Charactrization of CovR and VicRK in Streptococcus mutans

Author(s)

Rafael Nobrega Stipp

• Document type: Doctoral Thesis
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2010-02-19

Examining board members

Renata de Oliveira Mattos Graner; Marcia Pinto Alves Mayer; Frederico José Gueiros Filho; Pedro Luiz Rosalen; Ricardo Della Coletta

• Advisor: José Francisco Höfling; Renata de Oliveira Mattos Graner

Abstract

Streptococcus mutans, a major dental caries pathogen, expresses several virulence genes that mediate its growth and accumulation on tooth surfaces. In this process, GtfB and GtfC catalyze the extracellular synthesis of water-insoluble glucan matrix from sucrose, and are essential for accumulation of S. mutans in the dental biofilm. Glucan binding protein B might also mediate cell surface interaction with glucan. Two component transduction systems, such as CovR (Control of Virulence) and VicRK (Virulence Control), regulate, at least, part of S. mutans virulence genes. In this study we characterized CovR and VicRK functions and some phenotypic traits related to their absences. Knockouts strains covR- (SMU.1924) and vicK- (SMU.1517) strains were constructed and compared by differential microarray against wild type. Quantitative RT-PCR confirmed twenty-three up-regulated genes in covR-, while thirty down-regulated genes in vicKmutant. Recombinant CovR and VicR proteins were used in protein:DNA-promoter electrophoretic mobility shift assay (EMSA) to confirm direct regulator-promoter interaction and transcription. In addition to already-know CovR and VicR targets, qPCR and EMSA revealed that CovR acts as a negative regulator of four additional genes, and VicR controls five undescribed promoters. In both cases, genes controlled are mainly involved in biofilm growth and in cell wall biogenesis. Phenotypically, covR- mutants showed lower yield in culture, while vicK- mutants had altered cell morphology and long chains formation. Inactivation of covR significantly increased in vitro biofilm formation in all strains (3 to 17-fold increase, p<0.01), while vicK mutants showed poor biofilm growth (3.5 to 6.5-fold reduction; p<0.01). Cell hydrophobicity was increased by 40% in covR- mutants and by 10- fold in vicK- mutants, possibly due cell surface changes. Both knockoutsalso increased the autolysis resistance, in about 30% for the covR- mutants and in 2- fold for the vicK- mutant. It is concluded that CovR and VicR systems have important participation on S. mutans physiology, regulating genes that not only are involved in biofilm formation but that have functions in cell wall biogenesis. (AU)