CovR and VicRK Regulate the Collagen Binding Protein Cnm of Streptococcus mutans

Objectives: Determine whether the Two Component Systems CovR and VicRK regulate the Cnm adhesin and the protein glycosylation machinery Pgf in invasive Streptococcus mutans.
Methods: In silico analyzes identified putative binding sites of CovR and VicR in the cnm and pgfS promoters of S. mutans OMZ175. Isogenic covR and vicK mutants were obtained in OMZ175 (OMZ175ΔcovR and OMZ175ΔvicK, respectively) and their cnm and pfgS expression levels were analyzed by qRT-PCR and western blot (WB). Interactions of recombinant CovR and VicR with the promoters of cnm and pgfS were analyzed in Electrophoretic mobility shift assays (EMSA). Cnm-dependent phenotypes, i.e collagen- and laminin-binding, invasion of human coronary artery endothelial cells (HCAEC), and virulence in the in vivo model Galleria mellonella, were assessed in the covR and vicK mutant strains and compared to OMZ175.
Results: qRT-PCR revealed expression of cnm and pgfS were significantly down-regulated in OMZ175ΔcovR (>1.5-fold down), whereas these genes were up-regulated in OMZ175Δvick (>2.5-fold up). Transcriptional changes were compatible with changes in amounts of Cnm produced by these mutants as determined in WB analysis. EMSA confirmed that rCovR and rVicR directly bind to the promoter regions of cnm and pgfS. Collagen- and laminin-binding assays showed that the OMZ175ΔvicK has increased binding to either collagen or laminin (>250-fold). Unexpectedly, the OMZ175ΔcovR also showed enhanced ability to bind to collagen and laminin (>10-fold). Altered expression of cnm determined by qRT-PCR and WB analysis in OMZ175ΔcovR and OMZ175ΔvicK were consistently associated with altered abilities of the strains to invade HCAEC (p<0.05). When used to infect the invertebrate model G. mellonella, OMZ175Δvick resulted in enhanced virulence (p<0.01), whereas OMZ175ΔcovR behaved similarly to OMZ175.
Conclusions: CovR is an activator whereas VicK is a repressor of the systemic virulence gene cnm and of the protein glycosylation machinery Pgf in S. mutans OMZ175.