Transcriptomic analysis of Porphyromonas gingivalis-infected head and neck cancer cells: Identification of PLAU as a candidate prognostic biomarker.

Periodontal disease is a risk factor for head and neck squamous cell carcinoma (HNSCC), and Porphyromonas gingivalis, a major periodontal pathogen, has been identified as a specific and potentially independent microbial factor that increases the risk of cancer mortality. Gene expression in HNSCC due to P. gingivalis infection and how changes in gene expression affect the prognosis of HNSCC patients are not clarified. When P. gingivalis was cultured with HNSCC cells, it efficiently adhered to these cells and enhanced their invasive ability. A transcriptome analysis of P. gingivalis -infected HNSCC cells showed that genes related to migration, including CCL20, CITED2, CTGF, C8orf44-SGK3, DUSP10, EGR3, FUZ, HBEGF, IL1B, IL24, JUN, PLAU, PTGS2, P2RY1, SEMA7A, SGK1 and SIX2, were highly up- or down-regulated. The expression of up-regulated genes was examined using the expression data of HNSCC patients obtained from The Cancer Genome Atlas (TCGA) database, and the expression of 5 genes, including PLAU, was found to be higher in cancer tissue than in solid normal tissue. An analysis of protein-protein interactions revealed that these 5 genes formed a dense network. A Cox regression analysis showed that high PLAU expression levels were associated with a poor prognosis in patients with TCGA-HNSCC. Furthermore, the prognostic impact correlated with tumour size and the presence or absence of lymph node metastasis. Collectively, these results suggest the potential of PLAU as a molecular prognostic marker in HNSCC patients. Further in vivo and in vitro studies are needed to verify the findings of this study.

Potential Role of the Intratumoral Microbiota in Prognosis of Head and Neck Cancer.

The tumor microbiome, a relatively new research field, affects tumor progression through several mechanisms. The Cancer Microbiome Atlas (TCMA) database was recently published. In the present study, we used TCMA and The Cancer Genome Atlas and examined microbiome profiling in head and neck squamous cell carcinoma (HNSCC), the role of the intratumoral microbiota in the prognosis of HNSCC patients, and differentially expressed genes in tumor cells in relation to specific bacterial infections. We investigated 18 microbes at the genus level that differed between solid normal tissue (n = 22) and primary tumors (n = 154). The tissue microbiome profiles of Actinomyces, Fusobacterium, and Rothia at the genus level differed between the solid normal tissue and primary tumors of HNSCC patients. When the prognosis of groups with rates over and under the median for each microbe at the genus level was examined, rates for Leptotrichia which were over the median correlated with significantly higher overall survival rates. We then extracted 35 differentially expressed genes between the over- and under-the-median-for-Leptotrichia groups based on the criteria of >1.5 fold and p < 0.05 in the Mann-Whitney U-test. A pathway analysis showed that these Leptotrichia-related genes were associated with the pathways of Alzheimer disease, neurodegeneration-multiple diseases, prion disease, MAPK signaling, and PI3K-Akt signaling, while protein-protein interaction analysis revealed that these genes formed a dense network. In conclusion, probiotics and specific antimicrobial therapy targeting Leptotrichia may have an impact on the prognosis of HNSCC.

Green tea catechins inhibit Porphyromonas gulae LPS-induced inflammatory responses in human gingival epithelial cells.

OBJECTIVES: To determine the anti-inflammatory effects of green tea catechins in immortalized human gingival epithelial cells (Ca9-22) stimulated with Porphyromonas gulae lipopolysaccharide (LPS). METHODS: Ca9-22 cells were incubated with P. gulae LPS (10 µg/ml) with or without green tea catechins, epigallocatechin-3-gallate (EGCg), epigallocatechin (EGC), epicatechin-3-gallate (ECG), and epicatechin (EC) (each at 50 µM), for 6 or 24 h. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay were used to determine the induction of cyclooxygenase 2 (COX2), tumor necrosis factor alpha (TNF-ɑ), interleukin 6 (IL-6), and IL-8. Furthermore, the expression of toll-like receptors (TLRs) 2 and 4 was examined using real-time PCR and western blotting analysis, and phosphorylation of the p38 and ERK1/2 was examined using western blotting analysis. RESULTS: At the mRNA and protein levels, EGCg, EGC, ECG, and EC were found to significantly inhibit COX2, TNF-ɑ, IL-6, and IL-8. Furthermore, the levels of ERK1/2 and p38 phosphorylation induced by P. gulae LPS were decreased following the addition of each of the catechins, as well as TLR2 and 4 mRNA and protein. CONCLUSIONS: These findings indicate that green tea catechins are potent inhibitors of inflammatory responses induced by P. gulae LPS, and may also be useful for prevention and/or attenuation of periodontitis.

Prognostic association of starvation-induced gene expression in head and neck cancer.

Autophagy-related genes (ARGs) have been implicated in the initiation and progression of malignant tumor promotion. To investigate the dynamics of expression of genes, including ARGs, head and neck squamous cell carcinoma (HNSCC) cells were placed under serum-free conditions to induce growth retardation and autophagy, and these starved cells were subjected to transcriptome analysis. Among the 21 starvation-induced genes (SIGs) located in the autophagy, cell proliferation, and survival signaling pathways, we identified SIGs that showed prominent up-regulation or down-regulation in vitro. These included AGR2, BST2, CALR, CD22, DDIT3, FOXA2, HSPA5, PIWIL4, PYCR1, SGK3, and TRIB3. The Cancer Genome Atlas (TCGA) database of HNSCC patients was used to examine the expression of up-regulated genes, and CALR, HSPA5, and TRIB3 were found to be highly expressed relative to solid normal tissue in cancer and the survival rate was reduced in patients with high expression. Protein-protein interaction analysis demonstrated the formation of a dense network of these genes. Cox regression analysis revealed that high expression of CALR, HSPA5, and TRIB3 was associated with poor prognosis in patients with TCGA-HNSCC. Therefore, these SIGs up-regulated under serum starvation may be molecular prognostic markers in HNSCC patients.

Porphyromonas gulae lipopolysaccharide elicits inflammatory responses through toll-like receptor 2 and 4 in human gingivalis epithelial cells.

Porphyromonas gulae, a Gram-negative black-pigmented anaerobe, has been associated with periodontal disease in companion animals and its virulence has been attributed to various factors, including lipopolysaccharide (LPS), protease and fimbriae. Toll-like receptors (TLRs) recognise pathogen-associated molecular patterns, such as peptidoglycan, lipids, lipoproteins, nucleic acid and LPS. Following P. gulae infection, some inflammatory responses are dependent on both TLR2 and TLR4. In addition, a recent clinical study revealed that acute and persistent inflammatory responses enhance the expressions of TLR2 and TLR4 in the oral cavity. In this study, we investigated the interaction between P. gulae LPS and human gingivalis epithelial cells (Ca9-22 cells). P. gulae LPS was found to increase TLR2 and TLR4 mRNA expressions and protein productions, and enhanced inflammatory responses, such as COX2 , TNF-ɑ, IL-6 and IL-8. Stimulated Ca9-22 cells exhibited phosphorylation of ERK1/2 and p38, and their inhibitors diminished inflammatory responses, while knockdown of the TLR2 and/or TLR4 genes with small interfering RNA (siRNA) prevented inflammatory responses. Moreover, p38 and ERK1/2 phosphorylation was decreased in TLR2 and TLR4 gene knockdown cells. These findings suggest that P. gulae LPS activates p38 and ERK1/2 via TLR2 and TLR4, leading to inflammatory responses in human gingival epithelial cells.

Inhibition of Porphyromonas gulae and periodontal disease in dogs by a combination of clindamycin and interferon alpha.

Porphyromonas gulae is a major periodontal pathogen in dogs, which can be transmitted to their owners. A major virulence factor of P. gulae consists of a 41-kDa filamentous appendage (FimA) on the cell surface, which is classified into three genotypes: A, B, and C. Thus far, inhibition of periodontal disease in dogs remains difficult. The present study assessed the inhibitory effects of a combination of clindamycin and interferon alpha (IFN-α) formulation against P. gulae and periodontal disease. Growth of P. gulae was significantly inhibited by clindamycin; this inhibition had a greater effect on type C P. gulae than on type A and B isolates. In contrast, the IFN-α formulation inhibited the expression of IL-1ß and COX-2 elicited by type A and B isolates, but not that elicited by type C isolates. Furthermore, periodontal recovery was promoted by the administration of both clindamycin and IFN-α formulation to dogs undergoing periodontal treatment; moreover, this combined treatment reduced the number of FimA genotypes in oral specimens from treated dogs. These results suggest that a combination of clindamycin and IFN-α formulation inhibit P. gulae virulence and thus may be effective for the prevention of periodontal disease induced by P. gulae.

Adhesion and invasion of gingival epithelial cells by Porphyromonas gulae.

Porphyromonas gulae, an animal periodontal pathogen, possess fimbriae classified into three genotypes (A-C) based on the diversity of fimA genes encoding FimA. Accumulating evidence suggests that P. gulae strains with type C fimbriae are more virulent as compared to those with other types. The ability of these organisms to adhere to and invade gingival epithelial cells has yet to be examined. P. gulae showed the greatest levels of adhesion and invasion at a multiplicity of infection of 100 for 90 min. P. gulae type C and some type B strains invaded gingival epithelial cells at significantly greater levels than the other strains, at the same level of efficiency as P. gingivalis with type II fimbriae. Adhesion and invasion of gingival epithelial cells by P. gulae were inhibited by cytochalasin D and sodium azide, indicating the requirements of actin polymerization and energy metabolism for those activities. Invasion within gingival epithelial cells was blocked by staurosporine, whereas those inhibitors showed little effects on adhesion, while nocodazole and cycloheximide had negligible effects on either adhesion or invasion. P. gulae proteases were found to be essential for adhesion and invasion of gingival epithelial cells, while its DNA and RNA, and protein synthesis were unnecessary for those activities. Additionally, α5ß1 integrin antibodies significantly inhibited adhesion and invasion by P. gulae. This is the first report to characterize P. gulae adhesion and invasion of human gingival epithelial cells.

Cell Cycle Arrest and Apoptosis Induced by Porphyromonas gingivalis Require Jun N-Terminal Protein Kinase- and p53-Mediated p38 Activation in Human Trophoblasts.

Porphyromonas gingivalis, a periodontal pathogen, has been implicated as a causative agent of preterm delivery of low-birth-weight infants. We previously reported that P. gingivalis activated cellular DNA damage signaling pathways and ERK1/2 that lead to G1 arrest and apoptosis in extravillous trophoblast cells (HTR-8 cells) derived from the human placenta. In the present study, we further examined alternative signaling pathways mediating cellular damage caused by P. gingivalis. P. gingivalis infection of HTR-8 cells induced phosphorylation of p38 and Jun N-terminal protein kinase (JNK), while their inhibitors diminished both G1 arrest and apoptosis. In addition, heat shock protein 27 (HSP27) was phosphorylated through both p38 and JNK, and knockdown of HSP27 with small interfering RNA (siRNA) prevented both G1 arrest and apoptosis. Furthermore, regulation of G1 arrest and apoptosis was associated with p21 expression. HTR-8 cells infected with P. gingivalis exhibited upregulation of p21, which was regulated by p53 and HSP27. These results suggest that P. gingivalis induces G1 arrest and apoptosis via novel molecular pathways that involve p38 and JNK with its downstream effectors in human trophoblasts.

Apple- and Hop-Polyphenols Inhibit Porphyromonas gingivalis-Mediated Precursor of Matrix Metalloproteinase-9 Activation and Invasion of Oral Squamous Cell Carcinoma Cells.

BACKGROUND: Recent epidemiologic studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, periodontitis is markedly associated with orodigestive cancer mortality, whereas Porphyromonas gingivalis (Pg) infection has been identified as a specific and potentially independent microbial factor related to increased risk of orodigestive cancer death. The authors previously reported that Pg induced the precursor form of matrix metalloproteinase-9 (proMMP-9) production via proteinase-activated receptor (PAR)-related pathways, after which proMMP-9 was activated by gingipains to enhance cellular invasion of SAS cells. In the present study, effects of selected polyphenols as inhibitors of cellular invasion caused by Pg gingipains in SAS cells are examined. METHODS: OSCC cells were infected with Pg strains including gingipain mutants. To evaluate effects of inhibitors: 1) apple polyphenol (AP); 2) hop bract polyphenol (HBP); 3) high-molecular-weight fractions of HBP (HMW-HBP); 4) low-molecular-weight fractions of HBP (LMW-HBP); 5) epigallocatechin gallate (EGCg); 6) KYT-1 (Arg-gingipain inhibitor); and KYT-36 (Lys-gingipain inhibitor) in combination are used. PAR2 and PAR4 mRNA expressions are examined using real-time reverse transcription polymerase chain reaction, and signaling pathways are evaluated by western blotting analysis. RESULTS: KYT-1/KYT-36, AP, HBP, and HMW-HBP significantly inhibited PAR2 and PAR4 mRNA expressions, proMMP-9 activation, and cellular invasion. Furthermore, AP, HBP, and HMW-HBP reduced activation of heat shock protein 27 and Ets1 and nuclear translocation of nuclear factor-kappa B, whereas EGCg and LMW-HBP did not. CONCLUSION: These results suggest that AP, HBP, HMW-HBP are potent inhibitors of proMMP-9 activation and cellular invasion mediated with Pg in OSCC cells.

Noncanonical activation of ß-catenin by Porphyromonas gingivalis.

Porphyromonas gingivalis is an established pathogen in periodontal disease and an emerging pathogen in serious systemic conditions, including some forms of cancer. We investigated the effect of P. gingivalis on ß-catenin signaling, a major pathway in the control of cell proliferation and tumorigenesis. Infection of gingival epithelial cells with P. gingivalis did not influence the phosphorylation status of ß-catenin but resulted in proteolytic processing. The use of mutants deficient in gingipain production, along with gingipain-specific inhibitors, revealed that gingipain proteolytic activity was required for ß-catenin processing. The ß-catenin destruction complex components Axin1, adenomatous polyposis coli (APC), and GSK3ß were also proteolytically processed by P. gingivalis gingipains. Cell fractionation and Western blotting demonstrated that ß-catenin fragments were translocated to the nucleus. The accumulation of ß-catenin in the nucleus following P. gingivalis infection was confirmed by immunofluorescence microscopy. A luciferase reporter assay showed that P. gingivalis increased the activity of the ß-catenin-dependent TCF/LEF promoter. P. gingivalis did not increase Wnt3a mRNA levels, a finding consistent with P. gingivalis-induced proteolytic processing causing the increase in TCF/LEF promoter activity. Thus, our data indicate that P. gingivalis can induce the noncanonical activation of ß-catenin and disassociation of the ß-catenin destruction complex by gingipain-dependent proteolytic processing. ß-Catenin activation in epithelial cells by P. gingivalis may contribute to a proliferative phenotype.

Involvement of protease-activated receptor 4 in over-expression of matrix metalloproteinase 9 induced by Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontal pathogen, is epidemiologically associated with oral squamous cell carcinoma (OSCC). Matrix metalloproteinase 9 (MMP9) which degrades the extracellular matrix and basement membrane components has been implicated in invasion and metastasis of tumor cells. We previously reported that P. gingivalis promoted cellular invasion of carcinoma SAS cells, an established cell line from patients with squamous cell carcinoma of the tongue, by induction of MMP9 production via proteinase-activated receptor 2. In this study, we further examined alternative signaling pathways mediating inactive precursor of MMP9 (proMMP9) production induced by P. gingivalis in SAS cells. Following P. gingivalis infection, PAR4 mRNA expression was increased and proMMP9 production was enhanced, leading to acceleration of SAS cell invasion. Small interfering RNA knockdown of PAR4 gene abrogated both proMMP9 expression and cellular invasion induced by P. gingivalis in SAS cells. Moreover, the phosphorylation of p38 and ERK1/2 was reduced in PAR4 gene knockdown cells infected with P. gingivalis, whereas nuclear translocation of NF-kB was not inhibited. These results suggest that P. gingivalis activates PAR4 signaling pathways, leading proMMP9 over-expression and cellular invasion in OSCC cells.

Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation.

Recent epidemiological studies have revealed a significant association between periodontitis and oral squamous cell carcinoma (OSCC). Furthermore, matrix metalloproteinase 9 (MMP9) is implicated in the invasion and metastasis of tumour cells. We examined the involvement of Porphyromonas gingivalis, a periodontal pathogen, in OSCC invasion through induced expression of proMMP and its activation. proMMP9 was continuously secreted from carcinoma SAS cells, while P. gingivalis infection increased proenzyme expression and subsequently processed it to active MMP9 in culture supernatant, which enhanced cellular invasion. In contrast, Fusobacterium nucleatum, another periodontal organism, failed to demonstrate such activities. The effects of P. gingivalis were observed with highly invasive cells, but not with the low invasivetype. P. gingivalis also stimulated proteinase-activated receptor 2 (PAR2) and enhanced proMMP9 expression, which promoted cellular invasion. P. gingivalis mutants deficient in gingipain proteases failed to activate MMP9. Infected SAS cells exhibited activation of ERK1/2, p38, and NF-kB, and their inhibitors diminished both proMMP9-overexpression and cellular invasion. Together, our results show that P. gingivalis activates the ERK1/2-Ets1, p38/HSP27, and PAR2/NF-kB pathways to induce proMMP9 expression, after which the proenzyme is activated by gingipains to promote cellular invasion of OSCC cell lines. These findings suggest a novel mechanism of progression and metastasis of OSCC associated with periodontitis.

Distribution and molecular characterization of Porphyromonas gulae carrying a new fimA genotype.

Porphyromonas gulae is a gram-negative black-pigmented anaerobe which is known to be a pathogen for periodontitis in dogs. Approximately 41kDa filamentous appendages on the cell surface (FimA) encoded by the fimA gene are regarded as important factors associated with periodontitis. The fimA genotype was classified into two major types and strains in type B were shown to be more virulent than those in type A. In the present study, we characterized a strain with a novel fimA genotype and designated it as type C. The putative amino acid sequence was shown to be similar to the genotype IV fimA of Porphyromonas gingivalis, a major pathogen of human periodontitis. Analyses using an oral squamous cell carcinoma cell line derived from tongue primary lesions revealed that the type C strain inhibited proliferation and scratch closure more than genotype A and B strains. In addition, experiments using a mouse abscess model demonstrated that the type C strain could induce much higher systemic inflammation when compared with strains of the other genotypes. Furthermore, molecular analyses of oral swab specimens collected from dogs demonstrated that the detection frequencies of P. gulae and the genotype C in the periodontitis group were significantly higher than those in the periodontally healthy group. These results suggest that FimA of P. gulae is diverse with the virulence of genotype C strains the highest and that molecular identification of genotype C P. gulae could be a possible useful marker for identifying dogs at high risk of developing periodontitis.

Identification of signaling pathways mediating cell cycle arrest and apoptosis induced by Porphyromonas gingivalis in human trophoblasts.

Epidemiological and interventional studies of humans have revealed a close association between periodontal diseases and preterm delivery of low-birth-weight infants. Porphyromonas gingivalis, a periodontal pathogen, can translocate to gestational tissues following oral-hematogenous spread. We previously reported that P. gingivalis invades extravillous trophoblast cells (HTR-8) derived from the human placenta and inhibits proliferation through induction of arrest in the G(1) phase of the cell cycle. The purpose of the present study was to identify signaling pathways mediating cellular impairment caused by P. gingivalis. Following P. gingivalis infection, the expression of Fas was induced and p53 accumulated, responses consistent with response to DNA damage. Ataxia telangiectasia- and Rad3-related kinase (ATR), an essential regulator of DNA damage checkpoints, was shown to be activated together with its downstream signaling molecule Chk2, while the p53 degradation-related protein MDM2 was not induced. The inhibition of ATR prevented both G(1) arrest and apoptosis caused by P. gingivalis in HTR-8 cells. In addition, small interfering RNA (siRNA) knockdown of p53 abrogated both G(1) arrest and apoptosis. The regulation of apoptosis was associated with Ets1 activation. HTR-8 cells infected with P. gingivalis exhibited activation of Ets1, and knockdown of Ets1 with siRNA diminished both G(1) arrest and apoptosis. These results suggest that P. gingivalis activates cellular DNA damage signaling pathways that lead to G(1) arrest and apoptosis in trophoblasts.

Porphyromonas gingivalis SerB-mediated dephosphorylation of host cell cofilin modulates invasion efficiency.

Porphyromonas gingivalis, a host-adapted opportunistic pathogen, produces a serine phosphatase, SerB, known to affect virulence, invasion and persistence within the host cell. SerB induces actin filament rearrangement in epithelial cells, but the mechanistic basis of this is not fully understood. Here we investigated the effects of SerB on the actin depolymerizing host protein cofilin. P. gingivalis infection resulted in the dephosphorylation of cofilin in gingival epithelial cells. In contrast, a SerB-deficient mutant of P. gingivalis was unable to cause cofilin dephosphorylation. The involvement of cofilin in P. gingivalis invasion was determined by quantitative image analysis of epithelial cells in which cofilin had been knocked down or knocked in with various cofilin constructs. siRNA-silencing of cofilin led to a significant decrease in numbers of intracellular P. gingivalis marked by an absence of actin colocalization. Transfection with wild-type cofilin or constitutively active cofilin both increased numbers of intracellular bacteria, while constitutively inactive cofilin abrogated invasion. Expression of LIM kinase resulted in reduced P. gingivalis invasion, an effect that was reversed by expression of constitutively active cofilin. These results show that P. gingivalis SerB activity induces dephosphorylation of cofilin, and that active cofilin is required for optimal invasion into gingival epithelial cells.

[Cardiovascular diseases and periodontal diseases].

Accumulated evidence has strongly suggested that the long-term effects of periodontal diseases can be linked to more serious systemic conditions such as cardiovascular diseases, diabetes, and complications of pregnancy. Especially, a prevalence of coronary heart disease was found to be significantly increased in patients with periodontitis after adjusting for risk factors such as smoking, diabetes, alcohol intake, obesity, and blood pressure. Furthermore, various studies have shown that Porphyromonas gingivalis, a major periodontal pathogen, is able to exacerbate atherosclerosis following oral-hematogenous spread due to the bacteremia. By P. gingivalis, endothelial cells activate and upregulate various adhesion molecules, thus increasing the likelihood of macrophage diapedesis and subsequent conversion to foam cells thus furthering athroma progression. These findings likely indicate the tight relationship between periodontitis/periodontal pathogens and cardiovascular diseases.

Proliferation of smooth muscle cells stimulated by Porphyromonas gingivalis is inhibited by apple polyphenol.

BACKGROUND: Porphyromonas gingivalis (Pg) is thought to be involved in the progression of occlusive arterial lesions, whereas vascular smooth muscle cell (SMC) proliferation is considered to be involved in occlusive arterial disease. We previously showed that bacteremia caused by Pg infection induced proliferation of mouse aortic SMCs. Furthermore, human SMCs stimulated with human plasma incubated with Pg showed a marked transformation from the contractile to proliferative phenotype. In the present study, we examine the involvement of Pg gingipains and fimbriae in induction of the SMC transformation and proliferation, and effective inhibitors. METHODS: Pg strains including gingipain- and fimbria-null mutants were incubated in human plasma, after which the bacteria were removed and the supernatants were added to cultured SMCs. To evaluate the effects of inhibitors, Pg organisms were incubated in plasma in the presence of apple polyphenol (AP), epigallocatechin gallate, KYT-1 (Arg-gingipain inhibitor), and KYT-36 (Lys-gingipain inhibitor). RESULTS: Plasma supernatants from wild-type and fimbria-mutant cultures markedly stimulated cellular proliferation, whereas those containing gingipain-null mutants showed negligible effects. SMC proliferation was also induced by plasma treated with trypsin. Furthermore, plasma supernatants cultured in the presence of KYT-1/KYT-36 and AP showed significant inhibitory effects on SMC proliferation, whereas cultures with epigallocatechin gallate did not. CONCLUSION: Our results suggest that Pg gingipains are involved in the induction of SMC transformation and proliferation, whereas this was inhibited by AP.

Porphyromonas gingivalis invades human trophoblasts and inhibits proliferation by inducing G1 arrest and apoptosis.

Porphyromonas gingivalis is an oral pathogen that is also associated with serious systemic conditions such as preterm delivery. Here we investigated the interaction between P. gingivalis and a cell line of extravillous trophoblasts (HTR-8) derived from the human placenta. P. gingivalis internalized within HTR-8 cells and inhibited proliferation through induction of arrest in the G1 phase of the cell cycle. G1 arrest was associated with decreased expression of cyclin D and of CDKs 2, 4 and 6. In addition, levels of CDK inhibitors p15, p16, p18 and p21 were increased following P. gingivalis infection. The amount of Rb was diminished by P. gingivalis, and transient overexpression of Rb, with concomitant upregulation of phospho-Rb, relieved P. gingivalis-induced G1 arrest. HTR-8 cells halted in the G1 phase became apoptotic, and apoptosis was accompanied by an increase in the ratio of Bax/Bcl-2 and increased activity of caspases 3, 7 and 9. HTR-8 cells infected with P. gingivalis also exhibited a sustained activation of ERK1/2, and knock-down of ERK1/2 activity with siRNA abrogated both G1 arrest and apoptosis. Thus, P. gingivalis can invade placental trophoblasts and induce G1 arrest and apoptosis through pathways involving ERK1/2 and its downstream effectors, properties that provide a mechanistic basis for pathogenicity in complications of pregnancy.

Distinct roles of long/short fimbriae and gingipains in homotypic biofilm development by Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis, a periodontal pathogen, expresses a number of virulence factors, including long (FimA) and short (Mfa) fimbriae as well as gingipains comprised of arginine-specific (Rgp) and lysine-specific (Kgp) cysteine proteinases. The aim of this study was to examine the roles of these components in homotypic biofilm development by P. gingivalis, as well as in accumulation of exopolysaccharide in biofilms. RESULTS: Biofilms were formed on saliva-coated glass surfaces in PBS or diluted trypticase soy broth (dTSB). Microscopic observation showed that the wild type strain formed biofilms with a dense basal monolayer and dispersed microcolonies in both PBS and dTSB. A FimA deficient mutant formed patchy and small microcolonies in PBS, but the organisms proliferated and formed a cohesive biofilm with dense exopolysaccharides in dTSB. A Mfa mutant developed tall and large microcolonies in PBS as well as dTSB. A Kgp mutant formed markedly thick biofilms filled with large clumped colonies under both conditions. A RgpA/B double mutant developed channel-like biofilms with fibrillar and tall microcolonies in PBS. When this mutant was studied in dTSB, there was an increase in the number of peaks and the morphology changed to taller and loosely packed biofilms. In addition, deletion of FimA reduced the autoaggregation efficiency, whereas autoaggregation was significantly increased in the Kgp and Mfa mutants, with a clear association with alteration of biofilm structures under the non-proliferation condition. In contrast, this association was not observed in the Rgp-null mutants. CONCLUSION: These results suggested that the FimA fimbriae promote initial biofilm formation but exert a restraining regulation on biofilm maturation, whereas Mfa and Kgp have suppressive and regulatory roles during biofilm development. Rgp controlled microcolony morphology and biovolume. Collectively, these molecules seem to act coordinately to regulate the development of mature P. gingivalis biofilms.