CovR and VicRKX Regulate Transcription of the Collagen Binding Protein Cnm of Streptococcus mutans

Abstract

Cnm is a surface-associated protein present in a subset of Streptococcus mutans strains that mediates binding to extracellular matrices, intracellular invasion, and virulence. Here, we showed that cnm transcription is controlled by the global regulators CovR and VicRKX. In silico analysis identified multiple putative CovR- and VicR-binding motifs in the regulatory region of cnm as well as in the downstream gene pgfS, which is associated with the posttranslational modification of Cnm. Electrophoretic mobility shift assays revealed that CovR and VicR specifically and independently bind to the cnm and pgfS promoter regions. Quantitative real-time PCR and Western blot analyses of ΔcovR and ΔvicK strains as well as of a strain overexpressing vicRKX revealed that CovR functions as a positive regulator of cnm, whereas VicRKX acts as a negative regulator. In agreement with the role of VicRKX as a repressor, the ΔvicK strain showed enhanced binding to collagen and laminin and higher intracellular invasion rates. Overexpression of vicRKX was associated with decreased rates of intracellular invasion but did not affect collagen or lamin binding activities, suggesting that this system controls additional genes involved in binding to these extracellular matrix proteins. As expected, based on the role of CovR in cnm regulation, the ΔcovR strain showed decreased intracellular invasion rates, but, unexpectedly collagen and laminin binding activities were increased in this mutant strain. Collectively, the results presented here expand the repertoire of virulence-related genes regulated by CovR and VicRKX to include the core gene pgfS and the noncore gene cnmIMPORTANCEStreptococcus mutans is a major pathogen associated with dental caries and also implicated in systemic infections, in particular, infective endocarditis. The Cnm adhesin of S. mutans is an important virulence factor associated with systemic infections and caries severity. Despite its role in virulence, the regulatory mechanisms governing cnm expression are poorly understood. Here, we describe the identification of two independent regulatory systems controlling the transcription of cnm and the downstream pgfS-pgfM1-pgfE-pgfM2 operon. A better understanding of the mechanisms controlling expression of virulence factors like Cnm can facilitate the development of new strategies to treat bacterial infections.

Keywords: Streptococcus mutans; adhesins; host cell invasion; virulence regulation.

FIG 1

Analysis of the cnm and pgfS promoter regions and interactions of CovR and VicR with cnm and pgfS promoters. (A) Mapping of the transcription start site of cnm and pgfS genes by RACE-PCR. Translational start sites are marked with green letters, and transcription start sites are shown in bold black font. Putative CovR and VicR binding sites are shown in solid and dashed boxes, respectively. Consensus sequence matches for both CovR and VicR are shown in uppercase letters, whereas the mismatches present in the putative binding sites were assigned in lowercase letters. Underlined letters indicate −10 and −35 regions. (B) VicR and CovR directly interact with the promoter regions of cnm and pgfS. Recombinant CovR (rCovR) and VicR (rVicR) proteins bind to the promoter regions of cnm and pgfS, as determined by EMSA. The specificity of binding was confirmed in competitive assays using excess unlabeled specific DNA (cold+) or excess unlabeled unspecific DNA (cold−). The data shown are representative of those from at least three independent experiments.

FIG 2

Transcription levels of cnm and pgfS and abundance of Cnm in S. mutans OMZ175 and derivatives. The relative levels of gene transcripts of cnm (A) and pgfS (B) in ΔcovR and ΔvicK mutant strains of OMZ175 and pgfS in mutant strains of the cnm-negative strain UA159 (C) were determined by qRT-PCR. Columns and bars indicate averages and standard deviations from at least three independent experiments, respectively. Asterisks indicate significant differences compared to the parent strain (analysis of variance with a post hoc Dunnett's test; *, P < 0.05; **, P < 0.01). (D) Detection of Cnm in cell extracts of S. mutans strains by Western blotting. The Δcnm strain was used as a negative control. The blots shown are representative of those from three independent experiments.

FIG 3

Contribution of CovR and VicRKX to Cnm-mediated phenotypes associated with systemic virulence in OMZ175. (A, B) Relative collagen (A) and laminin (B) binding of covR and vicK mutants and their respective complemented strains, OMZ175 ΔcovR⁺ and OMZ175 ΔvicK⁺, compared to wild-type OMZ175. (C) Percent HCAEC invasion by covR and vicK mutants and the complemented strains in relation to that by OMZ175. Bacteria were recovered from the intracellular compartment of HCAEC after 3 h of infection. (A to C) Columns and bars represent means and standard deviations from at least three independent experiments, respectively. Asterisks indicate significant differences compared to the wild type (the Kruskal-Wallis test with a post hoc Dunn's test; *, P < 0.05; **, P < 0.01). (D, E) Percent survival of G. mellonella larvae infected with OMZ175 ΔvicK (OMZΔvicK) (D) and OMZ175 ΔcovR (OMZΔcovR) (E) mutants and their respective complemented strains. Heat-killed OMZ175 was used as a negative control of infection. The results are representative of those from experiments performed at least in triplicate. The results were plotted as Kaplan-Meier survival curves, and differences in survival were compared using the log-rank test (P < 0.05).

FIG 4

Overexpression of vicRKX results in reduced Cnm expression levels. (A) Western blot and densitometry analysis of Cnm expression in OMZ175, OMZ175 ΔvicK, the Δcnm strain, and OMZ175 vicRKX using an anti-Cnm-specific antibody. (B) qRT-PCR of cnm transcript levels in OMZ175 and OMZ175 vicRKX. (C to E) Collagen binding (C), laminin binding (D), and HCAEC invasion rates (E) in the vicRKX-overexpressing strain in relation to those in OMZ175. Columns and bars represent means and standard deviations from at least three independent experiments, respectively. Asterisks indicate significant differences in relation to the corresponding wild type (Kruskal-Wallis with post hoc Dunn's test; *, P < 0.05).

FIG 5

Determination of phosphorylation status of CovR. Western blot analysis of CovR protein of OMZ175, OMZ175 ΔvicK, and OMZ175 ΔcovR loaded (at 20 μg protein per lane) onto either a 10% Phos-tag SDS-PAGE gel (A) or 10% SDS-PAGE gel (B). Purified recombinant CovR (rCovR) was used as a control for unphosphorylated CovR. Dimer and Monomer, dimeric and monomeric CovR, respectively.