RESUMEN
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.
Asunto(s)
Biopelículas , Streptococcus mutans/metabolismo , Secuencia de Aminoácidos , Recuento de Células , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Streptococcus mutans/genética , Streptococcus mutans/crecimiento & desarrolloRESUMEN
The pH-inducible acid tolerance response (ATR) is believed to play a major role in acid adaptation and virulence of Streptococcus mutans. To study this phenomenon in S. mutans JH1005, differential display PCR was used to identify and clone 13 cDNA products that had increased expression in response to pH 5.0 compared to that of pH 7.5-grown cells. One of these products, confirmed to be pH inducible by RNA dot blot and reverse transcription-PCR analyses, had 67% identity to a uvrA-UV repair excinuclease gene in Bacillus subtilis. Further sequence analysis of the uvrA homologue using the S. mutans genome database revealed that the complete gene was encoded in an open reading frame (ORF) of 2,829 bp (944 amino acids; 104.67 kDa). Immediately 3' of uvrA was an ORF encoding a putative aminopeptidase gene (pepP). uvrA knockouts were constructed in S. mutans strains JH1005, NG8, and UA159 using allelic-exchange mutagenesis, replacing the entire gene with an erythromycin resistance cassette. As with uvrA mutants in other bacteria, the S. mutans uvrA mutants were extremely sensitive to UV irradiation. The uvrA mutant of S. mutans JH1005 was also more sensitive than the wild type to growth at pH 5.0, showing a 15% reduction in growth rate and a 14% reduction in final resting culture density. Acid-adapted S. mutans JH1005 uvrA mutants were shown to be more resistant to UV irradiation than was the parent but were unable to survive exposure to a killing pH of 3.0. Moreover, agarose gel electrophoretic analysis of chromosomal DNA isolated from uvrA-deficient cells exposed to low pH demonstrated more DNA damage than that for the wild-type strain. Here we suggest that uvrA and the nucleotide excision repair pathway are involved in the repair of acid-induced DNA damage and are associated with successful adaptation of S. mutans to low pH.