Phages are unrecognized players in the ecology of the oral pathogen Porphyromonas gingivalis.

BACKGROUND: Porphyromonas gingivalis (hereafter "Pg") is an oral pathogen that has been hypothesized to act as a keystone driver of inflammation and periodontal disease. Although Pg is most readily recovered from individuals with actively progressing periodontal disease, healthy individuals and those with stable non-progressing disease are also colonized by Pg. Insights into the factors shaping the striking strain-level variation in Pg, and its variable associations with disease, are needed to achieve a more mechanistic understanding of periodontal disease and its progression. One of the key forces often shaping strain-level diversity in microbial communities is infection of bacteria by their viral (phage) predators and symbionts. Surprisingly, although Pg has been the subject of study for over 40 years, essentially nothing is known of its phages, and the prevailing paradigm is that phages are not important in the ecology of Pg. RESULTS: Here we systematically addressed the question of whether Pg are infected by phages-and we found that they are. We found that prophages are common in Pg, they are genomically diverse, and they encode genes that have the potential to alter Pg physiology and interactions. We found that phages represent unrecognized targets of the prevalent CRISPR-Cas defense systems in Pg, and that Pg strains encode numerous additional mechanistically diverse candidate anti-phage defense systems. We also found that phages and candidate anti-phage defense system elements together are major contributors to strain-level diversity and the species pangenome of this oral pathogen. Finally, we demonstrate that prophages harbored by a model Pg strain are active in culture, producing extracellular viral particles in broth cultures. CONCLUSION: This work definitively establishes that phages are a major unrecognized force shaping the ecology and intra-species strain-level diversity of the well-studied oral pathogen Pg. The foundational phage sequence datasets and model systems that we establish here add to the rich context of all that is already known about Pg, and point to numerous avenues of future inquiry that promise to shed new light on fundamental features of phage impacts on human health and disease broadly. Video Abstract.

Comparative Pan-Genome Analysis of Oral Veillonella Species.

The genus Veillonella is a common and abundant member of the oral microbiome. It includes eight species, V. atypica, V. denticariosi, V. dispar, V. infantium, V. nakazawae, V. parvula, V. rogosae and V. tobetusensis. They possess important metabolic pathways that utilize lactate as an energy source. However, the overall metabolome of these species has not been studied. To further understand the metabolic framework of Veillonella in the human oral microbiome, we conducted a comparative pan-genome analysis of the eight species of oral Veillonella. Analysis of the oral Veillonella pan-genome revealed features based on KEGG pathway information to adapt to the oral environment. We found that the fructose metabolic pathway was conserved in all oral Veillonella species, and oral Veillonella have conserved pathways that utilize carbohydrates other than lactate as an energy source. This discovery may help to better understand the metabolic network among oral microbiomes and will provide guidance for the design of future in silico and in vitro studies.

The salivary microbiome of diabetic and non-diabetic adults with periodontal disease.

BACKGROUND: A comparison of the salivary microbiome of non-diabetic and diabetic cohorts having periodontal health, gingivitis and periodontitis could reveal microbial signatures unique to each group that will increase understanding of the role of oral microbiota in the pathogenesis of disease, and assist with diagnosis and risk assessment for both periodontal disease and diabetes. METHODS: A group of individuals diagnosed with type 2 diabetes (T2D) was compared with a group without T2D. For both the diabetic and non-diabetic cohorts, three subgroups were established: periodontal health, gingivitis, and periodontitis. Salivary DNA was extracted (n = 146), polymerase chain reaction was performed to amplify 16S rRNA hypervariable region V3-V4, and constructed libraries were sequenced and subjected to bioinformatic and statistical analyses. RESULTS: Microbiome analysis resulted in 88 different genus level operational taxonomic units (OTUs) for differential abundance testing. Results were largely described by two trends. Trend 1 showed OTUs that increased in abundance with increasing periodontal disease, and in diabetics relative to non-diabetics. Trend 1 OTUs comprised a mix of primarily anaerobic commensals and potential periodontopathogens. Trend 2 was driven primarily by genera that decreased in abundance in those with diabetes relative to those without diabetes, which included other anaerobes associated with periodontal disease. Overall, oral microbial diversity decreased in diabetics and increased with progression of periodontal disease compared with periodontally healthy controls. CONCLUSION: Although select microbiota increased in both diabetes and periodontal disease progression, these genera decreased in co-existing diabetes and periodontal disease. These findings suggest that the genera abundance continues to change with additional stress imposed by co-existing conditions.

Establishment of a species-specific primer pair for detecting Veillonella infantium based on the 70 kDa heat shock protein gene dnaK.

Recently, Veillonella infantium was isolated from tongue biofilm of a Thai child and established as a novel Veillonella species. In this study, a species-specific primer was designed to identify V. infantium on the basis of the sequence of the 70 kDa heat shock protein (dnaK) gene of Veillonella infantium JCM 31738T (= TSD-88T). The primer pair generated a specific PCR (Polymerase Chain Reaction) product specific for V. infantium, but not for other oral Veillonella species. This specific primer pair could detect dnaK even from 1 pg of genomic DNA extracted from the V. infantium type strain. To validate the primer pair, a number of strains of Veillonella species were isolated from tongue biofilm of 3 Japanese children, DNA was isolated from each strain, and PCR was performed using species-specific primers. All oral Veillonella species except V. infantium were identified by one-step PCR method reported previously. Four kinds of Veillonella species were detected in these subjects. V. rogosae was detected in all subjects and the most predominant species with an average prevalence of 82%. However, V. infantium was detected in 2 of 3 subjects and it was the second most predominant species of oral Veillonella detected in these subjects with an average prevalence of 9.4%. V. infantium appears to coexist with other oral Veillonella species in tongue biofilm. This species-specific primer pair established in this study could be useful to detect V. infantium and support the study of Veillonella for oral health in the future.

Draft Genome Sequences of Four Strains of Recently Established Novel Veillonella Species Isolated from Human Oral Cavities.

Veillonella species are known to contribute to the formation of early oral biofilms and tend to be prevalent in people with poor oral hygiene status. Here, we report the draft genome sequences of 4 oral Veillonella strains that were established recently as novel species.

Correction: Salivary inflammatory markers and microbiome in normoglycemic lean and obese children compared to obese children with type 2 diabetes.

[This corrects the article DOI: 10.1371/journal.pone.0172647.].

Comparative genomics and evolution of the amylase-binding proteins of oral streptococci.

BACKGROUND: Successful commensal bacteria have evolved to maintain colonization in challenging environments. The oral viridans streptococci are pioneer colonizers of dental plaque biofilm. Some of these bacteria have adapted to life in the oral cavity by binding salivary α-amylase, which hydrolyzes dietary starch, thus providing a source of nutrition. Oral streptococcal species bind α-amylase by expressing a variety of amylase-binding proteins (ABPs). Here we determine the genotypic basis of amylase binding where proteins of diverse size and function share a common phenotype. RESULTS: ABPs were detected in culture supernatants of 27 of 59 strains representing 13 oral Streptococcus species screened using the amylase-ligand binding assay. N-terminal sequences from ABPs of diverse size were obtained from 18 strains representing six oral streptococcal species. Genome sequencing and BLAST searches using N-terminal sequences, protein size, and key words identified the gene associated with each ABP. Among the sequenced ABPs, 14 matched amylase-binding protein A (AbpA), 6 matched amylase-binding protein B (AbpB), and 11 unique ABPs were identified as peptidoglycan-binding, glutamine ABC-type transporter, hypothetical, or choline-binding proteins. Alignment and phylogenetic analyses performed to ascertain evolutionary relationships revealed that ABPs cluster into at least six distinct, unrelated families (AbpA, AbpB, and four novel ABPs) with no phylogenetic evidence that one group evolved from another, and no single ancestral gene found within each group. AbpA-like sequences can be divided into five subgroups based on the N-terminal sequences. Comparative genomics focusing on the abpA gene locus provides evidence of horizontal gene transfer. CONCLUSION: The acquisition of an ABP by oral streptococci provides an interesting example of adaptive evolution.

Salivary inflammatory markers and microbiome in normoglycemic lean and obese children compared to obese children with type 2 diabetes.

BACKGROUND: There is emerging evidence linking diabetes with periodontal disease. Diabetes is a well-recognized risk factor for periodontal disease. Conversely, pro-inflammatory molecules released by periodontally-diseased tissues may enter the circulation to induce insulin resistance. While this association has been demonstrated in adults, there is little information regarding periodontal status in obese children with and without type 2 diabetes (T2D). We hypothesized that children with T2D have higher rates of gingivitis, elevated salivary inflammatory markers, and an altered salivary microbiome compared to children without T2D. METHODS: Three pediatric cohorts ages 10-19 years were studied: lean (normal weight-C), obese (Ob), and obese with T2D (T2D). Each subject completed an oral health survey, received a clinical oral examination, and provided unstimulated saliva for measurement of inflammatory markers and microbiome analysis. RESULTS: The diabetes group was less likely to have had a dental visit within the last six months. Body mass index (BMI) Z-scores and waist circumference/height ratios were similar between Ob and T2D cohorts. The number of carious lesions and fillings were similar for all three groups. The gingival index was greater in the T2D group compared to the Ob and C groups. Although salivary microbial diversity was minimal between groups, a few differences in bacterial genus composition were noted. CONCLUSIONS: Obese children with T2D show a trend toward poorer oral health compared to normal weight and obese children without T2D. This study characterizes the salivary microbiome of children with and without obesity and T2D. This study supports a modest link between T2D and periodontal inflammation in the pediatric population.

A distinct sortase SrtB anchors and processes a streptococcal adhesin AbpA with a novel structural property.

Surface display of proteins by sortases in Gram-positive bacteria is crucial for bacterial fitness and virulence. We found a unique gene locus encoding an amylase-binding adhesin AbpA and a sortase B in oral streptococci. AbpA possesses a new distinct C-terminal cell wall sorting signal. We demonstrated that this C-terminal motif is required for anchoring AbpA to cell wall. In vitro and in vivo studies revealed that SrtB has dual functions, anchoring AbpA to the cell wall and processing AbpA into a ladder profile. Solution structure of AbpA determined by NMR reveals a novel structure comprising a small globular α/ß domain and an extended coiled-coil heliacal domain. Structural and biochemical studies identified key residues that are crucial for amylase binding. Taken together, our studies document a unique sortase/adhesion substrate system in streptococci adapted to the oral environment rich in salivary amylase.

Draft genome sequences of 18 oral streptococcus strains that encode amylase-binding proteins.

A number of commensal oral streptococcal species produce a heterogeneous group of proteins that mediate binding of salivary α-amylase. This interaction likely influences streptococcal colonization of the oral cavity. Here, we present draft genome sequences of several strains of oral streptococcal species that bind human salivary amylase.

Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch.

Streptococcus gordonii, a primary colonizer of the tooth surface, interacts with salivary α-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the role of AbpA in gene regulation through comparative transcriptional profiling of wild-type KS1 and AbpA(-) mutant KS1ΩabpA under various environmental conditions. A portion of the total RNA isolated from mid-log-phase cells grown in 5% CO2 in (i) complex medium with or without amylase, (ii) defined medium (DM) containing 0.8% glucose with/without amylase, and (iii) DM containing 0.2% glucose and amylase with or without starch was reverse transcribed to cDNA and the rest used for RNA sequencing. Changes in the expression of selected genes were validated by quantitative reverse transcription-PCR. Maltodextrin-associated genes, fatty acid synthesis genes and competence genes were differentially expressed in a medium-dependent manner. Genes in another cluster containing a putative histidine kinase/response regulator, peptide methionine sulfoxide reductase, thioredoxin protein, lipoprotein, and cytochrome c-type protein were downregulated in KS1ΩabpA under all of the environmental conditions tested. Thus, AbpA appears to modulate genes associated with maltodextrin utilization/transport and fatty acid synthesis. Importantly, in all growth conditions AbpA was associated with increased expression of a potential two-component signaling system associated with genes involved in reducing oxidative stress, suggesting a role in signal transduction and stress tolerance.

MyPro: A seamless pipeline for automated prokaryotic genome assembly and annotation.

MyPro is a software pipeline for high-quality prokaryotic genome assembly and annotation. It was validated on 18 oral streptococcal strains to produce submission-ready, annotated draft genomes. MyPro installed as a virtual machine and supported by updated databases will enable biologists to perform quality prokaryotic genome assembly and annotation with ease.

Porphyromonas gingivalis modulates Pseudomonas aeruginosa-induced apoptosis of respiratory epithelial cells through the STAT3 signaling pathway.

Pseudomonas aeruginosa is an important opportunistic bacterial pathogen, causing infections of respiratory and other organ systems in immunocompromised hosts that may invade and proliferate in mucosal epithelial cells to induce apoptosis. Previous studies suggest that oral bacteria, especially gram-negative periodontal pathogens, may enhance P. aeruginosa invasion into respiratory epithelial cells to augment tissue destruction. In this study, we investigated the effect of the periodontopathogen Porphyromonas gingivalis on P. aeruginosa-induced epithelial cell apoptosis. P. gingivalis invasion transiently inhibited P. aeruginosa-induced apoptosis in respiratory epithelial cells via the signal transducer and activator of transcription 3 (STAT3) signaling pathway. The activated STAT3 up-regulated the downstream anti-apoptotic moleculars survivin and B-cell leukemia-2 (bcl-2). This process was accompanied by down-regulation of pro-apoptosis molecular Bcl-2-associated death promoter (bad) and caspase-3 activity inhibition. In addition, the activation of the STAT3 pathway was affected by P. gingivalis in a dose-dependent manner. Finally, co-invasion of P. aeruginosa and P. gingivalis led to greater cell death compared with P. aeruginosa challenge alone. These results suggest that regulation of P. aeruginosa-induced apoptosis by P. gingivalis contributes to the pathogenesis of respiratory disease. Interference with this process may provide a potential therapeutic strategy for the treatment and prevention of respiratory disease.

Collagen-binding streptococcal surface proteins influence the susceptibility of biofilm cells to endodontic antimicrobial solutions.

INTRODUCTION: Previous studies have indicated that the antimicrobial efficacy of endodontic irrigants may be diminished in the presence of patient tissues and fluids. With Streptococcus gordonii as a model microorganism, we used a genetic approach to investigate the hypothesis that bacterial surface proteins with collagen-binding abilities may function to protect biofilm cells from antiseptics commonly used in root canal treatment. METHODS: S. gordonii strain DL1 or isogenic mutant strains with deletions of genes encoding collagen-binding surface proteins were grown in microtiter plates to form 8-hour biofilms. Planktonic cells were aspirated, and the remaining biofilm cells were buffer-washed and then incubated with either pH-adjusted buffer or potentially protective solutions of type I collagen, serum, or saliva. Biofilms were rewashed, pulsed with sodium hypochlorite, chlorhexidine digluconate, or BioPure MTAD, and then rewashed. Fresh medium was added, and survivor cell growth was monitored for 24 hours. RESULTS: Buffer-treated biofilm cells of all 3 strains were similarly killed by sodium hypochlorite, chlorhexidine digluconate, and MTAD. Collagen, serum, and saliva significantly protected strain DL1 from all 3 antiseptics compared with buffer-treated cells (P ≤ .0004). However, preincubation with collagen, serum, or saliva left both mutant strain biofilms significantly more susceptible to all 3 antiseptics than were respectively treated strain DL1 biofilms (P ≤ .005). CONCLUSIONS: Interactions of S. gordonii surface proteins with collagen or similar components in serum and saliva may play roles in protecting biofilm cells from endodontic antiseptics. Elucidating molecular mechanisms underlying bacterial resistance to antimicrobials may facilitate the development of more effective treatments.

Host defense proteins derived from human saliva bind to Staphylococcus aureus.

Proteins in human saliva are thought to modulate bacterial colonization of the oral cavity. Yet, information is sparse on how salivary proteins interact with systemic pathogens that transiently or permanently colonize the oral environment. Staphylococcus aureus is a pathogen that frequently colonizes the oral cavity and can cause respiratory disease in hospitalized patients at risk. Here, we investigated salivary protein binding to this organism upon exposure to saliva as a first step toward understanding the mechanism by which the organism can colonize the oral cavity of vulnerable patients. By using fluorescently labeled saliva and proteomic techniques, we demonstrated selective binding of major salivary components by S. aureus to include DMBT1(gp-340), mucin-7, secretory component, immunoglobulin A, immunoglobulin G, S100-A9, and lysozyme C. Biofilm-grown S. aureus strains bound fewer salivary components than in the planctonic state, particularly less salivary immunoglobulins. A corresponding adhesive component on the S. aureus surface responsible for binding salivary immunoglobulins was identified as staphylococcal protein A (SpA). However, SpA did not mediate binding of nonimmunoglobulin components, including mucin-7, indicating the involvement of additional bacterial surface adhesive components. These findings demonstrate that a limited number of salivary proteins, many of which are associated with various aspects of host defense, selectively bind to S. aureus and lead us to propose a possible role of saliva in colonization of the human mouth by this pathogen.

Taking the starch out of oral biofilm formation: molecular basis and functional significance of salivary α-amylase binding to oral streptococci.

α-Amylase-binding streptococci (ABS) are a heterogeneous group of commensal oral bacterial species that comprise a significant proportion of dental plaque microfloras. Salivary α-amylase, one of the most abundant proteins in human saliva, binds to the surface of these bacteria via specific surface-exposed α-amylase-binding proteins. The functional significance of α-amylase-binding proteins in oral colonization by streptococci is important for understanding how salivary components influence oral biofilm formation by these important dental plaque species. This review summarizes the results of an extensive series of studies that have sought to define the molecular basis for α-amylase binding to the surface of the bacterium as well as the biological significance of this phenomenon in dental plaque biofilm formation.

Response of fatty acid synthesis genes to the binding of human salivary amylase by Streptococcus gordonii.

Streptococcus gordonii, an important primary colonizer of dental plaque biofilm, specifically binds to salivary amylase via the surface-associated amylase-binding protein A (AbpA). We hypothesized that a function of amylase binding to S. gordonii may be to modulate the expression of chromosomal genes, which could influence bacterial survival and persistence in the oral cavity. Gene expression profiling by microarray analysis was performed to detect genes in S. gordonii strain CH1 that were differentially expressed in response to the binding of purified human salivary amylase versus exposure to purified heat-denatured amylase. Selected genes found to be differentially expressed were validated by quantitative reverse transcription-PCR (qRT-PCR). Five genes from the fatty acid synthesis (FAS) cluster were highly (10- to 35-fold) upregulated in S. gordonii CH1 cells treated with native amylase relative to those treated with denatured amylase. An abpA-deficient strain of S. gordonii exposed to amylase failed to show a response in FAS gene expression similar to that observed in the parental strain. Predicted phenotypic effects of amylase binding to S. gordonii strain CH1 (associated with increased expression of FAS genes, leading to changes in fatty acid synthesis) were noted; these included increased bacterial growth, survival at low pH, and resistance to triclosan. These changes were not observed in the amylase-exposed abpA-deficient strain, suggesting a role for AbpA in the amylase-induced phenotype. These results provide evidence that the binding of salivary amylase elicits a differential gene response in S. gordonii, resulting in a phenotypic adjustment that is potentially advantageous for bacterial survival in the oral environment.

Genetic relationships between Candida albicans strains isolated from dental plaque, trachea, and bronchoalveolar lavage fluid from mechanically ventilated intensive care unit patients.

Candida albicans often resides in the oral cavity of healthy humans as a harmless commensal organism. This opportunistic fungus can cause significant disease in critically ill patients, such as those undergoing mechanical ventilation in the intensive care unit (ICU) having compromised local airway defense mechanisms. The goal of this study was to determine the intra- and inter-patient genetic relationship between strains of C. albicans recovered from dental plaque, tracheal secretions, and the lower airway by bronchoalveolar lavage of patients undergoing mechanical ventilation. Three pulsed-field gel electrophoresis (PFGE) typing methods were used to determine the genetic relatedness of the C. albicans strains, including electrophoretic karyotyping (EK) and restriction endonuclease analysis of the genome using SfiI (REAG-S) and BssHII (REAG-B). The C. albicans isolates from dental plaque and tracheo-bronchial sites from the same patient were genetically indistinguishable and retained over time, whereas strains from different patients usually separated into different genotypes. Among the three methods, REAG-B proved to be the most discriminatory method to differentiate isolates. The finding of genetically similar strains from the oral and tracheo-bronchial sites from the same patient supports the notion that the oral cavity may serve as an important source for C. albicans spread to the trachea and lung of mechanically ventilated patients.