Joel D. Rudney

Intracellular Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in Buccal Epithelial Cells Collected from Human Subjects

ABSTRACT The mouth may provide an accessible model for studying bacterial interactions with human cells in vivo. Using fluorescent in situ hybridization and laser scanning confocal microscopy, we found that human buccal epithelial cells from 23 of 24 subjects were infected with intracellular bacteria, including the periodontal pathogens Actinobacillus actinomycetemcomitans andPorphyromonas gingivalis, as well as other species which have yet to be identified. Buccal cell invasion may allow fastidious anaerobes to establish themselves in aerobic sites that otherwise present an unfavorable environment. Exfoliated buccal epithelial cells might provide a protected route for bacterial transmission between different oral sites within and between hosts.

Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis are Components of a Polymicrobial Intracellular Flora within Human Buccal Cells

Previously, we used in situ hybridization and confocal microscopy to detect the periodontal pathogens Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis within buccal epithelial cells taken directly from the mouth. This study tested the hypothesis that the intracellular flora of buccal cells is polymicrobial. Mixtures containing a red fluorescent universal probe paired with green fluorescent versions of either A. actinomycetemcomitans-, P. gingivalis-, or T. forsythensis-specific probes were hybridized with buccal cells collected from each of 38 healthy humans. We verified co-localization of probe pairs within cells by generating three-dimensional reconstructions. Intracellular bacteria were detected in every subject. Each cell that was labeled with a species-specific probe also contained bacteria recognized only by the universal probe. Bacteria labeled with specific probes often occupied smaller regions within larger masses of bacteria. Those findings suggest that future studies of invasion by oral bacteria may need to include microbial consortia.

Does Variability in Salivary Protein Concentrations Influence Oral Microbial Ecology and Oral Health

Salivary protein interactions with oral microbes in vitro include aggregation, adherence, cell-killing, inhibition of metabolism, and nutrition. Such interactions might be expected to influence oral ecology. However, inconsistent results have been obtained from in vivo tests of the hypothesis that quantitative variation in salivary protein concentrations will affect oral disease prevalence. Results may have been influenced by choices made during study design, including saliva source, stimulation status, control for flow rate, and assay methods. Salivary protein concentrations also may be subject to circadian variation. Values for saliva collected at the same time of day tend to remain consistent within subjects, but events such as stress, inflammation, infection, menstruation, or pregnancy may induce short-term changes. Long-term factors such as aging, systemic disease, or medication likewise may influence salivary protein concentrations. Such sources of variation may increase the sample size needed to find statistically significant differences. Clinical studies also must consider factors such as human population variation, strain and species differences in protein-microbe interactions, protein polymorphism, and synergistic or antagonistic interaction between proteins. Salivary proteins may form heterotypic complexes with unique effects, and different proteins may exert redundant effects. Patterns of protein-microbe interaction also may differ between oral sites. Future clinical studies must take those factors into account. Promising approaches might involve meta-analysis or multi-center studies, retrospective and prospective longitudinal designs, short-term measurement of salivary protein effects, and consideration of individual variation in multiple protein effects such as aggregation, adherence, and cell-killing.

THE PREDICTION OF SALIVA SWALLOWING FREQUENCY IN HUMANS FROM ESTIMATES OF SALIVARY FLOW RATE AND THE VOLUME OF SALIVA SWALLOWED

Saliva swallowing frequency is an important factor in models of oral clearance. It varies widely among individuals, and the basis for that variation has not been established. This study evaluated the use of unstimulated flow rate and the volume of saliva swallowed as predictors of swallowing frequency in 128 first-year dental students. A microphone was placed over the larynx, and swallowing activity was recorded for 30 min between 3-6:00 p.m. The average interval between swallows was determined, and individuals retained saliva in the mouth for a period equal to that time. Retained saliva was spat out, and volume was determined gravimetrically. Four replicate tubes were collected. Flow rate was determined as sample volume over average swallow time. A subset of 10 individuals was measured on two further occasions 2-3 months apart. An independent estimate of flow rate was taken on the second occasion. Repeat-measures analysis of variance and intraclass correlations were used to estimate the reproducibility of replicate volumes, and of measurements taken on different occasions. Associations between swallowing interval, saliva volume, and flow rate were evaluated by multiple regression. Replicate volumes were highly reproducible, as were measurements of volumes, swallow times, and flow rates on different occasions. Saliva volume and flow rate jointly accounted for 99% of variance in swallowing intervals. Swallowing intervals were shortest for individuals who combined high flow rates with small saliva volumes; current models suggest that their oral clearance might be most efficient. Swallowing intervals were longest for individuals with low flow rates and large volumes; their oral clearance might be the least efficient.

Fusobacterium nucleatum Transports Noninvasive Streptococcus cristatus into Human Epithelial Cells

ABSTRACT 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.

Antimicrobial proteins in human unstimulated whole saliva in relation to each other, and to measures of health status, dental plaque accumulation and composition

Saliva antimicrobial proteins may interact in a common system to influence the oral ecology. Clinical studies of antimicrobial protein action thus may require a multiple-protein approach. Multivariate statistical methods have been used to describe possible patterns of interaction for lysozyme, lactoferrin, salivary peroxidase and secretory IgA in stimulated parotid saliva. However, oral microbes are most likely to encounter antimicrobial proteins in mixed resting saliva. Relationships among levels of lysozyme, lactoferrin, salivary peroxidase, and secretory IgA therefore were investigated in whole saliva from 216 subjects, and an attempt made to relate interperson variation in those proteins to differences in health and status, and dental plaque accumulation and composition. All proteins were significantly (alpha = 0.05) correlated with each other (r = 0.38-0.52, p less than 0.001). There was only one axis of common variation among proteins, and that axis was significantly correlated (p less than 0.001) with total protein (r = 0.84) and flow rate (r = -0.56). That pattern deviated from the previous finding that proteins of acinar origin tended to vary independently from proteins of ductal origin in stimulated parotid saliva. The difference between parotid and whole saliva may reflect constitutive secretion of all proteins at low levels of stimulation. Common variation of unstimulated saliva proteins suggests that antimicrobial actions can be compared in subjects at population extremes. There were no significant associations between antimicrobial proteins in whole saliva and measures of health status or plaque accumulation. However, the proportions of Streptococcus sanguis were significantly correlated with lysozyme (r = -0.26), lactoferrin (r = -0.34), peroxidase (r = -0.30), total protein (r = -0.37), flow rate (r = 0.24) and principal-components scores (r = -0.33) in a subset of subjects (n = 85) where commercial biochemical tests were used to supplement species identification by colony morphology. Those findings may indicate that saliva antimicrobial proteins can affect the composition of dental plaque.

Saliva and Dental Plaque

Dental plaque is being redefined as oral biofilm. Diverse overlapping microbial consortia are present on all oral tissues. Biofilms are structured, displaying features like channels and projections. Constituent species switch back and forth between sessile and planktonic phases. Saliva is the medium for planktonic suspension. Several major functions can be defined for saliva in relation to oral biofilm. It serves as a medium for transporting planktonic bacteria within and between mouths. Bacteria in transit may be vulnerable to negative selection. Salivary agglutinins may prevent reattachment to surfaces. Killing by antimicrobial proteins may lead to attachment of dead cells. Salivary proteins form conditioning films on all oral surfaces. This contributes to positive selection for microbial adherence. Saliva carries chemical messengers which allow live adherent cells to sense a critical density of conspecifics. Growth begins, and thick biofilms may become resistant to antimicrobial substances. Salivary macromolecules may be catabolized, but salivary flow also may clear dietary substrates. Salivary proteins act in ways that benefit both host and microbe. All have multiple functions, and many do the same job. They form heterotypic complexes, which may exist in large micelle-like structures. These issues make it useful to compare subjects whose saliva functions differently. We have developed a simultaneous assay for aggregation, killing, live adherence, and dead adherence of oral species. Screening of 149 subjects has defined high killing/low adherence, low killing/high adherence, high killing/high adherence, and low killing/low adherence groups. These will be evaluated for differences in their flora.

Streptococci Dominate the Diverse Flora within Buccal Cells

Previously, we reported that intracellular Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythensis were present within buccal epithelial cells from human subjects, as lesser components of a polymicrobial flora. In this study, we further characterized that intracellular flora by using the same double-labeling techniques to identify Fusobacterium nucleatum, Prevotella intermedia, oral Campylobacter species, Eikenella corrodens, Treponema denticola, Gemella haemolysans, Granulicatella adiacens, and total streptococci within buccal epithelial cells. All those species were found within buccal cells. In every case, species recognized by green-labeled species-specific probes were accompanied by other bacteria recognized only by a red-labeled universal probe. Streptococci appeared to be a major component of the polymicrobial intracellular flora, being present at a level from one to two logs greater than the next most common species (G. adiacens). This is similar to what is observed in oral biofilms, where diverse species interact in complex communities that often are dominated by streptococci.

Potential biomarkers of human salivary function: a modified proteomic approach.

OBJECTIVE In previous studies, we defined groups of subjects with opposite salivary function. Group membership was associated with clinically relevant outcomes. High aggregation-adherence (HAA) groups showed lower levels of caries, supragingival plaque, total streptococci, and Tannerella forsythensis than low high aggregation-adherence (LAA) groups. In this study, we used a proteomic approach to search for biomarkers which could be useful as risk indicators for those outcomes. DESIGN Clarified resting whole saliva from each of 41 HAA and LAA subjects was separated by preparative isoelectric focusing. Fractions showing the most distinctive protein profiles were pooled into four sets (pI 3-3.5, pI 4-4.7, pI 5.7-7.7, pI 10-11.5). Each pool then was compared by SDS-PAGE. Image analysis software was used to quantify matched bands. Partial least squares analysis (PLS) was used to determine which of the 65 bands from all four pools were the best predictors of group membership, caries, total plaque, total streptococci, and T. forsythensis counts. Those bands were identified by mass spectroscopy (MS-MS). RESULTS Two bands consistently were strong predictors in separate PLS analyses of each outcome variable. In follow-up univariate analyses, those bands showed the strongest significant differences between the HAA and LAA groups. They also showed significant inverse correlations with caries and all the microbiological variables. MSMS identified those bands as statherin, and a truncated cystatin S missing the first eight N-terminal amino acids. CONCLUSIONS Levels of statherin and truncated cystatin S may be potential risk indicators for the development of caries and other oral diseases.

Correlations between human salivary levels of lysozyme, lactoferrin, salivary peroxidase and secretory immunoglobulin A with different stimulatory states and over time

Within-subject correlations for the levels of these salivary proteins were determined in unstimulated and stimulated parotid saliva collected from 8 subjects and for stimulated parotid saliva collected from the same subjects once a week for a 4-week period. Initial correlations between unstimulated and stimulated samples were high and statistically significant (p less than 0.05) for all four proteins. When data were adjusted for variation attributable to flow rate and total protein, some correlations remained the same and those for lysozyme, lactoferrin and salivary peroxidase increased. However, the correlation for secretory immunoglobulin A decreased to a point where it was no longer statistically significant. In the weekly comparison, within-subject correlations across weeks were significant (p less than or equal to 0.05) for lysozyme, lactoferrin and salivary peroxidase, but not for immunoglobulin A. After adjustment for flow rate and total protein, the pattern of correlation was unchanged. Thus the relative rankings of subjects for levels of lysozyme, lactoferrin or salivary peroxidase may be consistent across stimulatory states, even though absolute concentrations may change; levels of these proteins in stimulated parotid saliva may be maintained over time. Secretory immunoglobulin A appears to be more subject to short-term variation.