Deep metaproteomic analysis of human salivary supernatant.

The human salivary proteome is extremely complex, including proteins from salivary glands, serum, and oral microbes. Much has been learned about the host component, but little is known about the microbial component. Here we report a metaproteomic analysis of salivary supernatant pooled from six healthy subjects. For deep interrogation of the salivary proteome, we combined protein dynamic range compression (DRC), multidimensional peptide fractionation, and high-mass accuracy MS/MS with a novel two-step peptide identification method using a database of human proteins plus those translated from oral microbe genomes. Peptides were identified from 124 microbial species as well as uncultured phylotypes such as TM7. Streptococcus, Rothia, Actinomyces, Prevotella, Neisseria, Veilonella, Lactobacillus, Selenomonas, Pseudomonas, Staphylococcus, and Campylobacter were abundant among the 65 genera from 12 phyla represented. Taxonomic diversity in our study was broadly consistent with metagenomic studies of saliva. Proteins mapped to 20 KEGG pathways, with carbohydrate metabolism, amino acid metabolism, energy metabolism, translation, membrane transport, and signal transduction most represented. The communities sampled appear to be actively engaged in glycolysis and protein synthesis. This first deep metaproteomic catalog from human salivary supernatant provides a baseline for future studies of shifts in microbial diversity and protein activities potentially associated with oral disease.

Functional signatures of ex-vivo dental caries onset.

Background: The etiology of dental caries remains poorly understood. With the advent of next-generation sequencing, a number of studies have focused on the microbial ecology of the disease. However, taxonomic associations with caries have not been consistent. Researchers have also pursued function-centric studies of the caries microbial communities aiming to identify consistently conserved functional pathways. A major question is whether changes in microbiome are a cause or a consequence of the disease. Thus, there is a critical need to define conserved functional signatures at the onset of dental caries. Methods: Since it is unethical to induce carious lesions clinically, we developed an innovative longitudinal ex-vivo model integrated with the advanced non-invasive multiphoton second harmonic generation bioimaging to spot the very early signs of dental caries, combined with 16S rRNA short amplicon sequencing and liquid chromatography-mass spectrometry-based targeted metabolomics. Findings: For the first time, we induced longitudinally monitored caries lesions validated with the scanning electron microscope. Consequently, we spotted the caries onset and, associated with it, distinguished five differentiating metabolites - Lactate, Pyruvate, Dihydroxyacetone phosphate, Glyceraldehyde 3-phosphate (upregulated) and Fumarate (downregulated). Those metabolites co-occurred with certain bacterial taxa; Streptococcus, Veillonella, Actinomyces, Porphyromonas, Fusobacterium, and Granulicatella, regardless of the abundance of other taxa. Interpretation: These findings are crucial for understanding the etiology and dynamics of dental caries, and devising targeted interventions to prevent disease progression.

Persistence of extracrevicular bacterial reservoirs after treatment of aggressive periodontitis.

BACKGROUND: The purpose of this study was to test the hypothesis that periodontal pathogens associated with aggressive periodontitis persist in extracrevicular locations following scaling and root planing, systemic antibiotics, and antimicrobial rinses. METHODS: Eighteen patients with aggressive periodontitis received a clinical examination during which samples of subgingival plaque and buccal epithelial cells were obtained. Treatment consisted of full-mouth root planing, systemic antibiotics, and chlorhexidine rinses. Clinical measurements and sampling were repeated at 3 and 6 months. Quantitative polymerase chain reaction determined the number of Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia (previously T. forsythensis), and Treponema denticola in the plaque. Fluorescence in situ hybridization and confocal microscopy determined the extent of intracellular invasion in epithelial cells. RESULTS: Clinical measurements improved significantly following treatment. All bacterial species except P. gingivalis were significantly reduced in plaque between baseline and 3 months. However, all species showed a trend to repopulate between 3 and 6 months. This increase was statistically significant for log T. denticola counts. All species were detected intracellularly. The percentage of cells infected intracellularly was not affected by therapy. CONCLUSIONS: The 6-month increasing trend in the levels of plaque bacteria suggests that subgingival recolonization was occurring. Because the presence of these species within epithelial cells was not altered after treatment, it is plausible that recolonization may occur from the oral mucosa. Systemic antibiotics and topical chlorhexidine did not reduce the percentage of invaded epithelial cells. These data support the hypothesis that extracrevicular reservoirs of bacteria exist, which might contribute to recurrent or refractory disease in some patients.

Evaluating the potential of residual Pap test fluid as a resource for the metaproteomic analysis of the cervical-vaginal microbiome.

The human cervical-vaginal area contains proteins derived from microorganisms that may prevent or predispose women to gynecological conditions. The liquid Pap test fixative is an unexplored resource for analysis of microbial communities and the microbe-host interaction. Previously, we showed that the residual cell-free fixative from discarded Pap tests of healthy women could be used for mass spectrometry (MS) based proteomic identification of cervical-vaginal proteins. In this study, we reprocessed these MS raw data files for metaproteomic analysis to characterize the microbial community composition and function of microbial proteins in the cervical-vaginal region. This was accomplished by developing a customized protein sequence database encompassing microbes likely present in the vagina. High-mass accuracy data were searched against the protein FASTA database using a two-step search method within the Galaxy for proteomics platform. Data was analyzed by MEGAN6 (MetaGenomeAnalyzer) for phylogenetic and functional characterization. We identified over 300 unique peptides from a variety of bacterial phyla and Candida. Peptides corresponding to proteins involved in carbohydrate metabolism, oxidation-reduction, and transport were identified. By identifying microbial peptides in Pap test supernatants it may be possible to acquire a functional signature of these microbes, as well as detect specific proteins associated with cervical health and disease.

Oral bacteria in plaque and invading buccal cells of young orthodontic patients.

INTRODUCTION: In this longitudinal study, patients with fixed orthodontic appliances served as models to determine whether Actinobacillus actinomycetemcomitans, Tannerella forsythia, and total streptococci increased after treatment, and whether treatment affected bacterial invasion into the adjacent buccal epithelial cells (BEC). METHODS: Supragingival plaque, subgingival plaque, and BEC were collected from 27 patients before and at least 4 weeks after placement of orthodontic fixed appliances. Total sample DNA was determined, and bacteria were assayed by quantitative polymerase chain reaction. The BEC were also examined by confocal microscopy after fluorescent in-situ hybridization to visually detect the presence of each species bacteria in BEC. RESULTS: Total DNA in supragingival and subgingival plaque increased after appliance placement (P = .005). There was also a significant increase in supragingival streptococci (P = .0002). By confocal microscopy, a trend toward fewer buccal cells recovered was found after appliance placement, and there was a significant increase in the percentage of buccal cells containing A. actinomycetemcomitans (P = .0058). CONCLUSIONS: Appliance placement appeared to increase buccal cell susceptibility to A. actinomycetemcomitans invasion. This might be due to physical trauma or to leaching of metals from the appliances.

Protein relative abundance patterns associated with sucrose-induced dysbiosis are conserved across taxonomically diverse oral microcosm biofilm models of dental caries.

BACKGROUND: The etiology of dental caries is multifactorial, but frequent consumption of free sugars, notably sucrose, appears to be a major factor driving the supragingival microbiota in the direction of dysbiosis. Recent 16S rRNA-based studies indicated that caries-associated communities were less diverse than healthy supragingival plaque but still displayed considerable taxonomic diversity between individuals. Metagenomic studies likewise have found that healthy oral sites from different people were broadly similar with respect to gene function, even though there was an extensive individual variation in their taxonomic profiles. That pattern may also extend to dysbiotic communities. In that case, shifts in community-wide protein relative abundance might provide better biomarkers of dysbiosis that can be achieved through taxonomy alone. RESULTS: In this study, we used a paired oral microcosm biofilm model of dental caries to investigate differences in community composition and protein relative abundance in the presence and absence of sucrose. This approach provided large quantities of protein, which facilitated deep metaproteomic analysis. Community composition was evaluated using 16S rRNA sequencing and metaproteomic approaches. Although taxonomic diversity was reduced by sucrose pulsing, considerable inter-subject variation in community composition remained. By contrast, functional analysis using the SEED ontology found that sucrose induced changes in protein relative abundance patterns for pathways involving glycolysis, lactate production, aciduricity, and ammonia/glutamate metabolism that were conserved across taxonomically diverse dysbiotic oral microcosm biofilm communities. CONCLUSIONS: Our findings support the concept of using function-based changes in protein relative abundance as indicators of dysbiosis. Our microcosm model cannot replicate all aspects of the oral environment, but the deep level of metaproteomic analysis it allows makes it suitable for discovering which proteins are most consistently abundant during dysbiosis. It then may be possible to define biomarkers that could be used to detect at-risk tooth surfaces before the development of overt carious lesions.

Fatigue failure of dentin-composite disks subjected to cyclic diametral compression.

OBJECTIVE: Our aim was to establish the relationship between cyclic loading and fatigue life of the dentin-composite interface using the newly developed disk in diametral compression tests. The results were then used to estimate the fatigue life of restored teeth under occlusal loading. METHODS: Disk specimens (5mm dia.×2mm thick) were prepared using bovine incisors and restored with either a methacrylate-based composite Z100™ with Adper Single Bond Plus (Z100) or silorane-based composite Filtek™ LS with LS System adhesive (LS). The dentin-composite disks were tested under cyclic diametral compression to determine the number of cycles to failure (Nf) at three load levels (n=3 per group). Finite element analysis (FEA) was used to calculate the interfacial stresses (σ) within the specimen, to establish the σ vs. Nf curves, and those within a restored tooth under normal chewing forces (15N maximum). These were then used to estimate the lifetime of the restored tooth for the two restorative systems. RESULTS: The disks restored with LS had a higher fatigue resistance than those restored with Z100. The maximum interfacial stress in the restored tooth determined by FEA was ∼0.5MPa. Based on the estimate of 300,000 cycles of chewing per year, the predicted lifetime under occlusal loading for teeth restored with LS and Z100 was 33 and 10 years, respectively. SIGNIFICANCE: The disk in cyclic diametral compression has been used successfully to provide fatigue data which allows the lifetime of composite-restored teeth under occlusal loading to be predicted using numerical simulation.

Salivary CA130 with and without unilateral autonomic parotid denervation of rats fed different diets.

BACKGROUND: Tumor markers such as CA130 can be determined in human whole saliva. Saliva represents an attractive body fluid for longitudinal studies. MATERIALS AND METHODS: CA130 was determined in parotid saliva from 8 rats fed different diets, with or without autonomic denervation. RESULTS: CA130 could be determined in parotid saliva of rats, irrespective of diet and/or autonomic denervation. Whether the numerical decrease in CA130 observed after autonomic denervation is statistically significant requires further work. CONCLUSIONS: Since salivary CA130 has been shown to decrease following treatment with anti-cancer drugs in humans, the ability to determine this tumor marker in rat saliva opens new opportunities for optimizing cancer chronotherapy in the experimental laboratory.

Fusobacterium nucleatum transports noninvasive Streptococcus cristatus into human epithelial cells.

Analysis of human buccal epithelial cells frequently reveals an intracellular polymicrobial consortium of bacteria. Although several oral bacteria have been demonstrated to invade cultured epithelial cells, several others appear unable to internalize. We hypothesized that normally noninvasive bacteria may gain entry into epithelial cells via adhesion to invasive bacteria. Fusobacterium nucleatum is capable of binding to and invading oral epithelial cells. By contrast, Streptococcus cristatus binds weakly to host cells and is not internalized. F. nucleatum and S. cristatus coaggregate strongly via an arginine-sensitive interaction. Coincubation of KB or TERT-2 epithelial cells with equal numbers of F. nucleatum and S. cristatus bacteria led to significantly increased numbers of adherent and internalized streptococci. F. nucleatum also promoted invasion of KB cells by other oral streptococci and Actinomyces naeslundii. Dissection of fusobacterial or streptococcal adhesive interactions by using sugars, amino acids, or antibodies demonstrated that this phenomenon is due to direct attachment of S. cristatus to adherent and invading F. nucleatum. Inhibition of F. nucleatum host cell attachment and invasion with galactose, or fusobacterial-streptococcal coaggregation by the arginine homologue l-canavanine, abrogated the increased S. cristatus adhesion to, and invasion of, host cells. In addition, polyclonal antibodies to F. nucleatum, which inhibited fusobacterial attachment to both KB cells and S. cristatus, significantly decreased invasion by both species. Similar decreases were obtained when epithelial cells were pretreated with cytochalasin D, staurosporine, or cycloheximide. These studies indicate that F. nucleatum may facilitate the colonization of epithelial cells by bacteria unable to adhere or invade directly.