CcpA regulates central metabolism and virulence gene expression in Streptococcus mutans

Abstract

CcpA globally regulates transcription in response to carbohydrate availability in many gram-positive bacteria, but its role in Streptococcus mutans remains enigmatic. Using the fructan hydrolase (fruA) gene of S. mutans as a model, we demonstrated that CcpA plays a direct role in carbon catabolite repression (CCR). Subsequently, the expression of 170 genes was shown to be differently expressed (> or = 2-fold) in glucose-grown wild-type (UA159) and CcpA-deficient (TW1) strains (P < or = 0.001). However, there were differences in expression of only 96 genes between UA159 and TW1 when cells were cultivated with the poorly repressing substrate galactose. Interestingly, 90 genes were expressed differently in wild-type S. mutans when glucose- and galactose-grown cells were compared, but the expression of 515 genes was altered in the CcpA-deficient strain in a similar comparison. Overall, our results supported the hypothesis that CcpA has a major role in CCR and regulation of gene expression but revealed that in S. mutans there is a substantial CcpA-independent network that regulates gene expression in response to the carbohydrate source. Based on the genetic studies, biochemical and physiological experiments demonstrated that loss of CcpA impacts the ability of S. mutans to transport and grow on selected sugars. Also, the CcpA-deficient strain displayed an enhanced capacity to produce acid from intracellular stores of polysaccharides, could grow faster at pH 5.5, and could acidify the environment more rapidly and to a greater extent than the parental strain. Thus, CcpA directly modulates the pathogenic potential of S. mutans through global control of gene expression.

FIG. 1.

EMSAs. DNA fragments of the fruA promoter carrying wild-type (WT-CRE) or mutated CRE (MUT-CRE) sequences (42) were end labeled, and the abilities of different amounts of purified His₆-CcpA (the lanes contained 0, 27, 54, 109, and 218 pmol of protein) to induce a mobility shift in the presence (+) or absence (−) of 2 mM F-1,6-bP (Sigma) were examined. The arrow indicates the migration position of the shifted product.

FIG. 2.

Numbers of genes in functional categories differentially expressed in strain TW1 compared to UA159 when cells are cultivated in TV medium containing 0.5% glucose. AA, amino acid; TCS, two-component systems; PPNN, purines, pyrimidines, nucleosides, nucleotides.

FIG. 3.

Sugar transport by the PTS in S. mutans UA159 and the CcpA-deficient derivative TW1. Cells were cultivated in TV medium with glucose (A), fructose (B), or mannose (C) and harvested in the mid-exponential phase of growth, and PTS-dependent sugar transport was assayed as described in Materials and Methods. Data are expressed as means of at least three separate cultures that were assayed in triplicate. The error bars indicate standard deviations. Statistical significance (see text) was determined by the Student t test.

FIG. 4.

Glycolytic acidification by S. mutans UA159 and TW1 in the presence of added glucose (A) or using endogenous stores (B). Experiments were performed as described in Materials and Methods, and the data are representative of no fewer than three separate replicates that all produced the same results.