Dilani B. Senadheera

Bacterial biogeography of the human digestive tract

We present bacterial biogeography as sampled from the human gastrointestinal tract of four healthy subjects. This study generated >32 million paired-end sequences of bacterial 16S rRNA genes (V3 region) representing >95,000 unique operational taxonomic units (OTUs; 97% similarity clusters), with >99% Goods coverage for all samples. The highest OTU richness and phylogenetic diversity was found in the mouth samples. The microbial communities of multiple biopsy sites within the colon were highly similar within individuals and largely distinct from those in stool. Within an individual, OTU overlap among broad site definitions (mouth, stomach/duodenum, colon and stool) ranged from 32–110 OTUs, 25 of which were common to all individuals and included OTUs affiliated with Faecalibacterium prasnitzii and the TM7 phylum. This first comprehensive characterization of the abundant and rare microflora found along the healthy human digestive tract represents essential groundwork to investigate further how the human microbiome relates to health and disease.

Mechanistic Insights Revealed by the Crystal Structure of a Histidine Kinase with Signal Transducer and Sensor Domains

A crystal structure reveals an elegant mechanistic switch whereby helical bending and catalytic domain rotation allow self-activation of a histidine kinase during a bacterial stress response.

Inactivation of VicK Affects Acid Production and Acid Survival of Streptococcus mutans

The regulation of acid production in and the tolerance to low pH of the cariogenic bacterium Streptococcus mutans have garnered considerable attention since both of these properties contribute substantially to the virulence of this organism. Frequent or prolonged exposure to acid end products, mainly lactic acid, that are present following the consumption of dietary sugars erodes the dental enamel, thereby initiating dental caries. Here we report the involvement of the S. mutans VicK sensor kinase in both the acidogenicity and the aciduricity of this bacterium. When cultures were supplemented with glucose, the glycolytic rate of a VicK null mutant was significantly decreased compared to the glycolytic rate of the wild type (P < 0.05), suggesting that there was impaired acid production. Not surprisingly, the VicK deletion mutant produced less lactic acid, while an acid tolerance response assay revealed that loss of VicK significantly enhanced the survival of S. mutans (P < 0.05). Compared to the survival rates of the wild type, the survival rates of the VicK-deficient mutant were drastically increased when cultures were grown at pH 3.5 with or without preexposure to a signal pH (pH 5.5). Global transcriptional analysis using DNA microarrays and S. mutans wild-type UA159 and VicK deletion mutant strains grown at neutral and low pH values revealed that loss of VicK significantly affected expression of 89 transcripts more than twofold at pH 5.5 (P < 0.001). The affected transcripts included genes with putative functions in transport and maintenance of cell membrane integrity. While our results provide insight into the acid-inducible regulon of S. mutans, here we imply a novel role for VicK in regulating intracellular pH homeostasis in S. mutans.

Regulation of Bacteriocin Production and Cell Death by the VicRK Signaling System in Streptococcus mutans

The VicRK two-component signaling system modulates biofilm formation, genetic competence, and stress tolerance in Streptococcus mutans. We show here that the VicRK modulates bacteriocin production and cell viability, in part by direct modulation of competence-stimulating peptide (CSP) production in S. mutans. Global transcriptome and real-time transcriptional analysis of the VicK-deficient mutant (SmuvicK) revealed significant modulation of several bacteriocin-related loci, including nlmAB, nlmC, and nlmD (P < 0.001), suggesting a role for the VicRK in producing mutacins IV, V, and VI. Bacteriocin overlay assays revealed an altered ability of the vic mutants to kill related species. Since a well-conserved VicR binding site (TGTWAH-N(5)-TGTWAH) was identified within the comC coding region, we confirmed VicR binding to this sequence using DNA footprinting. Overexpression of the vic operon caused growth-phase-dependent repression of comC, comDE, and comX. In the vic mutants, transcription of nlmC/cipB encoding mutacin V, previously linked to CSP-dependent cell lysis, as well as expression of its putative immunity factor encoded by immB, were significantly affected relative to the wild type (P < 0.05). In contrast to previous reports that proposed a hyper-resistant phenotype for the VicK mutant in cell viability, the release of extracellular genomic DNA was significantly enhanced in SmuvicK (P < 0.05), likely as a result of increased autolysis compared with the parent. The drastic influence of VicRK on cell viability was also demonstrated using vic mutant biofilms. Taken together, we have identified a novel regulatory link between the VicRK and ComDE systems to modulate bacteriocin production and cell viability of S. mutans.

CHARACTERIZATION OF DNA BINDING SITES OF THE COME RESPONSE REGULATOR FROM STREPTOCOCCUS MUTANS

In Streptococcus mutans, both competence and bacteriocin production are controlled by ComC and the ComED two-component signal transduction system. Recent studies of S. mutans suggested that purified ComE binds to two 11-bp direct repeats in the nlmC-comC promoter region, where ComE activates nlmC and represses comC. In this work, quantitative binding studies and DNase I footprinting analysis were performed to calculate the equilibrium dissociation constant and further characterize the binding site of ComE. We found that ComE protects sequences inclusive of both direct repeats, has an equilibrium dissociation constant in the nanomolar range, and binds to these two direct repeats cooperatively. Furthermore, similar direct repeats were found upstream of cslAB, comED, comX, ftf, vicRKX, gtfD, gtfB, gtfC, and gbpB. Quantitative binding studies were performed on each of these sequences and showed that only cslAB has a similar specificity and high affinity for ComE as that seen with the upstream region of comC. A mutational analysis of the binding sequences showed that ComE does not require both repeats to bind DNA with high affinity, suggesting that single site sequences in the genome may be targets for ComE-mediated regulation. Based on the mutational analysis and DNase I footprinting analysis, we propose a consensus ComE binding site, TCBTAAAYSGT.

Characterization of a Glutamate Transporter Operon, glnQHMP, in Streptococcus mutans and Its Role in Acid Tolerance

Glutamate contributes to the acid tolerance response (ATR) of many Gram-negative and Gram-positive bacteria, but its role in the ATR of the oral bacterium Streptococcus mutans is unknown. This study describes the discovery and characterization of a glutamate transporter operon designated glnQHMP (Smu.1519 to Smu.1522) and investigates its potential role in acid tolerance. Deletion of glnQHMP resulted in a 95% reduction in transport of radiolabeled glutamate compared to the wild-type UA159 strain. The addition of glutamate to metabolizing UA159 cells resulted in an increased production of acidic end products, whereas the glnQHMP mutant produced less lactic acid than UA159, suggesting a link between glutamate metabolism and acid production and possible acid tolerance. To investigate this possibility, we conducted a microarray analysis with glutamate and under pH 5.5 and pH 7.5 conditions which showed that expression of the glnQHMP operon was downregulated by both glutamate and mild acid. We also measured the growth kinetics of UA159 and its glnQHMP-negative derivative at pH 5.5 and found that the mutant doubled at a much slower rate than the parent strain but survived at pH 3.5 significantly better than the wild type. Taken together, these findings support the involvement of the glutamate transporter operon glnQHMP in the acid tolerance response in S. mutans.

Bacterial community composition of chronic periodontitis and novel oral sampling sites for detecting disease indicators

BackgroundPeriodontitis is an infectious and inflammatory disease of polymicrobial etiology that can lead to the destruction of bones and tissues that support the teeth. The management of chronic periodontitis (CP) relies heavily on elimination or at least control of known pathogenic consortia associated with the disease. Until now, microbial plaque obtained from the subgingival (SubG) sites has been the primary focus for bacterial community analysis using deep sequencing. In addition to the use of SubG plaque, here, we investigated whether plaque obtained from supragingival (SupG) and tongue dorsum sites can serve as alternatives for monitoring CP-associated bacterial biomarkers.ResultsUsing SubG, SupG, and tongue plaque DNA from 11 healthy and 13 diseased subjects, we sequenced V3 regions (approximately 200 bases) of the 16S rRNA gene using Illumina sequencing. After quality filtering, approximately 4.1 million sequences were collapsed into operational taxonomic units (OTUs; sequence identity cutoff of >97%) that were classified to a total of 19 phyla spanning 114 genera. Bacterial community diversity and overall composition was not affected by health or disease, and multiresponse permutation procedure (MRPP) on Bray-Curtis distance measures only supported weakly distinct bacterial communities in SubG and tongue plaque depending on health or disease status (P < 0.05). Nonetheless, in SubG and tongue sites, the relative abundance of Firmicutes was increased significantly from health to disease and members of Synergistetes were found in higher abundance across all sites in disease. Taxa indicative of CP were identified in all three locations (for example, Treponema denticola, Porphyromonas gingivalis, Synergistes oral taxa 362 and 363).ConclusionsFor the first time, this study demonstrates that SupG and tongue dorsum plaque can serve as alternative sources for detecting and enumerating known and novel bacterial biomarkers of CP. This finding is clinically important because, in contrast with SubG sampling that requires trained professionals, obtaining plaque from SupG and tongue sites is convenient and minimally-invasive and offers a novel means to track CP-biomarker organisms during treatment outcome monitoring.

Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology

CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacterium Streptococcus mutans UA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of other S. mutans. The deletion of the cas genes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) in S. mutans UA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation of S. mutans by the plasmids matching the spacers 2 and 3. Functional analysis of the cas deletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology in S. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression of cas genes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we provide in vivo evidence that the type II-A CRISPR-Cas system of S. mutans may be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

Inter-personal diversity and temporal dynamics of dental, tongue, and salivary microbiota in the healthy oral cavity

Oral microbes form a complex and dynamic biofilm community, which is subjected to daily host and environmental challenges. Dysbiosis of the oral biofilm is correlated with local and distal infections and postulating a baseline for the healthy core oral microbiota provides an opportunity to examine such shifts during the onset and recurrence of disease. Here we quantified the daily, weekly, and monthly variability of the oral microbiome by sequencing the largest oral microbiota time-series to date, covering multiple oral sites in ten healthy individuals. Temporal dynamics of salivary, dental, and tongue consortia were examined by high-throughput 16S rRNA gene sequencing over 90 days, with four individuals sampled additionally 1 year later. Distinct communities were observed between dental, tongue, and salivary samples, with high levels of similarity observed between the tongue and salivary communities. Twenty-six core OTUs that classified within Streptococcus, Fusobacterium, Haemophilus, Neisseria, Prevotella, and Rothia genera were present in ≥95% samples and accounted for ~65% of the total sequence data. Phylogenetic diversity varied from person to person, but remained relatively stable within individuals over time compared to inter-individual variation. In contrast, the composition of rare microorganisms was highly variable over time, within most individuals. Using machine learning, an individuals oral microbial assemblage could be correctly assigned to them with 88–97% accuracy, depending on the sample site; 83% of samples taken a year after initial sampling could be confidently traced back to the source subject.Peering into the mouth: Bacterial turnover in plaque and salivaA study of bacteria in the mouth reveals insights into their diversity, stability, and variability among people and over time. By tracking daily, weekly, and monthly fluctuations of plaque and salivary bacteria in ten healthy volunteers, Dilani Senadheera at the Faculty of Dentistry, University of Toronto and co-researchers in Canada reveal significant differences in the “microbiome” present in dental, tongue and saliva samples over time. They found considerable variation in these communities between individuals, sufficient to identify a person with “bacterial fingerprints” using plaque or saliva even after 1 year. The researchers reveal a “core community” that spans different persons, oral sites, and time, suggesting some level of stability. This study is useful to understand the diversity and community drifts in different oral sites over time, which is important when plaque and saliva are used for bacterial analysis in diagnostic, risk-prediction, and forensic applications.

The copYAZ operon functions in copper efflux, biofilm formation, genetic transformation and stress tolerance in Streptococcus mutans

UNLABELLED In bacteria, copper homeostasis is closely monitored to ensure proper cellular functions while avoiding cell damage. Most Gram-positive bacteria utilize the copYABZ operon for copper homeostasis, where copA and copB encode copper-transporting P-type ATPases, whereas copY and copZ regulate the expression of the cop operon. Streptococcus mutans is a biofilm-forming oral pathogen that harbors a putative copper-transporting copYAZ operon. Here, we characterized the role of copYAZ operon in the physiology of S. mutans and delineated the mechanisms of copper-induced toxicity in this bacterium. We observed that copper induced toxicity in S. mutans cells by generating oxidative stress and disrupting their membrane potential. Deletion of the copYAZ operon in S. mutans strain UA159 resulted in reduced cell viability under copper, acid, and oxidative stress relative to the viability of the wild type under these conditions. Furthermore, the ability of S. mutans to form biofilms and develop genetic competence was impaired under copper stress. Briefly, copper stress significantly reduced cell adherence and total biofilm biomass, concomitantly repressing the transcription of the gtfB, gtfC, gtfD, gbpB, and gbpC genes, whose products have roles in maintaining the structural and/or functional integrity of the S. mutans biofilm. Furthermore, supplementation with copper or loss of copYAZ resulted in significant reductions in transformability and in the transcription of competence-associated genes. Copper transport assays revealed that the ΔcopYAZ strain accrued significantly large amounts of intracellular copper compared with the amount of copper accumulation in the wild-type strain, thereby demonstrating a role for CopYAZ in the copper efflux of S. mutans. The complementation of the CopYAZ system restored copper expulsion, membrane potential, and stress tolerance in the copYAZ-null mutant. Taking these results collectively, we have established the function of the S. mutans CopYAZ system in copper export and have further expanded knowledge on the importance of copper homeostasis and the CopYAZ system in modulating streptococcal physiology, including stress tolerance, membrane potential, genetic competence, and biofilm formation. IMPORTANCE S. mutans is best known for its role in the initiation and progression of human dental caries, one of the most common chronic diseases worldwide. S. mutans is also implicated in bacterial endocarditis, a life-threatening inflammation of the heart valve. The core virulence factors of S. mutans include its ability to produce and sustain acidic conditions and to form a polysaccharide-encased biofilm that provides protection against environmental insults. Here, we demonstrate that the addition of copper and/or deletion of copYAZ (the copper homeostasis system) have serious implications in modulating biofilm formation, stress tolerance, and genetic transformation in S. mutans. Manipulating the pathways affected by copper and the copYAZ system may help to develop potential therapeutics to prevent S. mutans infection in and beyond the oral cavity.