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Introduction: The human-derived amnion-chorion membrane (ACM) has endogenous antimicrobial properties, which are important for preventing the colonization and survival of oral bacteria on exposed membranes. This project aimed to decipher the underlying mechanism by identifying the components of ACM that confer antibacterial properties. In addition, the antimicrobial efficacy of these identified components on oral bacteria was assessed., Methods: Four antimicrobial proteins, histone H2A/H2B, cathelicidin LL-37, lactoferrin, and lysozyme, were identified via mass spectrometry in ACM. These proteins were then assessed for their efficacy in killing Streptococcus gordonii Challis. Log-phased bacterial cells were cultured with the commercially available proteins that were identified in ACM, either individually or in combination, at different concentrations. After incubation for 8 or 24 hours, the bacteria were stained with a live/dead viability kit and analyzed via confocal microscopy., Results: The combination of these proteins effectively killed S. gordonii in a dose-dependent fashion after 8 or 24 hours of incubation. When each protein was tested individually, it killed S. gordonii at a much lower efficacy relative to the combinations. The synergistic effects of the antimicrobial protein combinations were also observed in both the viable cell count recovery and minimum inhibitory concentration assays., Discussion: By shedding light on the mechanisms in the ACM's antimicrobial property, this study may raise more awareness of the potential benefit of utilization of a membrane with endogenous antimicrobial properties in regeneration surgeries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Brummerhop, Lee, Weltman, Tribble, van der Hoeven, Chiu, Hong and Wang.)

Peripheral Calcifying Odontogenic Cyst (PCOC) is the extraosseous form of calcifying odontogenic cyst that is limited to peripheral soft tissue without bony involvement. This case report presents a case of PCOC manifested as a progressive growth of gingival mass in a young male treated with excisional biopsy. Histological examination confirmed diagnosis of PCOC with presence of characteristic ghost cells and sporadic calcifications. No recurrence of the lesion and no complication were noted at three-year follow-up. Review of available literature on PCOC noted a predilection of occurrence in the mandible (61%) and in the anterior area of the jaws (58%). Mean age of patients was 41.7 ± SD25.43 (7-83) and 95% CI [33.6, 49.8] yrs. Mean size of the lesions was 1.38 ± SD1.1 (0.5-4.3) and 95% CI [0.93, 1.83] cm. Gender distribution was noted to be 51.3% male and 48.7% female., Competing Interests: The authors JC and GDT declared that they were editorial board members of Frontiers at the time of submission. This had no impact on the peer review process and the final decision. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Sheng, Tipton, Chang, Meng and Tribble.)

Periodontitis is a chronic inflammatory disease induced by dysbiotic dental biofilms. Management of periodontitis is primarily anti-bacterial via mechanical removal of bacterial biofilm. The successful resolution requires wound healing and tissue regeneration, which are not always achieved with these traditional methods. The discovery of specialized pro-resolving mediators (SPMs), a class of lipid mediators that induce the resolution of inflammation and promote local tissue homeostasis, creates another option for the treatment of periodontitis and other diseases of chronic inflammation. In this mini-review, we discuss the host-modulatory effects of SPMs on periodontal tissues and changes in the taxonomic composition of the gut and oral microbiome in the presence of SPMs and SPM precursor lipids. Further research into the relationship between host SPM production and microbiome-SPM modification has the potential to unveil new diagnostic markers of inflammation and wound healing. Expanding this field may drive the discovery of microbial-derived bioactive therapeutics to modulate immune responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Lee and Tribble.)

Objectives: Periodontitis disproportionately affects different racial and ethnic populations. We have previously reported the higher levels of Porphyromonas gingivalis and lower ratios of Streptococcus cristatus to P. gingivalis may contribute to periodontal health disparities. This prospective cohort study was designed to investigate if ethnic/racial groups responded differently to non-surgical periodontal treatment and if the treatment outcomes correlated to the bacterial distribution in patients with periodontitis before treatment., Methods: This prospective cohort pilot study was carried out in an academic setting, at the School of Dentistry, University of Texas Health Science Center at Houston. Dental plaque was collected from a total of 75 African Americans, Caucasians and Hispanics periodontitis patients in a 3-year period. Quantitation of P. gingivalis and S. cristatus was carried out using qPCR. Clinical parameters including probing depths and clinical attachment levels were determined before and after nonsurgical treatment. Data were analyzed using one-way ANOVA, the Kruskal-Wallis test, the paired samples t -test and the chi-square test., Results: The gains in clinical attachment levels after treatment significantly differed amongst the 3 groups-Caucasians responded most favorably, followed by African-Americans, lastly Hispanics, while numbers of P. gingivalis were highest in Hispanics, followed by African-Americans, and lowest in Caucasians ( p  = 0.015). However, no statistical differences were found in the numbers of S. cristatus amongst the 3 groups., Conclusion: Differential response to nonsurgical periodontal treatment and distribution of P. gingivalis are present in different ethnic/racial groups with periodontitis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Wang, Burgardt, Parthasarathy, Ho, Weltman, Tribble, Hong, Cron and Xie.)

Objective: The objective of this cross-sectional study is to investigate alveolar bone gene expression in health and diabetes through ribonucleic acid (RNA) sequencing and bioinformatics analysis., Background: It is relatively unknown how type 2 diabetes modulates gene expression in alveolar bone in humans. Clinical concern regarding increased implant failure rate in patients with diabetes has been discussed in the literature. Previous studies in animal models and humans have suggested an imbalance between the genes regulating bone formation with data suggesting bone resorption in diabetes. However, there is lack of data regarding a comprehensive gene expression from human alveolar bone in diabetes., Methods: Alveolar bone was collected from healthy and type 2 diabetic subjects undergoing periodontal and implant surgeries. The homogenized RNA sample was then extracted and analyzed for quantity and quality. RNA samples were further purified using ribosomal RNA depletion technique and processed for RNA sequencing and analysis. Expression levels for mRNAs were performed by calculating FPKM ([total_exon_fragments/mapped reads (millions) × exon length (kB)]), and differentially expressed mRNAs were selected with log 2 (fold change) >1 or log 2 (fold change) ≤1 and with a parametric F test comparing nested linear models., Results: Eighteen bone samples (10 healthy, 8 patients with diabetes) were analyzed for gene expression. The mean age and HbA1c% of healthy versus diabetic subjects were as follows: age (55.3 ± 17.5 vs 63.9 ± 8.7 years) and HbA1c% (5.6 ± 0.29 vs 7.3 ± 2.4), respectively. Sequencing analysis showed that expression of genes that regulate bone turnover like TGFB1, LTBP4, IGF1, BMP2, BMP4, BMP6, SMAD1, RUNX2, MCSF, and THRA was significantly downregulated in diabetes samples compared with healthy controls with overall reduced expression of genes in the bone regulation pathway in patients with diabetes. Bioinformatics analysis for the altered genes highlighted several pathways related to bone homeostasis and inflammation in diabetes. Periodontitis did not affect the gene expression pattern based on diabetes status., Conclusions: Altered expression of genes due to downregulation of certain pathways that are involved in bone turnover and inflammation suggests that overall wound healing and bone homeostasis may be compromised in type 2 diabetes., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Patients with Parkinson's disease (PD) are at increased risk of aspiration pneumonia, their primary cause of death. Their oral microbiota differs from healthy controls, exacerbating this risk. Our goal was to explore if poor oral health, poor oral hygiene, and dysphagia status affect the oral microbiota composition of these patients. In this cross-sectional case-control study, the oral microbiota from hard and soft tissues of patients with PD ( n = 30) and age-, gender-, and education-matched healthy controls ( n = 30) was compared using 16S rRNA gene sequencing for bacterial identification. Study participants completed dietary, oral hygiene, drooling, and dysphagia questionnaires, and an oral health screening. Significant differences in soft tissue beta-diversity ( p < 0.005) were found, and a higher abundance of opportunistic oral pathogens was detected in patients with PD. Factors that significantly influenced soft tissue beta-diversity and microbiota composition include dysphagia, drooling (both p < 0.05), and salivary pH ( p < 0.005). Thus, patients with PD show significant differences in their oral microbiota compared to the controls, which may be due, in part, to dysphagia, drooling, and salivary pH. Understanding factors that alter their oral microbiota could lead to the development of diagnostic and treatment strategies that improve the quality of life and survivability of these patients.

Failure of resolution pathways in periodontitis is reflected in levels of specialized pro-resolving lipid mediators (SPMs) and SPM pathway markers but their relationship with the subgingival microbiome is unclear. This study aimed to analyze and integrate lipid mediator level, SPM receptor gene expression and subgingival microbiome data in subjects with periodontitis vs. healthy controls. The study included 13 periodontally healthy and 15 periodontitis subjects that were evaluated prior to or after non-surgical periodontal therapy. Samples of gingival tissue and subgingival plaque were collected prior to and 8 weeks after non-surgical treatment; only once in the healthy group. Metabololipidomic analysis was performed to measure levels of SPMs and other relevant lipid mediators in gingiva. qRT-PCR assessed relative gene expression (2 -ΔΔCT ) of known SPM receptors. 16S rRNA sequencing evaluated the relative abundance of bacterial species in subgingival plaque. Correlations between lipid mediator levels, receptor gene expression and bacterial abundance were analyzed using the Data Integration Analysis for Biomarker discovery using Latent cOmponents (DIABLO) and Sparse Partial Least Squares (SPLS) methods. Profiles of lipid mediators, receptor genes and the subgingival microbiome were distinct in the three groups. The strongest correlation existed between lipid mediator profile and subgingival microbiome profile. Multiple lipid mediators and bacterial species were highly correlated (correlation coefficient ≥0.6) in different periodontal conditions. Comparing individual correlated lipid mediators and bacterial species in periodontitis before treatment to healthy controls revealed that one bacterial species, Corynebacterium durum , and five lipid mediators, 5(S)6(R)-DiHETE, 15(S)-HEPE, 7-HDHA, 13-HDHA and 14-HDHA, were identified in both conditions. Comparing individual correlated lipid mediators and bacterial species in periodontitis before treatment to after treatment revealed that one bacterial species, Anaeroglobus geminatus , and four lipid mediators, 5(S)12(S)-DiHETE, RvD1, Maresin 1 and LTB4, were identified in both conditions. Four Selenomonas species were highly correlated with RvD1, RvE3, 5(S)12(S)-DiHETE and proinflammatory mediators in the periodontitis after treatment group. Profiles of lipid mediators, receptor gene and subgingival microbiome are associated with periodontal inflammation and correlated with each other, suggesting inflammation mediated by lipid mediators influences microbial composition in periodontitis. The role of correlated individual lipid mediators and bacterial species in periodontal inflammation have to be further studied., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lee, Li, Zhu, Tribble, Zheng, Ferguson, Maddipati, Angelov and Van Dyke.)

Oral ulcers are lesions that occur due to disruption of epithelial integrity of the mucosa of the oral cavity. Intraoral ulcers are often associated with pain, redness, symptoms of discomfort, and blood hemorrhage. The etiology for many oral ulcers is local trauma, systemic health conditions, or medication; for other ulcers the cause is less clear. This pilot study aims to evaluate the salivary components and microbiome in patients with atraumatic pre-ulcerous and ulcerous oral lesions compared to control individuals, while considering three common risk factors for atraumatic ulcers, smoking, stress, and gender. This study uses matched age, sex, and ethnicity samples from healthy otherwise and oral lesion patients to investigate the changes in salivary surfactant protein A (SP-A) and examines the prevalence and diversity of the salivary oral microflora. The goal is to determine if there are factors in saliva that have the potential to be used as biomarkers for risk of developing atraumatic oral ulcers. Our data show that the average level of SP-A is significantly reduced in female smokers compared to non-smoker healthy females. The average level of SP-A in female oral lesion patients is reduced compared to controls. The microbiome composition is significantly affected by smoking and the level of SP-A. Comparing the control participants and oral lesion patients, there are 16 species of bacteria that are significantly different, and all of these bacteria are significantly affected by smoking and SP-A. LEfSe analysis identified five bacteria that may represent potential biomarkers. This preliminary study demonstrates the potential of the oral microbiome to act as a biomarker for oral ulcer risk and infers potential mechanistic links between risk factors and alterations in innate immune mechanisms such as SP-A levels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Adibi, Seferovic, Tribble, Alcorn and Fakhouri.)

Human derived composite amnion-chorion membrane (ACM) has been used to facilitate wound healing due to reported anti-inflammatory properties and promotion of cell proliferation. This study aimed to assess the antimicrobial properties of the ACM using novel methods to visualize the antimicrobial efficacy of membranes in situ at different time points. Porcine Pericardium Collagen Membranes (PPCM) served as membrane controls. Circular pieces of the membranes were used in three different assays: insert, agar contact and glass-bottom well assays. Streptococcus gordonii were spotted onto the membranes and the plates were subsequently centrifuged to ensure direct bacterial contact with the membranes in the insert and agar contact assays, thus better mimicking bacterial adherence in the oral cavity. After incubation at 37 °C for 8, 24, and 48 hours, the membranes were dyed with the Live/Dead BacLight Bacterial Viability fluorescence stain and analyzed via confocal microscopy. The results demonstrated that the ACM completely inhibited bacterial growth at all time points, whereas the PPCM did not demonstrate any antimicrobial properties. Within the limits of this study, the ACM showed extremely high antimicrobial efficacy against oral streptococci. In addition, our methods may be useful in assessing antimicrobial properties for biomaterials with minimum diffusion ability, when traditional assessment methods are not applicable.

The oral microbiome has the potential to provide an important symbiotic function in human blood pressure physiology by contributing to the generation of nitric oxide (NO), an essential cardiovascular signaling molecule. NO is produced by the human body via conversion of arginine to NO by endogenous nitric oxide synthase (eNOS) but eNOS activity varies by subject. Oral microbial communities are proposed to supplement host NO production by reducing dietary nitrate to nitrite via bacterial nitrate reductases. Unreduced dietary nitrate is delivered to the oral cavity in saliva, a physiological process termed the enterosalivary circulation of nitrate. Previous studies demonstrated that disruption of enterosalivary circulation via use of oral antiseptics resulted in increases in systolic blood pressure. These previous studies did not include detailed information on the oral health of enrolled subjects. Using 16S rRNA gene sequencing and analysis, we determined whether introduction of chlorhexidine antiseptic mouthwash for 1 week was associated with changes in tongue bacterial communities and resting systolic blood pressure in healthy normotensive individuals with documented oral hygiene behaviors and free of oral disease. Tongue cleaning frequency was a predictor of chlorhexidine-induced changes in systolic blood pressure and tongue microbiome composition. Twice-daily chlorhexidine usage was associated with a significant increase in systolic blood pressure after 1 week of use and recovery from use resulted in an enrichment in nitrate-reducing bacteria on the tongue. Individuals with relatively high levels of bacterial nitrite reductases had lower resting systolic blood pressure. These results further support the concept of a symbiotic oral microbiome contributing to human health via the enterosalivary nitrate-nitrite-NO pathway. These data suggest that management of the tongue microbiome by regular cleaning together with adequate dietary intake of nitrate provide an opportunity for the improvement of resting systolic blood pressure.

Porphyromonas gingivalis is considered a keystone pathogen in adult periodontitis but has also been associated with systemic diseases. It has a myriad of virulence factors that differ between strains. Genetic exchange and intracellular genome rearrangements may be responsible for the variability in the virulence of P. gingivalis . The present review discusses how the exchange of alleles can convert this bacterium from commensalistic to pathogenic and potentially shapes the host-microbe environment from homeostasis to dysbiosis. It is likely that genotypes of P. gingivalis with increased pathogenic adaptations may spread in the human population with features acquired from a common pool of alleles. The exact molecular mechanisms that trigger this exchange are so far unknown but they may be elicited by environmental pressure.

Objective: In the Mexican-American population, the prevalence of Type 2 diabetes mellitus (T2DM) is as high as 50% of the population. This randomized controlled clinical trial was designed to elucidate how treatment of periodontal disease affects HbA1c values in this population., Materials and Methods: One hundred and fifty-four T2DM patients with periodontal disease were enrolled in the study. The test group was treated with scaling and root planing (SRP); the control group received oral hygiene instructions. At baseline and 4-6 weeks after therapy, a complete periodontal examination was performed. Blood was collected at baseline and 4 months later for HbA1c levels., Results: One hundred and twenty-six individuals completed the study. Baseline mean ± SD HbA1c for the test and control groups were 9.0 ± 2.3% and 8.4 ± 2.0% respectively. Non-significant difference in HbA1c reductions (0.6 ± 2.1% and 0.3 ± 1.7%) was found between test and control groups at 4 months. Comparisons of the periodontal clinical parameters between the test and control groups found significant differences with improved results in the test subjects., Conclusions: No statistically significant differences were found in the changes of HbA1c levels between test and control groups. Non-surgical periodontal therapy improved the magnitude of change in periodontal parameters as compared to the control subjects. ClinicalTrials.gov Identifier: NCT01128374., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Porphyromonas gingivalis is a gram-negative anaerobic bacterium, a member of the human oral microbiome, and a proposed "keystone" pathogen in the development of chronic periodontitis, an inflammatory disease of the gingiva. P. gingivalis is a genetically diverse species, and is able to exchange chromosomal DNA between strains by natural competence and conjugation. In this study, we investigate the role of horizontal DNA transfer as an adaptive process to modify behavior, using the major fimbriae as our model system, due to their critical role in mediating interactions with the host environment. We show that P. gingivalis is able to exchange fimbrial allele types I and IV into four distinct strain backgrounds via natural competence. In all recombinants, we detected a complete exchange of the entire fimA allele, and the rate of exchange varies between the different strain backgrounds. In addition, gene exchange within other regions of the fimbrial genetic locus was identified. To measure the biological implications of these allele swaps we compared three genotypes of fimA in an isogenic background, strain ATCC 33277. We demonstrate that exchange of fimbrial allele type results in profound phenotypic changes, including the quantity of fimbriae elaborated, membrane blebbing, auto-aggregation and other virulence-associated phenotypes. Replacement of the type I allele with either the type III or IV allele resulted in increased invasion of gingival fibroblast cells relative to the isogenic parent strain. While genetic variability is known to impact host-microbiome interactions, this is the first study to quantitatively assess the adaptive effect of exchanging genes within the pan genome cloud. This is significant as it presents a potential mechanism by which opportunistic pathogens may acquire the traits necessary to modify host-microbial interactions.

Genes are transferred between bacteria in dental plaque by transduction, conjugation, and transformation. Membrane vesicles can also provide a mechanism for horizontal gene transfer. DNA transfer is considered bacterial sex, but the transfer is not parallel to processes that we associate with sex in higher organisms. Several examples of bacterial gene transfer in the oral cavity are given in this review. How frequently this occurs in dental plaque is not clear, but evidence suggests that it affects a number of the major genera present. It has been estimated that new sequences in genomes established through horizontal gene transfer can constitute up to 30% of bacterial genomes. Gene transfer can be both inter- and intrageneric, and it can also affect transient organisms. The transferred DNA can be integrated or recombined in the recipient's chromosome or remain as an extrachromosomal inheritable element. This can make dental plaque a reservoir for antimicrobial resistance genes. The ability to transfer DNA is important for bacteria, making them better adapted to the harsh environment of the human mouth, and promoting their survival, virulence, and pathogenicity.

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that colonizes the human oral cavity. It is implicated in the development of periodontitis, a chronic periodontal disease affecting half of the adult population in the USA. To survive in the oral cavity, these bacteria must colonize dental plaque biofilms in competition with other bacterial species. Long-term survival requires P. gingivalis to evade host immune responses, while simultaneously adapting to the changing physiology of the host and to alterations in the plaque biofilm. In reflection of this highly variable niche, P. gingivalis is a genetically diverse species and in this review the authors summarize genetic diversity as it relates to pathogenicity in P. gingivalis. Recent studies revealing a variety of mechanisms by which adaptive changes in genetic content can occur are also reviewed. Understanding the genetic plasticity of P. gingivalis will provide a better framework for understanding the host-microbe interactions associated with periodontal disease.

This review covers developments in the study of polymicrobial communities, biofilms and selected areas of host response relevant to dental plaque and related areas of oral biology. The emphasis is on recent studies in which proteomic methods, particularly those using mass spectrometry as a readout, have played a major role in the investigation. The last 5-10 years have seen a transition of such methods from the periphery of oral biology to the mainstream, as in other areas of biomedical science. For reasons of focus and space, the authors do not discuss biomarker studies relevant to improved diagnostics for oral health, as this literature is rather substantial in its own right and deserves a separate treatment. Here, global gene regulation studies of plaque-component organisms, biofilm formation, multispecies interactions and host-microbe interactions are discussed. Several aspects of proteomics methodology that are relevant to the studies of multispecies systems are commented upon.

Unlabelled: Porphyromonas gingivalis is a Gram-negative anaerobe that resides exclusively in the human oral cavity. Long-term colonization by P. gingivalis requires the bacteria to evade host immune responses while adapting to the changing host physiology and alterations in the composition of the oral microflora. The genetic diversity of P. gingivalis appears to reflect the variability of its habitat; however, little is known about the molecular mechanisms generating this diversity. Previously, our research group established that chromosomal DNA transfer occurs between P. gingivalis strains. In this study, we examine the role of putative DNA transfer genes in conjugation and transformation and demonstrate that natural competence mediated by comF is the dominant form of chromosomal DNA transfer, with transfer by a conjugation-like mechanism playing a minor role. Our results reveal that natural competence mechanisms are present in multiple strains of P. gingivalis, and DNA uptake is not sensitive to DNA source or modification status. Furthermore, extracellular DNA was observed for the first time in P. gingivalis biofilms and is predicted to be the major DNA source for horizontal transfer and allelic exchange between strains. We propose that exchange of DNA in plaque biofilms by a transformation-like process is of major ecological importance in the survival and persistence of P. gingivalis in the challenging oral environment., Importance: P. gingivalis colonizes the oral cavities of humans worldwide. The long-term persistence of these bacteria can lead to the development of chronic periodontitis and host morbidity associated with tooth loss. P. gingivalis is a genetically diverse species, and this variability is believed to contribute to its successful colonization and survival in diverse human hosts, as well as evasion of host immune defenses and immunization strategies. We establish here that natural competence is the major driving force behind P. gingivalis DNA exchange and that conjugative DNA transfer plays a minor role. Furthermore, we reveal for the first time the presence of extracellular DNA in P. gingivalis biofilms, which is most likely the source of DNA exchanged between strains within dental plaque. These studies expand our understanding of the mechanisms used by this important member of the human oral flora to transition its relationship with the host from a commensal to a pathogenic relationship.

Background: Porphyromonas gingivalis is etiologically associated with chronic periodontitis. The major fimbriae of this periodontal pathogen mediate binding to host gingival epithelial cells and fibroblasts, a critical function in the initiation of periodontitis. However, the role of fimbriae in P. gingivalis-osteoblast interactions remains unknown. In the present study, the involvement of major fimbriae in the initial and long-term interactions between P. gingivalis and osteoblasts is investigated., Methods: Primary mouse calvarial osteoblast cultures were established and inoculated with P. gingivalis ATCC 33277 or YPF1, a major fimbriae-deficient mutant of P. gingivalis. Confocal microscopy images were acquired to assess bacterial invasion. DNA content measurement, real-time polymerase chain reaction, and alizarin red S staining and calcium content analysis were used to study the impact of bacteria on the proliferation, differentiation, and mineralization of osteoblasts, respectively., Results: Compared to the parent strain, YPF1 was significantly reduced in invasion of osteoblasts after 3 hours interaction. However, extended culture of infected osteoblasts did not reveal significant differences in persistence between the two strains. Proliferation of osteoblasts was not affected by either strain, and differentiation and mineralization of osteoblasts were inhibited by both strains to comparable levels., Conclusion: This study reveals that major fimbriae are involved in the initial invasion of osteoblasts by P. gingivalis, but are not essential for the subsequent inhibition of osteoblast differentiation and mineralization in long-term culture.

Prevotella species are members of the bacterial oral flora and are opportunistic pathogens in polymicrobial infections of soft tissues. Antibiotic resistance to tetracyclines is common in these bacteria, and the gene encoding this resistance has been previously identified as tetQ. The tetQ gene is also found on conjugative transposons in the intestinal Bacteroides species; whether these related bacteria have transmitted tetQ to Prevotella is unknown. In this study, we describe our genetic analysis of mobile tetQ elements in oral Prevotella species. Our results indicate that the mobile elements encoding tetQ in oral species are distinct from those found in the Bacteroides. The intestinal bacteria may act as a reservoir for the tetQ gene, but Prevotella has incorporated this gene into an IS21-family transposon. This transposon is present in Prevotella species from more than one geographical location, implying that the mechanism of tetQ spread between oral Prevotella species is highly conserved., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Porphyromonas gingivalis is etiologically associated with adult periodontitis, but it is unclear how P. gingivalis long-term interactions with bone cells contribute to this disease. This study investigates P. gingivalis interactions with osteoblasts over an extended time course. A primary mouse calvarial osteoblast culture was established and inoculated with P. gingivalis 33277 repeatedly every other day for up to four weeks. Invasion of osteoblasts by P. gingivalis, and the resulting effects on the proliferation, differentiation, and mineralization of osteoblasts were evaluated. P. gingivalis was found to invade osteoblasts in a dose-dependent manner, and repetitive inoculation increased the percentage of osteoblasts with internalized P. gingivalis. P. gingivalis did not affect osteoblast proliferation, but inhibited their differentiation and mineralization, partially via an inhibition of the differentiation regulatory transcription factors Cbfa-1 and osterix. In conclusion, P. gingivalis invades osteoblasts and inhibits bone formation, which likely contributes to alveolar bone loss in chronic periodontitis., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)

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.

Low Molecular Weight Tyrosine Phosphatases (LMWTP) are widespread in prokaryotes; however, understanding of the signalling cascades controlled by these enzymes is still emerging. Porphyromonas gingivalis, an opportunistic oral pathogen, expresses a LMWTP, Ltp1, that is differentially regulated in biofilm communities. Here we characterize the enzymatic activity of Ltp1 and, through the use of mutants that lack Ltp1 or expresses catalytically defective Ltp1, show that tyrosine phosphatase activity constrains both monospecies biofilm development and community development with the antecedent oral biofilm constituent Streptococcus gordonii. Exopolysaccharide production is downregulated by Ltp1 through transcriptional regulation of multiple genes involved in biosynthesis and transport. Furthermore, Ltp1 regulates transcriptional activity of luxS and thus impacts AI-2-dependent signalling in biofilm communities. In the absence of Ltp1 transcription across the hmu haemin uptake locus is reduced, and consequently uptake of haemin is impaired in the Ltp1 mutant. The gingipain proteinases Kgp and RgpA/B remain phosphorylated in the Ltp1 mutant. Phosphorylated Rgps are poorly secreted, whereas cell surface activity of phosphorylated Kgp is enhanced. By controlling the activity of several virulence-associated properties, Ltp1 may restrain the pathogenic potential of P. gingivalis and maintain a commensal interaction with the host.

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.

Clp proteases and chaperones are ubiquitous among prokaryotes and eukaryotes, and in many pathogenic bacteria the Clp stress response system is also involved in regulation of virulence properties. In this study, the roles of ClpB, ClpC, and ClpXP in stress resistance, homotypic and heterotypic biofilm formation, and intracellular invasion in the oral opportunistic pathogen Porphyromonas gingivalis were investigated. Absence of ClpC and ClpXP, but not ClpB, resulted in diminished tolerance to high temperatures. Response to oxidative stress was not affected by the loss of any of the Clp proteins. The clpC and clpXP mutants demonstrated elevated monospecies biofilm formation, and the absence of ClpXP also enhanced heterotypic P. gingivalis-Streptococcus gordonii biofilm formation. All clp mutants adhered to gingival epithelial cells to the same level as the wild type; however, ClpC and ClpXP were found to be necessary for entry into host epithelial cells. ClpB did not play a role in entry but was required for intracellular replication and survival. ClpXP negatively regulated the surface exposure of the minor fimbrial (Mfa) protein subunit of P. gingivalis, which stimulates biofilm formation but interferes with epithelial cell entry. Collectively, these results show that the Clp protease complex and chaperones control several processes that are important for the colonization and survival of P. gingivalis in the oral cavity.

Porphyromonas gingivalis is a major oral pathogen that contributes to the development of periodontal disease. There is a significant degree of genetic variation among strains of P. gingivalis, and the population structure has been predicted to be panmictic, indicating that horizontal DNA transfer and recombination between strains are likely. The molecular events underlying this genetic exchange are not understood, although a putative type IV secretion system is present in the genome sequence of strain W83, implying that DNA conjugation may be responsible for genetic transfer in these bacteria. In this study, we provide in vitro evidence for the horizontal transfer of DNA using plasmid- and chromosome-based assays. In the plasmid assays, Bacteroides-derived shuttle vectors were tested for transfer from P. gingivalis strains into Escherichia coli. Of the eight strains tested, five were able to transfer DNA into E. coli by a mechanism most consistent with conjugation. Additionally, strains W83 and 33277 tested positive for the transfer of chromosomally integrated antibiotic resistance markers. Ten chimeras resulting from the chromosomal transfer assay were further analyzed by Southern hybridization and were shown to have exchanged DNA fragments of between 1.1 and 5.6 kb, but the overall strain identity remained intact. Chimeras showed phenotypic changes in the ability to accrete into biofilms, implying that DNA transfer events are sufficient to generate measurable changes in complex behaviors. This ability to transfer chromosomal DNA between strains may be an adaptation mechanism in the complex environment of the host oral cavity.

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.

Porphyromonas gingivalis, one of the causative agents of adult periodontitis, develops biofilm microcolonies on substrata of Streptococcus gordonii but not on Streptococcus mutans. P. gingivalis genome microarrays were used to identify genes differentially regulated during accretion of P. gingivalis in heterotypic biofilms with S. gordonii. Thirty-three genes showed up- or downregulation by array analysis, and differential expression was confirmed by quantitative reverse transcription-PCR. The functions of the regulated genes were predominantly related to metabolism and energy production. In addition, many of the genes have no current known function. The roles of two upregulated genes, ftsH (PG0047) encoding an ATP-dependent zinc metallopeptidase and ptpA (PG1641) encoding a putative tyrosine phosphatase, were investigated further by mutational analysis. Strains with mutations in these genes developed more abundant biofilms with S. gordonii than the parental strain developed. ftsH and ptpA may thus participate in a regulatory network that constrains P. gingivalis accumulation in heterotypic biofilms. This study provided a global analysis of P. gingivalis transcriptional responses in an oral microbial community and also provided insight into the regulation of heterotypic biofilm development.

Haloacid dehalogenase (HAD) family phosphatases are widespread in prokaryotes and are generally involved in metabolic processes. Porphyromonas gingivalis, an invasive periodontal pathogen, secretes the HAD family phosphoserine phosphatase SerB653 when in contact with gingival epithelial cells. Here we characterize the structure and enzymatic activity of SerB653 and show that a SerB653 allelic replacement mutant of P. gingivalis is deficient in internalization and persistence in gingival epithelial cells. In contrast, mutation of a second HAD family serine phosphatase of P. gingivalis (SerB1170), or of a serine transporter, did not affect invasion. A pull-down assay identified GAPDH and heat-shock protein 90 as potential substrates for SerB653. Furthermore, exogenous phosphatase regulated microtubule dynamics in host cells. These data indicate that P. gingivalis has adapted a formerly metabolic enzyme to facilitate entry into host cells by modulating host cytoskeletal architecture. Our findings define a virulence-related role of a HAD family phosphatase and reveal an invasin of an important periodontal pathogen.

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.

The oral pathogen Porphyromonas gingivalis expresses a homolog of the internalin family protein InlJ. Inactivation of inlJ reduced monospecies biofilm formation by P. gingivalis. In contrast, heterotypic P. gingivalis-Streptococcus gordonii biofilm formation was enhanced in the InlJ-deficient mutant. The results indicate a nuanced role for InlJ in regulating biofilm accumulations of P. gingivalis.

Dental plaque biofilm formation proceeds through a developmental pathway initiated by the attachment of pioneer organisms, such as Streptococcus gordonii, to tooth surfaces. Through a variety of synergistic interactions, pioneer organisms facilitate the colonization of later arrivals including Porphyromonas gingivalis, a potential periodontal pathogen. We have investigated genes of S. gordonii required to support a heterotypic biofilm community with P. gingivalis. By screening a plasmid integration library of S. gordonii, genes were identified that are crucial for the accumulation of planktonic P. gingivalis cells into a multispecies biofilm. These genes were further investigated by specific mutation and complementation analyses. The biofilm-associated genes can be grouped into broad categories based on putative function as follows: (i) intercellular or intracellular signalling (cbe and spxB), (ii) cell wall integrity and maintenance of adhesive proteins (murE, msrA and atf), (iii) extracellular capsule biosynthesis (pgsA and atf), and (iv) physiology (gdhA, ccmA and ntpB). In addition, a gene for a hypothetical protein was identified. Biofilm visualization and quantification by confocal microscopy confirmed the role of these genes in the maturation of the multispecies community, including biofilm architectural development. The results suggest that S. gordonii governs the development of heterotypic oral biofilms through multiple genetic pathways.

Conjugative transposons have been identified in several bacterial species, most notably the Gram-positive Enterococci and the Gram-negative Bacteroides. In Bacteroides species, these elements encode a complete conjugative machinery, which mediates their own intercellular transfer, and they can mobilize in trans co-resident elements. One such mobilizable element is the antibiotic resistance transposon, Tn4555, which was previously found to integrate into a specific genome target site via a site-specific recombination mechanism. In this work, we demonstrate that three Tn4555 genes were involved in integration of the element. These were int encoding a lambda-type integrase, which was absolutely required for integration of the transposon, and two accessory genes, which increased the frequency of integration. Interestingly, one of these accessory gene products, TnpA, directed the insertion of Tn4555 into the genome target site; in the absence of tnpA, the insertion pattern was essentially random. This is the first example of a site-specific recombinase that uses a specific targeting protein.

Tn4555 is a 12.1-kb Bacteroides antibiotic resistance transposon representative of a novel class of transmissible genetic elements that can be transferred by resident conjugative tetracycline resistance transposons (Tc(r)-elements) but are not capable of self-transfer. Previously it was shown that Tn4555 transposes by a site-specific recombination mechanism that utilizes a circular intermediate. This circular form is induced by tetracycline and it also is the substrate for conjugation. To better understand the mechanism of transposition, the entire nucleotide sequence of Tn4555 was determined and a set of genes potentially involved in transposition was identified. The transposon was 12,105 bp including a variable 6-bp coupling sequence associated with one of the transposon termini. The element had a 44.3% G + C composition and nine potential protein coding regions were observed, eight of which were encoded on the forward strand. Two putative transposition genes were found. The int gene product had significant C-terminal homology to the lambda family of integrases and the xis gene product was similar to several excisionase proteins encoded by both plasmids and conjugative transposons. The mobA mobilization gene and cfxA beta-lactamase gene of Tn4555 had been previously identified, and the remaining five open reading frames had no significant matches with sequences in the available databases. Northern hybridization analysis revealed that all Tn4555 genes except for orf-9 were expressed and two sets of genes, tnpA, int and xis, orf-5, orf-6 were organized in operons. None of the genes seemed to be induced significantly by the addition of tetracycline to cultures. Although a small 0.4-kb xis-specific transcript appeared in tetracycline-treated cultures it was not clear if this was due to an induction or if it was a specific degradation product., (Copyright 1999 Academic Press.)

The Bacteroides mobilizable transposon Tn4555 is a 12.2-kb molecule that encodes resistance to cefoxitin. Conjugal transposition is hypothesized to occur via a circular intermediate and is stimulated by coresident tetracycline resistance elements and low levels of tetracycline. In this work, the ends of the transposon were identified and found to consist of 12-bp imperfect inverted repeats, with an extra base at one end. In the circular form, the ends were separated by a 6-bp "coupling sequence" which was associated with either the left or the right transposon terminus when the transposon was inserted into the chromosome. Tn4555 does not duplicate its target site upon insertion. Using a conjugation-based transposition assay, we showed that the coupling sequence originated from 6 bases of genomic DNA flanking either side of the transposon prior to excision. Tn4555 preferentially transposed into a 589-bp genomic locus containing a 207-bp direct repeat. Integration occurred before or after the repeated sequence, with one integration site between the two repeats. These observations are consistent with a transposition model based on site-specific recombination. In the bacteriophage lambda model for site-specific recombination, the bacteriophage recombines with the Escherichia coli chromosome via a 7-bp "crossover" region. We propose that the coupling sequence of Tn4555 is analogous in function to the crossover region of lambda but that unlike the situation in lambda, recombination occurs between regions of nonhomologous DNA. This ability to recombine into divergent target sites is also a feature of the gram-positive bacterial transposon Tn916.