The Streptococcus mutans CesSR System Responds to Cell Envelope Stress

Background: Two-component signal transduction systems (TCSs) allow cells to monitor their environment and respond to transient environmental stimuli.  Streptococcus mutans, a major etiological agent of human dental caries, causes demineralization of tooth enamel through acid production.  Genetic studies have demonstrated that the CesSR TCS regulates important S. mutans virulence traits such as acid tolerance and biofilm formation.  Although not examined in streptococci, homologues of CesSR are widely disseminated in Firmicutes (low G+C Gram-positive bacteria) as part of the cell envelope stress response network.  Objective: To characterize the role of CesSR in cell envelope stress tolerance in S. mutans.  Methods: Isogenic mutants were constructed by insertional mutagenesis.  Susceptibility to cell envelope biosynthesis inhibitors was examined by growth kinetic assays.  Transcriptional studies were performed by reverse transcription (RT)-PCR and real-time RT-PCR.  Results: The CesSR TCS is part of a five-gene operon, cesFSR-ppiB-pnpB, that is transcribed as a polycistronic mRNA.  The cesF and cesSR defective mutants were successfully generated.  The susceptibility of ces mutants to the inhibitory action of a panel of eight antimicrobials, each active at different steps in peptidoglycan biosynthesis, was examined.  Relative to wild-type strain, ces mutants were at least 2- to 8-fold more sensitive to Lipid II-interfering antibiotics vancomycin and bacitracin, and to compounds that compromise cell membrane integrity such as chlorhexidine and SDS.  Lipid II-inhibitors vancomycin, bacitracin and nisin, along with membrane pore-former chlorhexidine induced cesR gene expression 3- to 5-fold in a concentration-dependent manner.  In contrast, other beta-lactam and D-cycloserine antibiotics had no discernable effect on gene expression and on the survival of the ces mutants compared to parent strain.  Conclusion: The cesFSR genes play a role in cell envelope stress response, and therefore serve an important function in maintaining the cell membrane integrity of S. mutans. Acknowledgements: CIHR (grant MT-15431) and NIH (grant R01DE013230).