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

Abstract

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-celldensity 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.

Inhibition ofSteptococcus mutansbiofilm formation by extracts ofTenacibaculumsp. 20J, a bacterium with wide-spectrum quorum quenching activity

“…Biofilm formation by S. mutans is regulated by two QS systems, although the hierarchy of their organization is unknown. The QS system typical of streptococci is regulated by a peptide pheromone signal called competence stimulating peptide (CSP) [9], which is species-specific and detected by a histidine-kinase [10]. The inactivation of any of the genes related to the CSP QS system results in the alteration of biofilm architecture [11].…”

Global transcriptional analysis of acid-inducible genes in Streptococcus mutans: multiple two-component systems involved in acid adaptation

“…To identify acid-inducible genes, we grew exponential-phase cells of S. mutans UA159 for 2 h in TYG broth at pH 5.5, a signal pH that has been demonstrated to induce acid adaptation of S. mutans effectively (Li et al, 2001b;Svensäter et al, 1997); cells grown at pH 7.5 were used as a control. Following acid adaptation, the cell pellets were collected by centrifugation at 8000 g for 15 min at 4 uC and resuspended in 1 ml cool Trizol (Invitrogen).…”

“…To determine the effect of inactivation of the histidine kinase sensor protein on acid adaptation by S. mutans, all the mutants and the parent were assayed for their survival at pH 3.5 with or without pre-exposure to pH 5.5 for 2 h using a protocol described by Li et al (2001b). Briefly, exponential-phase cells of each strain were divided into two groups: one resuspended in fresh TYG broth at pH 5.5 and another resuspended in TYG broth at pH 7.5.…”

“…In the S. mutans genome, there are 14 TCSs, and at least one orphan response regulator has been identified (Ajdic et al, 2002;Biswas et al, 2008;Idone et al, 2003). Some of these systems, such as LiaSR (formerly HK/ RR11), CiaHR, ComDE and CovR, have been found to play a role in acid resistance and considered as a major attribute to the virulence of S. mutans (Ahn et al, 2006;Idone et al, 2003;Li et al, 2001bLi et al, , 2002Lévesque et al, 2007;Kawada-Matsuo et al, 2009). However, little is known of the actual mechanisms of how these TCSs sense and integrate pH signals to regulate gene expression for acid adaptation.…”

“…Other mechanisms of acid resistance in S. mutans include induction of stress proteins (Hamilton & Svensäter, 1998;Len et al, 2004;Svensäter et al, 1997;Wilkins et al, 2002), changes in membrane-associated proteins and fatty acid composition (Fozo et al, 2007;Hasona et al, 2007), DNA repair enzymes (Hahn et al, 1999;Hanna et al, 2001) and increase in alkali production through several metabolic pathways (Burne et al, 1999;Griswold et al, 2006). Moreover, the ComCDE quorum-sensing system has been found to play a role in cell-density-dependent acid tolerance by S. mutans (Li et al, 2001b). S. mutans is also capable of mounting an adaptive acid tolerance response (ATR), since pre-exposure of S. mutans to pH 5.0-6.0 for 1-2 h (adaptation) renders the organism less susceptible to lethal acidification, as opposed to a quick pH drop by adding HCl to growing cultures (Nascimento et al, 2004;Svensäter et al, 1997).…”