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Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

ABSTRACT: Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans.The point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation.SrtA?N40(UA159) and the mutant enzymes, SrtA?N40(D56E) and SrtA?N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtA?N40(D56E) and SrtA?N40(R157H) remained approximately equal to the affinity of SrtA?N40(UA159), as determined by the Michaelis constant (K m ). However, the catalytic rate constant (k cat ) and catalytic efficiency (k cat /K m ) of SrtA?N40(D56E) were reduced compared with those of SrtA?N40(R157H) and SrtA?N40(UA159), whereas the k cat and k cat /K m values of SrtA?N40(R157H) were slightly lower than those of SrtA?N40(UA159).The findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.

SUBMITTER: Zhuang PL

PROVIDER: S-EPMC4827206 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

Zhuang P L PL Yu L X LX Tao Y Y Zhou Y Y Zhi Q H QH Lin H C HC

BMC oral health 20160411

<h4>Background</h4>Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans.<h4>Methods</h4>The point mutated recombinant S.mutans T16 ...[more]

PMID: 27068451

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Project description:Streptococcus mutans is a common constituent of oral biofilms and a primary etiologic agent of human dental caries. The bacteria associated with dental caries have potent abilities to produce organic acids from dietary carbohydrates and to grow and metabolize in acidic conditions. By contrast, many commensal bacteria produce ammonia through the arginine deiminase system (ADS), which moderates the pH of oral biofilms. Arginine metabolism by the ADS is a significant deterrent to the initiation and progression of dental caries. In this study, we observed how exogenously provided l-arginine affects the growth, the virulence properties, and the tolerance of environmental stresses of S. mutans Supplementation with 1.5% arginine (final concentration) had an inhibitory effect on the growth of S. mutans in complex and chemically defined media, particularly when cells were exposed to acid or oxidative stress. The genes encoding virulence factors required for attachment/accumulation (gtfB and spaP), bacteriocins (nlmA, nlmB, nlmD, and cipB), and the sigma factor required for competence development (comX) were downregulated during growth with 1.5% arginine. Deep sequencing of RNA (RNA-Seq) comparing the transcriptomes of S. mutans growing in chemically defined media with and without 1.5% arginine revealed differential expression of genes encoding ATP-binding cassette transporters, metal transporters, and constituents required for survival, metabolism, and biofilm formation. Therefore, the mechanisms of action by which arginine inhibits dental caries include direct adverse effects on multiple virulence-related properties of the most common human dental caries pathogen.IMPORTANCE Metabolism of the amino acid arginine by the arginine deiminase system (ADS) of certain oral bacteria raises the pH of dental plaque and provides a selective advantage to health-associated bacteria, thereby protecting the host from dental caries (cavities). Here, we examine the effects of arginine on the cavity-causing bacterium Streptococcus mutans We find that arginine negatively impacts the growth, the pathogenic potential, and the tolerance of environmental stresses in a way that is likely to compromise the ability of S. mutans to cause disease. Using genetic and genomic techniques, multiple mechanisms by which arginine exerts its influence on virulence-related properties of S. mutans are discovered. This report demonstrates that a primary mechanism of action by which arginine inhibits the initiation and progression of dental caries may be by reducing the pathogenic potential of S. mutans.

Dataset Information

Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

Publications

Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

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