Richard P. Ellen

A Quorum-Sensing Signaling System Essential for Genetic Competence in Streptococcus mutans Is Involved in Biofilm Formation

In a previous study, a quorum-sensing signaling system essential for genetic competence in Streptococcus mutans was identified, characterized, and found to function optimally in biofilms (Li et al., J. Bacteriol. 183:897-908, 2001). Here, we demonstrate that this system also plays a role in the ability of S. mutans to initiate biofilm formation. To test this hypothesis, S. mutans wild-type strain NG8 and its knockout mutants defective in comC, comD, comE, and comX, as well as a comCDE deletion mutant, were assayed for their ability to initiate biofilm formation. The spatial distribution and architecture of the biofilms were examined by scanning electron microscopy and confocal scanning laser microscopy. The results showed that inactivation of any of the individual genes under study resulted in the formation of an abnormal biofilm. The comC mutant, unable to produce or secrete a competence-stimulating peptide (CSP), formed biofilms with altered architecture, whereas the comD and comE mutants, which were defective in sensing and responding to the CSP, formed biofilms with reduced biomass. Exogenous addition of the CSP and complementation with a plasmid containing the wild-type comC gene into the cultures restored the wild-type biofilm architecture of comC mutants but showed no effect on the comD, comE, or comX mutant biofilms. The fact that biofilms formed by comC mutants differed from the comD, comE, and comX mutant biofilms suggested that multiple signal transduction pathways were affected by CSP. Addition of synthetic CSP into the culture medium or introduction of the wild-type comC gene on a shuttle vector into the comCDE deletion mutant partially restored the wild-type biofilm architecture and further supported this idea. We conclude that the quorum-sensing signaling system essential for genetic competence in S. mutans is important for the formation of biofilms by this gram-positive organism.

Quorum sensing and biofilm formation in Streptococcal infections

Members of the bacterial genus Streptococcus are responsible for causing a wide variety of infections in humans. Many Streptococci use quorum-sensing systems to regulate several physiological properties, including the ability to incorporate foreign DNA, tolerate acid, form biofilms, and become virulent. These quorum-sensing systems are primarily made of small soluble signal peptides that are detected by neighboring cells via a histidine kinase/response regulator pair.

A VicRK Signal Transduction System in Streptococcus mutans Affects gtfBCD, gbpB, and ftf Expression, Biofilm Formation, and Genetic Competence Development

Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the vicRK signal transduction system in Streptococcus mutans. A vicK (putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a vicR (putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the vicRKX operon (Smuvic+). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the vicK mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic+. Based on real-time PCR, Smuvic+ showed increased gtfBCD, gbpB, and ftf expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the gtfB, gtfC, and ftf genes. Also, transformation efficiency in the presence or absence of the S. mutans competence-stimulating peptide was altered for the vic mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of vicK did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the vicRK signal transduction system in regulating several important physiological processes in S. mutans.

Novel Two-Component Regulatory System Involved in Biofilm Formation and Acid Resistance in Streptococcus mutans

The abilities of Streptococcus mutans to form biofilms and to survive acidic pH are regarded as two important virulence determinants in the pathogenesis of dental caries. Environmental stimuli are thought to regulate the expression of several genes associated with virulence factors through the activity of two-component signal transduction systems. Yet, little is known of the involvement of these systems in the physiology and pathogenicity of S. mutans. In this study, we describe a two-component regulatory system and its involvement in biofilm formation and acid resistance in S. mutans. By searching the S. mutans genome database with tblastn with the HK03 and RR03 protein sequences from S. pneumoniae as queries, we identified two genes, designated hk11 and rr11, that encode a putative histidine kinase and its cognate response regulator. To gain insight into their function, a PCR-mediated allelic-exchange mutagenesis strategy was used to create the hk11 (Em(r)) and rr11 (Em(r)) deletion mutants from S. mutans wild-type NG8 named SMHK11 and SMRR11, respectively. The mutants were examined for their growth rates, genetic competence, ability to form biofilms, and resistance to low-pH challenge. The results showed that deletion of hk11 or rr11 resulted in defects in biofilm formation and resistance to acidic pH. Both mutants formed biofilms with reduced biomass (50 to 70% of the density of the parent strain). Scanning electron microscopy revealed that the biofilms formed by the mutants had sponge-like architecture with what appeared to be large gaps that resembled water channel-like structures. The mutant biofilms were composed of longer chains of cells than those of the parent biofilm. Deletion of hk11 also resulted in greatly diminished resistance to low pH, although we did not observe the same effect when rr11 was deleted. Genetic competence was not affected in either mutant. The results suggested that the gene product of hk11 in S. mutans might act as a pH sensor that could cross talk with one or more response regulators. We conclude that the two-component signal transduction system encoded by hk11 and rr11 represents a new regulatory system involved in biofilm formation and acid resistance in S. mutans.

Cell Density Modulates Acid Adaptation in Streptococcus mutans: Implications for Survival in Biofilms

Streptococcus mutans normally colonizes dental biofilms and is regularly exposed to continual cycles of acidic pH during ingestion of fermentable dietary carbohydrates. The ability of S. mutans to survive at low pH is an important virulence factor in the pathogenesis of dental caries. Despite a few studies of the acid adaptation mechanism of this organism, little work has focused on the acid tolerance of S. mutans growing in high-cell-density biofilms. It is unknown whether biofilm growth mode or high cell density affects acid adaptation by S. mutans. This study was initiated to examine the acid tolerance response (ATR) of S. mutans biofilm cells and to determine the effect of cell density on the induction of acid adaptation. S. mutans BM71 cells were first grown in broth cultures to examine acid adaptation associated with growth phase, cell density, carbon starvation, and induction by culture filtrates. The cells were also grown in a chemostat-based biofilm fermentor for biofilm formation. Adaptation of biofilm cells to low pH was established in the chemostat by the acid generated from excess glucose metabolism, followed by a pH 3.5 acid shock for 3 h. Both biofilm and planktonic cells were removed to assay percentages of survival. The results showed that S. mutans BM71 exhibited a log-phase ATR induced by low pH and a stationary-phase acid resistance induced by carbon starvation. Cell density was found to modulate acid adaptation in S. mutans log-phase cells, since pre-adapted cells at a higher cell density or from a dense biofilm displayed significantly higher resistance to the killing pH than the cells at a lower cell density. The log-phase ATR could also be induced by a neutralized culture filtrate collected from a low-pH culture, suggesting that the culture filtrate contained an extracellular induction component(s) involved in acid adaptation in S. mutans. Heat or proteinase treatment abolished the induction by the culture filtrate. The results also showed that mutants defective in the comC, -D, or -E genes, which encode a quorum sensing system essential for cell density-dependent induction of genetic competence, had a diminished log-phase ATR. Addition of synthetic competence stimulating peptide (CSP) to the comC mutant restored the ATR. This study demonstrated that cell density and biofilm growth mode modulated acid adaptation in S. mutans, suggesting that optimal development of acid adaptation in this organism involves both low pH induction and cell-cell communication.

Streptococcus mutans and Lactobacillus Detection in the Assessment of Dental Root Surface Caries Risk

Forty-five subjects contributing 150 root surfaces with gingival recession were sampled seven times over a 32-month period. We calculated isolation frequencies of lactobacilli on selective Rogosa SL agar and S. mutans in a sensitive enrichment broth and on mitis salivarius agar. Both S. mutans and lactobacilli were isolated more frequently from surfaces which became carious than from those remaining caries-free. Isolation frequencies were also higher for caries-free surfaces in caries-active subjects than for caries-free surfaces in caries-inactive subjects. The presence or absence of S. mutans and lactobacilli in samples taken at baseline could discriminate between subjects who were to be root-caries-active and those who were to remain root-caries-inactive during the subsequent observation period. Moreover, if both bacteria were detected or only S. mutans was detected on a root surface at its entry into the study, that surface had a greater risk for developing a root lesion. However, the tests could not predict which root surfaces within the mouths of caries-active subjects were to become carious. Analysis of the data suggests that simple microbiological detection tests may be useful in identifying patients at high risk of root caries.

CLINICAL SCIENCE Longitudinal Microbiological Investigation of a Hospitalized Population of Older Adults with a High Root Surface Caries Risk

Intact root surfaces of chronically hospitalized subjects were sampled periodically to enumerate bacterial species believed to be associated with root caries. Bacteria were cultivated and enumerated using a series of selective and enriched media. Microbial counts, isolation frequencies, and percent cultivable flora data were analyzed for caries-active and caries-free surfaces and subjects. S. mutans, S. sanguis, A. viscosus, A. naeslundii, total lactobacilli, and Veillonella accounted for a mean of less than 20% of the cultivated flora, with mitis salivarius agar cultivable streptococci averaging less than 5%. The microbial count data were highly variable, precluding the finding of significant differences in caries association for either subjects or sites. Streptococci, especially S. mutans, correlated highly with lactobacilli in the samples.

Clinical Science A Longitudinal Study of Root Caries: Baseline and Incidence Data

A 34-month clinical investigation of root caries was conducted using a hospitalized, chronically-ill population. Forty-five subjects with 150 caries-free surfaces were observed for at least 12 consecutive months. Clinical measurements included diagnosis of new root and coronal caries, gingival inflammation, pocket depth, recession, and distance of new lesions from the gingival margin. The overall root caries attack rate was 1.90 lesions per 100 person-months at risk, with the greatest rate occurring during the first year of study. Mesial and facial surfaces accounted for 84% of all new lesions. Sixteen subjects accounted for 26 new lesions; nine subjects had two or more new lesions. Subjects with new root lesions were found to have lower enamel caries experience but higher root caries experience at baseline and higher scores for gingival inflammation, pocket depth, and recession compared with those who did not develop root caries.

Involvement of Sortase Anchoring of Cell Wall Proteins in Biofilm Formation by Streptococcus mutans

ABSTRACT Streptococcus mutans is one of the best-known biofilm-forming organisms associated with humans. We investigated the role of the sortase gene (srtA) in monospecies biofilm formation and observed that inactivation of srtA caused a decrease in biofilm formation. Genes encoding three putative sortase-dependent proteins were also found to be up-regulated in biofilms versus planktonic cells and mutations in these genes resulted in reduced biofilm biomass.

New insights into the emerging role of oral spirochaetes in periodontal disease

Spirochaetes are prominent in the polymicrobial infections that cause periodontal diseases. Periodontitis is a chronic inflammatory condition of the periodontium, characterized by proinflammatory soft tissue damage and alveolar bone loss. Treponema denticola is the most well-understood oral spirochaete, expressing a wealth of virulence factors that mediate tissue penetration and destruction as well as evasion of host immune responses. This review focuses on emerging knowledge of virulence mechanisms of Treponema denticola as well as mechanisms of other less-studied oral treponemes.