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RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans.

ABSTRACT: Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In Streptococcus mutans, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in S. mutans biology have not been well investigated. The present study characterizes the impact of RGP disruption as a result of the deletion of rgpF, the gene encoding a rhamnosyltransferase involved in the construction of the core polyrhamnose backbone of RGP. The ?rgpF mutant strain displayed an overall reduced fitness compared to the wild type, with heightened sensitivities to various stress-inducing culture conditions and an inability to tolerate acid challenge. The loss of rgpF caused a perturbation of membrane-associated functions known to be critical for aciduricity, a hallmark of S. mutans acid tolerance. The proton gradient across the membrane was disrupted, and the ?rgpF mutant strain was unable to induce activity of the F₁F_o ATPase in cultures grown under low-pH conditions. Further, the virulence potential of S. mutans was also drastically reduced following the deletion of rgpF The ?rgpF mutant strain produced significantly less robust biofilms, indicating an impairment in its ability to adhere to hydroxyapatite surfaces. Additionally, the ?rgpF mutant lost competitive fitness against oral peroxigenic streptococci, and it displayed significantly attenuated virulence in an in vivoGalleria mellonella infection model. Collectively, these results highlight a critical function of the RGP in the maintenance of overall stress tolerance and virulence traits in S. mutansIMPORTANCE The cell wall of Streptococcus mutans, the bacterium most commonly associated with tooth decay, is abundant in rhamnose-glucose polysaccharides (RGP). While these structures are antigenically distinct to S. mutans, the process by which they are formed and the enzymes leading to their construction are well conserved among streptococci. The present study describes the consequences of the loss of RgpF, a rhamnosyltransferase involved in RGP construction. The deletion of rgpF resulted in severe ablation of the organism's overall fitness, culminating in significantly attenuated virulence. Our data demonstrate an important link between the RGP and cell wall physiology of S. mutans, affecting critical features used by the organism to cause disease and providing a potential novel target for inhibiting the pathogenesis of S. mutans.

SUBMITTER: Kovacs CJ

PROVIDER: S-EPMC5686612 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature

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RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans.

Kovacs C J CJ Faustoferri R C RC Quivey R G RG

Journal of bacteriology 20171114 24

Bacterial cell wall dynamics have been implicated as important determinants of cellular physiology, stress tolerance, and virulence. In <i>Streptococcus mutans</i>, the cell wall is composed primarily of a rhamnose-glucose polysaccharide (RGP) linked to the peptidoglycan. Despite extensive studies describing its formation and composition, the potential roles for RGP in <i>S. mutans</i> biology have not been well investigated. The present study characterizes the impact of RGP disruption as a resu ...[more]

PMID: 28924033

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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.

Project description:Aerobic microorganisms have evolved different strategies to withstand environmental oxidative stresses generated by various reactive oxygen species (ROS). For the facultative anaerobic human oral pathogen Streptococcus mutans, the mechanisms used to protect against ROS are not fully understood, since it does not possess catalase, an enzyme that degrades hydrogen peroxide. In order to elucidate the genes that are essential for superoxide stress response, methyl viologen (MV)-sensitive mutants of S. mutans were generated via ISS1 mutagenesis. Screening of approximately 2,500 mutants revealed six MV-sensitive mutants, each containing an insertion in one of five genes, including a highly conserved hypothetical gene, SMU.1297. Sequence analysis suggests that SMU.1297 encodes a hypothetical protein with a high degree of homology to the Bacillus subtilis YtqI protein, which possesses an oligoribonuclease activity that cleaves nano-RNAs and a phosphatase activity that degrades 3'-phosphoadenosine-5'-phosphate (pAp) and 3'-phosphoadenosine-5'-phosphosulfate (pApS) to produce AMP; the latter activity is similar to the activity of the Escherichia coli CysQ protein, which is required for sulfur assimilation. SMU.1297 was deleted using a markerless Cre-loxP-based strategy; the SMU.1297 deletion mutant was just as sensitive to MV as the ISS1 insertion mutant. Complementation of the deletion mutant with wild-type SMU.1297, in trans, restored the parental phenotype. Biochemical analyses with purified SMU.1297 protein demonstrated that it has pAp phosphatase activity similar to that of YtqI but apparently lacks an oligoribonuclease activity. The ability of SMU.1297 to dephosphorylate pApS in vivo was confirmed by complementation of an E. coli cysQ mutant with SMU.1297 in trans. Thus, our results suggest that SMU.1297 is involved in superoxide stress tolerance in S. mutans. Furthermore, the distribution of homologs of SMU.1297 in streptococci indicates that this protein is essential for superoxide stress tolerance in these organisms.

Project description:BackgroundThe S. mutans LrgA/B holin-like proteins have been shown to affect biofilm formation and oxidative stress tolerance, and are regulated by oxygenation, glucose levels, and by the LytST two-component system. In this study, we sought to determine if LytST was involved in regulating lrgAB expression in response to glucose and oxygenation in S. mutans.ResultsReal-time PCR revealed that growth phase-dependent regulation of lrgAB expression in response to glucose metabolism is mediated by LytST under low-oxygen conditions. However, the effect of LytST on lrgAB expression was less pronounced when cells were grown with aeration. RNA expression profiles in the wild-type and lytS mutant strains were compared using microarrays in early exponential and late exponential phase cells. The expression of 40 and 136 genes in early-exponential and late exponential phase, respectively, was altered in the lytS mutant. Although expression of comYB, encoding a DNA binding-uptake protein, was substantially increased in the lytS mutant, this did not translate to an effect on competence. However, a lrgA mutant displayed a substantial decrease in transformation efficiency, suggestive of a previously-unknown link between LrgA and S. mutans competence development. Finally, increased expression of genes encoding antioxidant and DNA recombination/repair enzymes was observed in the lytS mutant, suggesting that the mutant may be subjected to increased oxidative stress during normal growth. Although the intracellular levels of reaction oxygen species (ROS) appeared similar between wild-type and lytS mutant strains after overnight growth, challenge of these strains with hydrogen peroxide (H2O2) resulted in increased intracellular ROS in the lytS mutant.ConclusionsOverall, these results: (1) Reinforce the importance of LytST in governing lrgAB expression in response to glucose and oxygen, (2) Define a new role for LytST in global gene regulation and resistance to H2O2, and (3) Uncover a potential link between LrgAB and competence development in S. mutans.

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RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans.

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RgpF Is Required for Maintenance of Stress Tolerance and Virulence in Streptococcus mutans.

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