Two-component system vicrk regulates functions associated with establishment of streptococcus sanguinis in biofilms

Streptococcus sanguinis is a commensal pioneer colonizer of teeth and an opportunistic pathogen of infectious endocarditis. The establishment of S. sanguinis in host sites likely requires dynamic fitting of the cell wall in response to local stimuli. In this study, we investigated the two-component system (TCS) VicRK in S. sanguinis (VicRKSs), which regulates genes of cell wall biogenesis, biofilm formation, and virulence in opportunistic pathogens. A vicK knockout mutant obtained from strain SK36 (SKvic) showed slight reductions in aerobic growth and resistance to oxidative stress but an impaired ability to form biofilms, a phenotype restored in the complemented mutant. The biofilm-defective phenotype was associated with reduced amounts of extracellular DNA during aerobic growth, with reduced production of H2O2, a metabolic product associated with DNA release, and with inhibitory capacity of S. sanguinis competitor species. No changes in autolysis or cell surface hydrophobicity were detected in SKvic. Reverse transcription-quantitative PCR (RT-qPCR), electrophoretic mobility shift assays (EMSA), and promoter sequence analyses revealed that VicR directly regulates genes encoding murein hydrolases (SSA_0094, cwdP, and gbpB) and spxB, which encodes pyruvate oxidase for H2O2 production. Genes previously associated with spxB expression (spxR, ccpA, ackA, and tpK) were not transcriptionally affected in SKvic. RT-qPCR analyses of S. sanguinis biofilm cells further showed upregulation of VicRK targets (spxB, gbpB, and SSA_0094) and other genes for biofilm formation (gtfP and comE) compared to expression in planktonic cells. This study provides evidence that VicRKSs regulates functions crucial for S. sanguinis establishment in biofilms and identifies novel VicRK targets potentially involved in hydrolytic activities of the cell wall required for these functions.Streptococcus sanguinis is a commensal pioneer colonizer of teeth and an opportunistic pathogen of infectious endocarditis. The establishment of S. sanguinis in host sites likely requires dynamic fitting of the cell wall in response to local stimuli. In t821249414951FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOCAPES - COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL E NÍVEL SUPERIOR2009/54182-7; 2008/58333-7; 2009/50547-0sem informaçã

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

EMSA analysis with promoter regions of genes potentially regulated by VicR and/or CovR.

<p>Genes which showed significant fold-changes in expression in UAvic and/or UAcov mutants compared to UA159 were selected. (A) Recombinant VicR protein (rVicR) specifically bound to the promoter regions of <i>wapE</i>, <i>lysM</i>, <i>smaA</i> and <i>SMU.367</i>; <i>covR</i> was a negative control fragment of similar size. (B) rCovR specifically bound to the promoters of <i>wapE</i>, <i>lysM</i>, <i>epsC</i> and <i>gbpB</i>, but not to <i>gtfD</i>. (C) EMSA assays performed in the presence of the two regulators (rCovR and rVicR) showed co-binding to the promoter regions of <i>lysM</i>, <i>gbpB</i> and <i>gtfC</i>. Co-binding was not observed with the promoter fragments of <i>smaA</i> and <i>epsC</i>, which are exclusively regulated by VicR and CovR, respectively. Specificity of binding was confirmed in competitive assays with excess of cold DNA.</p

Comparisons of cell surface properties of UA159 and mutant strains.

<p>Columns represent means of: (A) mureinolytic activity, (B) autolysis, (C) amounts of extracellular DNA in culture supernatants of 8 h biofilms, and (D) cell surface hydrophobicities. Data were obtained from three independent experiments performed in triplicate. Bars represent standard deviations. Asterisks indicate significant differences (* p<0.01; **p<0.05) compared to control strains (parent or complemented), as tested by ANOVA with <i>post hoc</i> Dunnett. In B, only strains with significant differences compared to parent are shown.</p

Quantitative comparisons of SEM images of 2 h biofilms using Image J software.

<p>(A) Columns represent mean numbers of matrix-based microcolonies, and (B) mean coverage areas (µm²) determined in 32 pre-determined areas per strain in one representative experiment. Bars indicate standard deviations. Asterisks indicate statistically significant differenced compared to parent UA159 (* p<0.05; Kruskal Wallis with <i>post hoc</i> Dunn's multiple comparison).</p

CovR and VicRK Regulate Cell Surface Biogenesis Genes Required for Biofilm Formation in <em>Streptococcus mutans</em>

<div><p>The two-component system VicRK and the orphan regulator CovR of <i>Streptococcus mutans</i> co-regulate a group of virulence genes associated with the synthesis of and interaction with extracellular polysaccharides of the biofilm matrix. Knockout mutants of v<i>icK</i> and <i>covR</i> display abnormal cell division and morphology phenotypes, although the gene function defects involved are as yet unknown. Using transcriptomic comparisons between parent strain UA159 with <i>vicK</i> (UAvic) or <i>covR</i> (UAcov) deletion mutants together with electrophoretic motility shift assays (EMSA), we identified genes directly regulated by both VicR and CovR with putative functions in cell wall/surface biogenesis, including <i>gbpB, wapE, smaA, SMU.2146c</i>, and <i>lysM.</i> Deletion mutants of genes regulated by VicR and CovR (<i>wapE, lysM, smaA</i>), or regulated only by VicR (<i>SMU.2146c</i>) or CovR (<i>epsC</i>) promoted significant alterations in biofilm initiation, including increased fragility, defects in microcolony formation, and atypical cell morphology and/or chaining. Significant reductions in mureinolytic activity and/or increases in DNA release during growth were observed in knockout mutants of <i>smaA</i>, <i>wapE, lysM</i>, SMU.<i>2146c</i> and <i>epsC</i>, implying roles in cell wall biogenesis. <i>WapE</i> and <i>lysM</i> mutations also affected cell hydrophobicity and sensitivity to osmotic or oxidative stress. Finally, <i>vicR, covR</i> and VicRK/CovR-targets (<i>gbpB, wapE, smaA</i>, <i>SMU.2146c, lysM, epsC</i>) are up-regulated in UA159 during biofilm initiation, in a sucrose-dependent manner. These data support a model in which VicRK and CovR coordinate cell division and surface biogenesis with the extracellular synthesis of polysaccharides, a process apparently required for formation of structurally stable biofilms in the presence of sucrose.</p> </div

Comparison of biomasses of 8 h biofilms formed in the presence of sucrose.

<p>Columns represent means of three independent experiments performed in six replicates. Bars indicate standard deviations. Significant differences compared to parent strain UA159 (dashed column) are indicated by asterisks (* p<0.05; ANOVA with <i>post hoc</i> Dunnett‘s test).</p

WebLogo representation of the position weight matrices derived from VicR or CovR regulated promoters.

<p>(A) VicR and (B) CovR consensus sequences. (C) Sequence and position of the VicR/CovR binding sites in each gene promoter. Consensus for VicR: TGTWAHNNNNNTGTWAH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058271#pone.0058271-Dubrac1" target="_blank">[9]</a>, and consensus for CovR AWATTTTTAAWAAAAR where W is A or T and R is C or A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058271#pone.0058271-Miller1" target="_blank">[30]</a>. Lower case indicates mismatch. * Distance from putative translation start site.</p

Downregulation Of Gbpb, A Component Of The Vicrk Regulon, Affects Biofilm Formation And Cell Surface Characteristics Of Streptococcus Mutans.

The virulence of the dental caries pathogen Streptococcus mutans relies in part on the sucrose-dependent synthesis of and interaction with glucan, a major component of the extracellular matrix of tooth biofilms. However, the mechanisms by which secreted and/or cell-associated glucan-binding proteins (Gbps) produced by S. mutans participate in biofilm growth remain to be elucidated. In this study, we further investigate GbpB, an essential immunodominant protein with similarity to murein hydrolases. A conditional knockdown mutant that expressed gbpB antisense RNA under the control of a tetracycline-inducible promoter was constructed in strain UA159 (UACA2) and used to investigate the effects of GbpB depletion on biofilm formation and cell surface-associated characteristics. Additionally, regulation of gbpB by the two-component system VicRK was investigated, and phenotypic analysis of a vicK mutant (UAvicK) was performed. GbpB was directly regulated by VicR, and several phenotypic changes were comparable between UACA2 and UAvicK, although differences between these strains existed. It was established that GbpB depletion impaired initial phases of sucrose-dependent biofilm formation, while exogenous native GbpB partially restored the biofilm phenotype. Several cellular traits were significantly affected by GbpB depletion, including altered cell shape, decreased autolysis, increased cell hydrophobicity, and sensitivity to antibiotics and osmotic and oxidative stresses. These data provide the first experimental evidence for GbpB participation in sucrose-dependent biofilm formation and in cell surface properties.79786-9

RT-qPCR analysis of gene expression in strain UA159 in 4 h biofilms and in planktonic phase.

<p>Biofilm (B) and planktonic (P) cells derived from the same cultures were grown in medium with or without 0.1% sucrose as indicated. Levels of transcripts in cells from sucrose-grown biofilms were set to 100% in order to calculate relative amounts of transcripts from cells grown under other conditions (biofilms w/o sucrose and planktonic cells). Columns represent means of three independent experiments performed in duplicate. Bars represent standard deviations. Statistically significant differences in gene expression in biofilm or planktonic cells grown without sucrose compared to the respective sucrose-grown cells are indicated above columns of cells without sucrose. Differences between biofilm and planktonic cells grown in the presence of sucrose are indicated above brackets. Statistical comparisons between biofilm <i>versus</i> planktonic cells in absence of sucrose are not shown. ANOVA with <i>post hoc</i> Dunnett’s test: * p<0.01; § p<0.05.</p