Abstract
The microbes that inhabit the human oral cavity are subjected to constant fluctuations in their environment. To overcome these challenges and gain a competitive advantage, oral streptococci employ numerous adaptive strategies, many of which appear to be intertwined with the development of genetic competence. Here, we demonstrate that the regulatory circuits that control development of competence in Streptococcus mutans, a primary etiological agent of human dental caries, are integrated with key stress tolerance pathways by the molecular alarmone (p)ppGpp. We first observed that the growth of a strain that does not produce (p)ppGpp (ΔrelAPQ, (p)ppGpp0) is not sensitive to growth inhibition by comX inducing peptide (XIP), unlike the wild-type strain UA159, even though XIP-dependent activation of the alternative sigma factor comX by the ComRS pathway is not impaired in the (p)ppGpp0 strain. Overexpression of a (p)ppGpp synthase gene (relP) in the (p)ppGpp0 mutant restored growth inhibition by XIP. We also demonstrate that exposure to micromolar concentrations of XIP elicited changes in (p)ppGpp accumulation in UA159. Loss of the RelA/SpoT homolog (RSH) enzyme, RelA, lead to higher basal levels of (p)ppGpp accumulation, but to decreased sensitivity to XIP and to decreases in comR promoter activity and ComX protein levels. By introducing single amino acid substitutions into the RelA enzyme, the hydrolase activity of the enzyme was shown to be crucial for full com gene induction and transformation by XIP. Finally, loss of relA resulted in phenotypic changes to ΔrcrR mutants, highlighted by restoration of transformation and ComX protein production in the otherwise non-transformable ΔrcrR-NP mutant. Thus, RelA activity and its influence on (p)ppGpp pools appears to modulate competence signaling and development through RcrRPQ and the peptide effectors encoded within rcrQ. Collectively, this study provides new insights into the molecular mechanisms that integrate intercellular communication with the physiological status of the cells and the regulation of key virulence-related phenotypes in S. mutans.
Highlights
The human oral cavity typically hosts over six billion microbes, with as many as 1,200 distinct taxa being identified in metagenomic studies (Jenkinson, 2011)
While previous communications indicated that a deletion of the relA gene of S. mutans does not appear to have effects on transformation efficiency (Lemos et al, 2004), those experiments were performed in the complex medium brain heart infusion (BHI) in the presence of competence stimulating peptide (CSP)
To determine if (p)ppGpp influenced the development of competence by ComRS in a chemically defined medium (FMC), we compared the growth kinetics between the S. mutans WT strain UA159 and a (p)ppGpp0 mutant in the presence of various concentrations of sXIP, which was dissolved in dimethyl sulfoxide (DMSO)
Summary
The human oral cavity typically hosts over six billion microbes, with as many as 1,200 distinct taxa being identified in metagenomic studies (Jenkinson, 2011). Oral microbes contend with constant fluctuations in environmental conditions, including pH, oxygen tension, nutrient availability, and carbohydrate source (Lemos and Abranches, 2005). These dynamic perturbations can alter the composition and biochemical activities of the biofilms by disrupting a homeostatic environment and shifting the oral microbiome from a state of health to one of disease. RelP is responsible for the majority of (p)ppGpp produced during exponential growth (Lemos et al, 2007) Another synthetase, RelQ, plays an important role in acid and oxidative stress tolerance. Acetate metabolism and (p)ppGpp accumulation were recently shown to be tightly linked via the Pta-Acetate kinase pathway, and acetylphosphate accumulates under conditions of oxidative stress (Kim et al, 2015)
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