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1.  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.
PMCID: PMC4828917  PMID: 25911337
Bioinformatics; Prokaryote; Whole genome sequencing; Assembly; Annotation
2.  Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch 
Applied and Environmental Microbiology  2015;81(16):5363-5374.
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.
PMCID: PMC4510181  PMID: 26025889
3.  History of periodontal disease diagnosis and lung cancer incidence in the Women’s Health Initiative Observational Study 
Cancer causes & control : CCC  2014;25(8):1045-1053.
While some evidence suggests that periodontal disease (PD) might be positively associated with lung cancer, prospective studies in women are limited. Previous findings may reflect residual confounding by smoking. The study aims to determine whether history of PD diagnosis is associated with incident lung cancer in a large cohort of postmenopausal women.
Prospective analyses were conducted in a cohort of 77,485 postmenopausal women enrolled in the Women’s Health Initiative Observational Study. History of PD (prevalence of 26.1%) was self-reported and 754 incident lung cancer cases occurred during an average 6.8 (SD ±2.6) years of follow-up. Cox regression analysis was used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs).
Overall, PD was positively associated with lung cancer risk after adjusting for detailed smoking history including smoking status and pack-years of smoking (HR=1.24, 95% CI: 1.07–1.45). There was a positive additive interaction between PD with pack-years of smoking (P=0.02), suggesting a potential synergistic effect between PD and smoking intensity on lung cancer. The association between PD and lung cancer was stronger in former smokers. When restricted to never-smokers, PD was not associated with lung cancer (HR=1.02, 95% CI: 0.68–1.53).
PD was not independently associated with lung cancer in non-smoking postmenopausal women. However, smoking and PD jointly increased lung cancer risk beyond that expected from the sum of the each effect separately. The potential synergism between PD and smoking on lung cancer warrants further examination.
PMCID: PMC4117379  PMID: 24913780
lung cancer; periodontal disease; postmenopause; smoking; chronic inflammation; interaction
4.  Local Inflammation and Human Papillomavirus Status of Head and Neck Cancers 
To determine whether periodontitis is associated with human papillomavirus (HPV) status of head and neck squamous cell carcinoma (HNSCC).
Design and Setting
Hospital-based case-control study in a comprehensive cancer center.
Evaluation included all patients diagnosed with incident primary squamous cell carcinoma of the oral cavity, oropharynx, and larynx between 1999 and 2007 for whom tissue samples and dental records were available (N = 124). Patients younger than 21 years and those with a history of cancer were excluded. Periodontitis history was assessed by alveolar bone loss in millimeters from panoramic radiographs by one examiner blinded to cancer status.
Main Outcome Measure
The presence of HPV-16 DNA in paraffin-embedded tumor samples was identified by polymerase chain reaction.
The prevalence of HPV-positive HNSCC was 50 of 124 patients (40.3%). A higher proportion of oropharyngeal cancers were HPV-positive (32 of 49 [65.3%]) compared with oral cavity (9 of 31 [29.0%]) and laryngeal (9 of 44 [20.5%]) cancers. Each millimeter of alveolar bone loss was associated with 2.6 times increased odds (odds ratio [OR], 2.61; 95% CI, 1.58-4.30) of HPV-positive tumor status after adjustment for age at diagnosis, sex, and smoking status. The strength of the association was greater among patients with oropharyngeal SCC (OR, 11.70; 95% CI, 2.09-65.53) compared with those with oral cavity SCC (OR,2.32; 95% 0,0.65-8.27) and laryngeal SCC (OR, 3.89; 95% CI, 0.95-15.99).
A history of chronic inflammatory disease in the oral cavity may be associated with tumor HPV status in patients with HNSCC. This association seems to be stronger among palienLs with oropharyngeal cancer compared with those who have oral cavity or laryngeal SCC.
PMCID: PMC4477511  PMID: 22710409
5.  Draft Genome Sequences of 18 Oral Streptococcus Strains That Encode Amylase-Binding Proteins 
Genome Announcements  2015;3(3):e00510-15.
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.
PMCID: PMC4440966  PMID: 25999552
6.  Implications of salivary protein binding to commensal and pathogenic bacteria 
An important function of salivary proteins is to interact with microorganisms that enter the oral cavity. For some microbes, these interactions promote microbial colonization. For others, these interactions are deleterious and result in the elimination of the microbe from the mouth, This paper reviews recent studies of the interaction of salivary proteins with two model bacteria; the commensal species Streptococcus gordonii, and the facultative pathogen Staphylococcus aureus. These organisms selectively interact with a variety of salivary proteins to influence important functions such as bacterial adhesion to surfaces, evasion of host defense, bacterial nutrition and metabolism and gene expression.
PMCID: PMC3974197  PMID: 24707190
Saliva; microbial adhesion; biofilm; oral diseases
7.  Global Metabolomic Analysis of Human Saliva and Plasma from Healthy and Diabetic Subjects, with and without Periodontal Disease 
PLoS ONE  2014;9(8):e105181.
Recent studies suggest that periodontal disease and type 2 diabetes mellitus are bi-directionally associated. Identification of a molecular signature for periodontitis using unbiased metabolic profiling could allow identification of biomarkers to assist in the diagnosis and monitoring of both diabetes and periodontal disease. This cross-sectional study identified plasma and salivary metabolic products associated with periodontitis and/or diabetes in order to discover biomarkers that may differentiate or demonstrate an interaction of these diseases. Saliva and plasma samples were analyzed from 161 diabetic and non-diabetic human subjects with a healthy periodontium, gingivitis and periodontitis. Metabolite profiling was performed using Metabolon's platform technology. A total of 772 metabolites were found in plasma and 475 in saliva. Diabetics had significantly higher levels of glucose and α-hydroxybutyrate, the established markers of diabetes, for all periodontal groups of subjects. Comparison of healthy, gingivitis and periodontitis saliva samples within the non-diabetic group confirmed findings from previous studies that included increased levels of markers of cellular energetic stress, increased purine degradation and glutathione metabolism through increased levels of oxidized glutathione and cysteine-glutathione disulfide, markers of oxidative stress, including increased purine degradation metabolites (e.g. guanosine and inosine), increased amino acid levels suggesting protein degradation, and increased ω-3 (docosapentaenoate) and ω-6 fatty acid (linoleate and arachidonate) signatures. Differences in saliva between diabetic and non-diabetic cohorts showed altered signatures of carbohydrate, lipid and oxidative stress exist in the diabetic samples. Global untargeted metabolic profiling of human saliva in diabetics replicated the metabolite signature of periodontal disease progression in non-diabetic patients and revealed unique metabolic signatures associated with periodontal disease in diabetics. The metabolites identified in this study that discriminated the periodontal groups may be useful for developing diagnostics and therapeutics tailored to the diabetic population.
PMCID: PMC4136819  PMID: 25133529
8.  Novel Iron- and Fur-Regulated Small Regulatory RNAs in Aggregatibacter actinomycetemcomitans 
Molecular oral microbiology  2012;27(5):327-349.
Iron can regulate biofilm formation via non-coding small RNA (sRNA). To determine if iron-regulated sRNAs are involved in biofilm formation by the periodontopathogen Aggregatibacter actinomycetemcomitans, total RNA was isolated from bacteria cultured with iron supplementation or chelation. Transcriptional analysis demonstrated that the expression of four sRNA molecules (JA01-JA04) identified by bioinformatics was significantly up-regulated in iron-stressed medium compared to iron-rich medium. A DNA fragment encoding each sRNA promoter was able to titrate E. coli Fur from a Fur-repressible reporter fusion in an iron uptake regulator titration assay. Cell lysates containing recombinant AaFur shifted the mobility of sRNA-specific DNAs in a gel shift assay. Potential targets of these sRNAs, determined in silico, included genes involved in biofilm formation. A. actinomycetemcomitans overexpressing JA03 sRNA maintained a rough phenotype on agar, but no longer adhered to uncoated polystyrene or glass, although biofilm determinant gene expression was only modestly decreased. In summary, these sRNA have the ability to modulate biofilm formation, but their functional targets genes remain to be confirmed.
PMCID: PMC3442931  PMID: 22958383
iron; Fur; sRNA; biofilm; regulation; transcription
9.  Host Defense Proteins Derived from Human Saliva Bind to Staphylococcus aureus 
Infection and Immunity  2013;81(4):1364-1373.
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 DMBT1gp-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.
PMCID: PMC3639616  PMID: 23403559
10.  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.
PMCID: PMC3553756  PMID: 23144140
11.  Performance of Multiplex Cytokine Assays in Serum and Saliva among Community-Dwelling Postmenopausal Women 
PLoS ONE  2013;8(4):e59498.
Multiplexing arrays increase the throughput and decrease sample requirements for studies employing multiple biomarkers. The goal of this project was to examine the performance of Multiplex arrays for measuring multiple protein biomarkers in saliva and serum. Specimens from the OsteoPerio ancillary study of the Women’s Health Initiative Observational Study were used. Participants required the presence of at least 6 teeth and were excluded based on active cancer and certain bone issues but were not selected on any specific condition. Quality control (QC) samples were created from pooled serum and saliva. Twenty protein markers were measured on five multiplexing array panels. Sample pretreatment conditions were optimized for each panel. Recovery, lower limit of quantification (LLOQ) and imprecision were determined for each analyte. Statistical adjustment at the plate level was used to reduce imprecision estimates and increase the number of usable observations. Sample pre-treatment improved recovery estimates for many analytes. The LLOQ for each analyte agreed with manufacturer specifications except for MMP-1 and MMP-2 which were significantly higher than reported. Following batch adjustment, 17 of 20 biomarkers in serum and 9 of 20 biomarkers in saliva demonstrated acceptable precision, defined as <20% coefficient of variation (<25% at LLOQ). The percentage of cohort samples having levels within the reportable range for each analyte varied from 10% to 100%. The ratio of levels in saliva to serum varied from 1∶100 to 28∶1. Correlations between saliva and serum were of moderate positive magnitude and significant for CRP, MMP-2, insulin, adiponectin, GM-CSF and IL-5. Multiplex arrays exhibit high levels of analytical imprecision, particularly at the batch level. Careful sample pre-treatment can enhance recovery and reduce imprecision. Following statistical adjustments to reduce batch effects, we identified biomarkers that are of acceptable quality in serum and to a lesser degree in saliva using Multiplex arrays.
PMCID: PMC3618114  PMID: 23577067
12.  Genetic Relationships between Respiratory Pathogens Isolated from Dental Plaque and Bronchoalveolar Lavage Fluid from Patients in the Intensive Care Unit Undergoing Mechanical Ventilation 
Ventilator-associated pneumonia (VAP) is a leading cause of morbidity and mortality in patients hospitalized in intensive care units. Recent studies suggest that dental plaque biofilms serve as a reservoir for respiratory pathogens. The goal of this study was to determine the genetic relationship between strains of respiratory pathogens first isolated from the oral cavity and later isolated from bronchoalveolar lavage fluid from the same patient undergoing mechanical ventilation with suspected VAP.
Plaque and tracheal secretion samples were obtained on the day of hospital admission and every other day thereafter until discharge from the intensive care unit from 100 patients who underwent mechanical ventilation. Bronchoalveolar lavage was performed for 30 patients with suspected VAP. Pulse-field gel electrophoresis and multilocus sequence typing were used to determine the genetic relatedness of strains obtained from oral, tracheal, and bronchoalveolar lavage samples.
Isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter species, and enteric species recovered from plaque from most patients were indistinguishable from isolates recovered from bronchoalveolar lavage fluid (i.e., had >95% similarity of pulse-field gel electrophoresis patterns). Nearly one-half of the Pseudomonas strains showed identical genetic profiles between patients, which suggested a common environmental source of infection.
Respiratory pathogens isolated from the lung are often genetically indistinguishable from strains of the same species isolated from the oral cavity in patients who receive mechanical ventilation who are admitted to the hospital from the community. Thus, dental plaque serves as an important reservoir for respiratory pathogens in patients who undergo mechanical ventilation.
Trial registration identifier: NCT00123123
PMCID: PMC3582026  PMID: 18991508
13.  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.
PMCID: PMC3298122  PMID: 22247133
14.  Identification and characterization of amylase binding protein C (AbpC) from Streptococcus mitis NS51 
Molecular oral microbiology  2010;25(2):150-156.
A substantial proportion of the streptococcal species found in dental plaque biofilms are able to interact with the abundant salivary enzyme α-amylase. These streptococci produce proteins that specifically bind amylase. An important plaque species, Streptococcus mitis, secretes a 36-kDa amylase binding protein into the extracellular milieu. Proteins precipitated from S. mitis NS51 cell culture supernatant by the addition of purified salivary amylase were separated by SDS-PAGE, transferred to a membrane, and a prominent 36-kDa band was cut from the membrane and sequencedto yield N-terminal amino acid sequence DSQAQYSNGV. Search of the S. mitis genome sequence database revealed a single open reading frame containing this sequence, and the gene was amplified from S. mitis genomic DNA polymerase chain reaction. The coding region of this ORF, designated amylase-binding protein C (AbpC), was cloned into an Escherichia coli expression vectorand the recombinant AbpC protein (rAbpC) was purified from the soluble fraction of E. coli cell lysate. Purified AbpC was found to interact with immobilized amylase, thus confirming AbpC as a new streptococcal amylase-binding protein.
PMCID: PMC2862347  PMID: 20331802
Dental plaque; Saliva
15.  Genetic relationships between Candida albicans strains isolated from dental plaque, trachea, and bronchoalveolar lavage fluid from mechanically ventilated intensive care unit patients 
Journal of Oral Microbiology  2011;3:10.3402/jom.v3i0.6362.
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.
PMCID: PMC3124833  PMID: 21731911
yeast; pulsed-field gel electrophoresis (PFGE); molecular epidemiology; mechanical ventilation; oral cavity
16.  Transcriptional and Translational Analysis of Biofilm Determinants of Aggregatibacter actinomycetemcomitans in Response to Environmental Perturbation ▿  
Infection and Immunity  2009;77(7):2896-2907.
Fimbriae, lipopolysaccharide (LPS), and extracellular polymeric substance (EPS) all contribute to biofilm formation by the periodontopathogen Aggregatibacter actinomycetemcomitans. To understand how individual biofilm determinants respond to changing environmental conditions, the transcription of genes responsible for fimbria, LPS, and EPS production, as well as the translation of these components, was determined in rough (Rv) and isogenic smooth (Sv) variants of A. actinomycetemcomitans cultured in half-strength and full-strength culture medium under anaerobic or aerobic conditions, and in iron-supplemented and iron-chelated medium. The transcription of tadV (fimbrial assembly), pgaC (extracellular polysaccharide synthesis), and orf8 or rmlB (lipopolysaccharide synthesis) was measured by real-time PCR. The amounts of fimbriae, LPS, and EPS were also estimated from stained sodium dodecyl sulfate-polyacrylamide gels and verified by Western blotting and enzyme-linked immunoadsorbent assay using specific antibodies. Each gene was significantly upregulated in the Rv compared to in the Sv. The transcription of fimbrial, LPS, and EPS genes in the Rv was increased approximately twofold in cells cultured in full-strength medium under anaerobic conditions compared to that in cells cultured under aerobic conditions. Under anaerobic conditions, the transcription of fimbrial and EPS enzymes was elevated in both Rv and Sv cells cultured in half-strength medium, compared to that in full-strength medium. Iron chelation also increased the transcription and translation of all biofilm determinants compared to their expression with iron supplementation, yet the quantity of biofilm was not significantly changed by any environmental perturbation except iron limitation. Thus, anaerobic conditions, nutrient stress, and iron limitation each upregulate known biofilm determinants of A. actinomycetemcomitans to contribute to biofilm formation.
PMCID: PMC2708548  PMID: 19433550
17.  A randomized trial of chlorhexidine gluconate on oral bacterial pathogens in mechanically ventilated patients 
Critical Care  2009;13(4):R117.
Dental plaque biofilms are colonized by respiratory pathogens in mechanically-ventilated intensive care unit patients. Thus, improvements in oral hygiene in these patients may prevent ventilator-associated pneumonia. The goal of this study was to determine the minimum frequency (once or twice a day) for 0.12% chlorhexidine gluconate application necessary to reduce oral colonization by pathogens in 175 intubated patients in a trauma intensive care unit.
A randomized, double-blind, placebo-controlled clinical trial tested oral topical 0.12% chlorhexidine gluconate or placebo (vehicle alone), applied once or twice a day by staff nurses. Quantitation of colonization of the oral cavity by respiratory pathogens (teeth/denture/buccal mucosa) was measured.
Subjects were recruited from 1 March, 2004 until 30 November, 2007. While 175 subjects were randomized, microbiologic baseline data was available for 146 subjects, with 115 subjects having full outcome assessment after at least 48 hours. Chlorhexidine reduced the number of Staphylococcus aureus, but not the total number of enterics, Pseudomonas or Acinetobacter in the dental plaque of test subjects. A non-significant reduction in pneumonia rate was noted in groups treated with chlorhexidine compared with the placebo group (OR = 0.54, 95% CI: 0.23 to 1.25, P = 0.15). No evidence for resistance to chlorhexidine was noted, and no adverse events were observed. No differences were noted in microbiologic or clinical outcomes between treatment arms.
While decontamination of the oral cavity with chlorhexidine did not reduce the total number of potential respiratory pathogens, it did reduce the number of S. aureus in dental plaque of trauma intensive care patients.
Trial Registration NCT00123123.
PMCID: PMC2750165  PMID: 19765321
18.  Amylase-Binding Protein B of Streptococcus gordonii Is an Extracellular Dipeptidyl-Peptidase▿  
Infection and Immunity  2008;76(10):4530-4537.
The oral commensal bacterium Streptococcus gordonii interacts with salivary amylase via two amylase-binding proteins, AbpA and AbpB. Based on sequence analysis, the 20-kDa AbpA protein is unique to S. gordonii, whereas the 82-kDa AbpB protein appears to share sequence homology with other bacterial dipeptidases. The aim of this study was to verify the peptidase activity of AbpB and further explore its potential functions. The abpB gene was cloned, and histidine-tagged AbpB (His-AbpB) was expressed in Escherichia coli and purified. Its amylase-binding activity was verified in an amylase ligand binding assay, and its cross-reactivity was verified with an anti-AbpB antibody. Both recombinant His-AbpB and partially purified native AbpB displayed dipeptidase activity and degraded human type VI collagen and fibrinogen, but not salivary amylase. Salivary amylase precipitates not only AbpA and AbpB but also glucosyltransferase G (Gtf-G) from S. gordonii supernatants. Since Streptococcus mutans also releases Gtf enzymes that could also be involved in multispecies plaque interactions, the effect of S. gordonii AbpB on S. mutans Gtf-B activity was also tested. Salivary amylase and/or His-AbpB caused a 1.4- to 2-fold increase of S. mutans Gtf-B sucrase activity and a 3- to 6-fold increase in transferase activity. An enzyme-linked immunosorbent assay verified the interaction of His-AbpB and amylase with Gtf-B. In summary, AbpB demonstrates proteolytic activity and interacts with and modulates Gtf activity. These activities may help explain the crucial role AbpB appears to play in S. gordonii oral colonization.
PMCID: PMC2546850  PMID: 18678669
20.  Candidate salivary biomarkers associated with alveolar bone loss: cross-sectional and in vitro studies 
This cross-sectional study evaluated the association between radiographic evidence of alveolar bone loss and the concentration of host-derived bone resorptive factors (interleukin-1 beta, tumor necrosis factor-alpha, interleukin-6, prostaglandin-E2), and markers of bone turnover [pyridinoline cross-linked carboxyterminal telopeptide of type I collagen (ICTP), osteocalcin, osteonectin] in stimulated human whole saliva collected from 110 untreated dental patients. Alveolar bone loss scores for each patient were derived from radiographic examination. Variables positively associated with increased bone loss score were: age, current smoking, use of bisphosphonate drugs, and salivary interleukin-1beta levels above the median. Salivary osteonectin levels above the median were associated with a decreased bone loss score. Additional in vitro studies were carried out to determine the fate of interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha added to whole and parotid saliva. All cytokines added to saliva were detected in significantly lower concentrations than when added to buffer alone. Protease inhibitors added to saliva did not prevent the reduction in detection of biomarkers. Variation in time of incubation, repeated cycles of freezing and thawing, or exposure to dimethylsulfoxide did not appreciably affect the measurement of cytokines in saliva. These results suggest that detection of biomarkers by conventional immunoassays may underestimate the actual quantity of molecules in saliva.
PMCID: PMC2258090  PMID: 17328758
periodontal disease; salivary biomarkers; pro-inflammatory cytokines; bone turnover
21.  Interaction of Salivary alpha-Amylase and Amylase-Binding-Protein A (AbpA) of Streptococcus gordonii with Glucosyltransferase of S. gordonii and Streptococcus mutans 
BMC Microbiology  2007;7:60.
Glucosyltransferases (Gtfs), enzymes that produce extracellular glucans from dietary sucrose, contribute to dental plaque formation by Streptococcus gordonii and Streptococcus mutans. The alpha-amylase-binding protein A (AbpA) of S. gordonii, an early colonizing bacterium in dental plaque, interacts with salivary amylase and may influence dental plaque formation by this organism. We examined the interaction of amylase and recombinant AbpA (rAbpA), together with Gtfs of S. gordonii and S. mutans.
The addition of salivary alpha-amylase to culture supernatants of S. gordonii precipitated a protein complex containing amylase, AbpA, amylase-binding protein B (AbpB), and the glucosyltransferase produced by S. gordonii (Gtf-G). rAbpA was expressed from an inducible plasmid, purified from Escherichia coli and characterized. Purified rAbpA, along with purified amylase, interacted with and precipitated Gtfs from culture supernatants of both S. gordonii and S. mutans. The presence of amylase and/or rAbpA increased both the sucrase and transferase component activities of S. mutans Gtf-B. Enzyme-linked immunosorbent assay (ELISA) using anti-Gtf-B antibody verified the interaction of rAbpA and amylase with Gtf-B. A S. gordonii abpA-deficient mutant showed greater biofilm growth under static conditions than wild-type in the presence of sucrose. Interestingly, biofilm formation by every strain was inhibited in the presence of saliva.
The results suggest that an extracellular protein network of AbpA-amylase-Gtf may influence the ecology of oral biofilms, likely during initial phases of colonization.
PMCID: PMC3225810  PMID: 17593303
22.  Role of Streptococcus gordonii Amylase-Binding Protein A in Adhesion to Hydroxyapatite, Starch Metabolism, and Biofilm Formation 
Infection and Immunity  2001;69(11):7046-7056.
Interactions between bacteria and salivary components are thought to be important in the establishment and ecology of the oral microflora. α-Amylase, the predominant salivary enzyme in humans, binds to Streptococcus gordonii, a primary colonizer of the tooth. Previous studies have implicated this interaction in adhesion of the bacteria to salivary pellicles, catabolism of dietary starches, and biofilm formation. Amylase binding is mediated at least in part by the amylase-binding protein A (AbpA). To study the function of this protein, an erythromycin resistance determinant [erm(AM)] was inserted within the abpA gene of S. gordonii strains Challis and FAS4 by allelic exchange, resulting in abpA mutant strains Challis-E1 and FAS4-E1. Comparison of the wild-type and mutant strains did not reveal any significant differences in colony morphology, biochemical metabolic profiles, growth in complex or defined media, surface hydrophobicity, or coaggregation properties. Scatchard analysis of adhesion isotherms demonstrated that the wild-type strains adhered better to human parotid-saliva- and amylase-coated hydroxyapatite than did the AbpA mutants. In contrast, the mutant strains bound to whole-saliva-coated hydroxyapatite to a greater extent than did the wild-type strains. While the wild-type strains preincubated with purified salivary amylase grew well in defined medium with potato starch as the sole carbohydrate source, the AbpA mutants did not grow under the same conditions even after preincubation with amylase. In addition, the wild-type strain produced large microcolonies in a flow cell biofilm model, while the abpA mutant strains grew much more poorly and produced relatively small microcolonies. Taken together, these results suggest that AbpA of S. gordonii functions as an adhesin to amylase-coated hydroxyapatite, in salivary-amylase-mediated catabolism of dietary starches and in human saliva-supported biofilm formation by S. gordonii.
PMCID: PMC100085  PMID: 11598080
23.  RegG, a CcpA Homolog, Participates in Regulation of Amylase-Binding Protein A Gene (abpA) Expression in Streptococcus gordonii 
Journal of Bacteriology  2001;183(11):3521-3525.
The amylase-binding protein A (AbpA) of Streptococcus gordonii was found to be undetectable in supernatants of mid-log-phase cultures containing >1% glucose but abundant in supernatants of cultures made with brain heart infusion (BHI), which contains 0.2% glucose. A 10-fold decrease in the level of abpA mRNA in S. gordonii cells cultured in BHI was noted after the addition of glucose to 1%. Analysis of the abpA sequence revealed a potential catabolite responsive element CRE 153 bp downstream of the putative translational start site. A catabolite control protein A gene (ccpA) homolog from S. gordonii, designated regG, was cloned. A regG mutant strain demonstrated moderately less repression of abpA transcription in the presence of 1% glucose. Diauxic growth with glucose and lactose was not affected in the RegG mutant compared to the wild-type parental strain. These results suggest that while RegG plays a role in abpA expression, other mechanisms of catabolite repression are present.
PMCID: PMC99651  PMID: 11344161
24.  Prevalence of the Amylase-Binding Protein A Gene (abpA) in Oral Streptococci 
Journal of Clinical Microbiology  1999;37(12):4081-4085.
Salivary amylase binds specifically to a number of oral streptococcal species. This interaction may play an important role in dental plaque formation. Recently, a 585-bp gene was cloned and sequenced from Streptococcus gordonii Challis encoding a 20.5-kDa amylase-binding protein (AbpA). The goal of this study was to determine if related genes are present in other species of oral streptococci. Biotinylated abpA was used in Southern blot analysis to screen genomic DNA from several strains representing eight species of oral streptococci. This probe hybridized with a 4.0-kb HindIII restriction fragment from all 13 strains of S. gordonii tested. The probe did not appear to bind to any restriction fragments from other species of amylase-binding oral streptococci including Streptococcus mitis (with the exception of 1 of 14 strains), Streptococcus crista (3 strains), Streptococcus anginosus (1 strain), and Streptococcus parasanguinis (1 strain), or to non-amylase-binding oral streptococci including Streptococcus sanguinis (3 strains), Streptococcus oralis (4 strains), and Streptococcus mutans (1 strain). Primers homologous to sequences within the 3′ and 5′ ends of abpA yielded products of 400 bp following PCR of genomic DNA from the Southern blot-positive strains. Several of these PCR products were cloned and sequenced. The levels of similarity of these cloned products to the abpA of S. gordonii Challis ranged from 91 to 96%. These studies reveal that the abpA gene appears to be specific to S. gordonii and differs from genes encoding amylase-binding proteins from other species of amylase-binding streptococci.
PMCID: PMC85885  PMID: 10565935
25.  Identification and Molecular Analysis of Rough-Colony-Specific Outer Membrane Proteins of Actinobacillus actinomycetemcomitans 
Infection and Immunity  1999;67(6):2901-2908.
Actinobacillus actinomycetemcomitans, a gram-negative bacterium isolated from the human mouth, has been implicated in the pathogenesis of early-onset periodontitis. Primary isolates cultured from subgingival plaque exhibit an adherent, rough colony phenotype which spontaneously converts to a nonadherent, smooth phenotype upon in vitro subculture. The rough colony variant produces abundant fimbriae and autoaggregates, while the smooth colony variant is planktonic and produces scant fimbriae. To begin to understand the significance of colony variation in biofilm formation by A. actinomycetemcomitans, outer membrane protein profiles of four isogenic rough and smooth colony variants were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two proteins with relative molecular masses of 43 and 20 kDa were expressed by the rough colony variants exclusively. Expression of these proteins was not found to be dependent on growth phase, oxygen tension, or type of complex medium. N-terminal amino acid sequences of these proteins obtained by Edman degradation were compared with sequences from the University of Oklahoma A. actinomycetemcomitans genome database. Two contiguous open reading frames (ORFs) encoding proteins having sequence homology with these proteins were identified. The 43-kDa protein (RcpA [rough colony protein A]) was similar to precursor protein D of the general secretion pathway of gram-negative bacilli, while the 20-kDa protein (RcpB [rough colony protein B]) appeared to be unique. The genes encoding these proteins have been cloned from A. actinomycetemcomitans 283 and sequenced. A BLASTX (gapped BLAST) search of the surrounding ORFs revealed homology with other fimbria-related proteins. These data suggest that the genes encoding the 43-kDa (rcpA) and 20-kDa (rcpB) proteins may be functionally related to each other and to genes that may encode fimbria-associated proteins.
PMCID: PMC96598  PMID: 10338497

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