingivalis included those involving cyclins, p53 and PI3K. The predicted infection-dependent phenotype was confirmed by cytofluorimetry that showed an enhanced proliferation rate of gingival epithelial cells infected with P. gingivalis associated with accelerated progression through the S-phase. Elevated cell proliferation was dependent on the presence of the long fimbriae of P. gingivalis. The ability of P. gingivalis, a common inhabitant of the subgingival crevice, to accelerate cell cycling could have biological consequences for barrier and signaling functions, and for physiological status, of the gingival epithelium.

Intrinsic apoptotic pathways of gingival epithelial cells modulated by Porphyromonas gingivalis.

Porphyromonas gingivalis can inhibit chemically induced apoptosis in primary cultures of gingival epithelial cells through blocking activation of the effector caspase-3. The anti-apoptotic phenotype of P. gingivalis is conserved across strains and does not depend on the presence of fimbriae, as fimbriae-deficient mutants and a naturally occurring non-fimbriated strain were able to impede apoptosis. To dissect the survival pathways modulated by P. gingivalis, protein and gene expression of a number of components of apoptotic death pathways were investigated. P. gingivalis infection of epithelial cells resulted in the phosphorylation of JAK1 and Stat3. Quantitative real-time reverse transcription polymerase chain reaction showed that expression of Survivin and Stat3 itself, targets of activated Stat3, were elevated in P. gingivalis-infected cells. siRNA knockdown of JAK1, in combination with knockdown of Akt, abrogated the ability of P. gingivalis to block apoptosis. In contrast, cIAP-1 and cIAP-2 were not differentially regulated at either the protein or mRNA levels by P. gingivalis. One mechanism by which P. gingivalis can block apoptotic pathways in gingival epithelial cells therefore is through manipulation of the JAK/Stat pathway that controls the intrinsic mitochondrial cell death pathways. Induction of a pro-survival phenotype may prevent programmed host cell death and aid survival of P. gingivalis within gingival epithelial cells.

Gingival epithelial cell transcriptional responses to commensal and opportunistic oral microbial species.

Transcriptional profiling and ontology tools were utilized to define the biological pathways of gingival epithelial cells modulated by coculture with the oral commensal Streptococcus gordonii and the opportunistic commensal Fusobacterium nucleatum. Overall, F. nucleatum and S. gordonii perturbed the gingival epithelial cell transcriptome much less significantly than the oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans perturbed the transcriptome, indicating that there was a greater degree of host adaptation by the commensal species (M. Handfield, J. J. Mans, G. Zheng, M. C. Lopez, S. Mao, A. Progulske-Fox, G. Narasimhan, H. V. Baker, and R. J. Lamont, Cell. Microbiol. 7:811-823, 2005). The biological pathways significantly impacted by F. nucleatum and S. gordonii included the mitogen-activated protein kinase (MAPK) and Toll-like receptor signaling pathways. Differential regulation of GADD45 and DUSP4, key components of the MAPK pathway, was confirmed at the protein level by Western blotting. Modulation of the MAPK pathway is likely to affect host cell proliferation and differentiation. In addition, both the MAPK and Toll-like receptor pathways ultimately converge on cytokine gene expression. An enzyme-linked immunosorbent assay of secreted interleukin-6 (IL-6) and IL-8 demonstrated that F. nucleatum induced production of these cytokines, whereas S. gordonii inhibited secretion from the epithelial cells. Stimulation of secretion of proinflammatory cytokines from epithelial cells may reflect the invasive phenotype of F. nucleatum and contribute to the greater pathogenic potential of F. nucleatum than of S. gordonii.

LuxS involvement in the regulation of genes coding for hemin and iron acquisition systems in Porphyromonas gingivalis.

The periodontal pathogen Porphyromonas gingivalis employs a variety of mechanisms for the uptake of hemin and inorganic iron. Previous work demonstrated that hemin uptake in P. gingivalis may be controlled by LuxS-mediated signaling. In the present study, the expression of genes involved in hemin and iron uptake was determined in parent and luxS mutant strains by quantitative real-time reverse transcription-PCR. Compared to the parental strain, the luxS mutant showed reduced levels of transcription of genes coding for the TonB-linked hemin binding protein Tlr and the lysine-specific protease Kgp, which can degrade host heme-containing proteins. In contrast, there was up-regulation of the genes for another TonB-linked hemin binding protein, HmuR; a hemin binding lipoprotein, FetB; a Fe(2+) ion transport protein, FeoB1; and the iron storage protein ferritin. Differential expression of these genes in the luxS mutant was maximal in early-exponential phase, which corresponded with peak expression of luxS and AI-2 signal activity. Complementation of the luxS mutation with wild-type luxS in trans rescued expression of hmuR. Mutation of the GppX two-component signal transduction pathway caused an increase in expression of luxS along with tlr and lower levels of message for hmuR. Moreover, expression of hmuR was repressed, and expression of tlr stimulated, when the luxS mutant was incubated with AI-2 partially purified from the culture supernatant of wild-type cells. A phenotypic outcome of the altered expression of genes involved in hemin uptake was impairment of growth of the luxS mutant in hemin-depleted medium. The results demonstrate a role of LuxS/AI-2 in the regulation of hemin and iron acquisition pathways in P. gingivalis and reveal a novel control pathway for luxS expression.