Construction and phenotypic characterization of Streptococcus mutans mutants strains of genes related to virulence

Abstract

Dental caries is an infectious and multifactorial disease, biofilm dependent, which has Streptococcus mutans as the main etiological agent. The virulence of S. mutans resides in its abilities to adhere and form biofilms on tooth surfaces, to produce organic acids and to tolerate environmental stresses, particularly low pH. As S. mutans is able to emerge as the dominant flora in the biofilm, even under stress conditions, it is the target of numerous studies. It is known that Clp system (ATP-dependent caseinolytic protease) has a central role in tolerance against environment stress by this microorganism. Clp plays an important role in cellular homeostasis, and has ability to control the stability of regulatory proteins of S. mutans. Furthermore, it was observed that Spx protein is used as a substrate for the Clp proteolytic system. Thus, it was done a detailed phenotypic characterization of Spx, which was shown as a global regulator able to regulate the transcription of many genes that are known to be related to stress in S. mutans and others hypothetical. Thereby, the aim of this study will be to continue with the research line in progress through construction and phenotypical characterization, in vitro and in vivo, of S. mutans mutants with deletion of hypothetical genes that showed altered expression on mutants with spxA and spxAB deleted genes. For this, it will be constructed mutants of selected genes, smu144c, smu1116c and smu1784c, which showed altered expression on mutants with spxA and spxAB deleted genes and are strong candidates to be genes related to survival of S. mutans under stress conditions, and its construction will be confirmed. It will be done transcriptional analysis of selected genes during oxidative and acid stress conditions through Real Time PCR. Moreover, the constructed mutants will be phenotypically characterized by assessing their susceptibility, survival and growth under acid and oxidative stress conditions. Enzymatic assays will also be done evaluating activity of protective enzymes against oxidative stress, which are NADH oxidase (Nox), glutathione oxidoreductase (Gor) e Superoxide dismutase (Sod). Finally, the mutants will be analyzed as their infection ability in animal models (Infection in Galleria mellonella and experimental dental caries in rats). It is expected, after phenotypical characterization, that will be identified new genes regulated by Spx and related to oxidative stress in S. mutans, to facilitate understanding about the mechanisms by which this microorganism is able to survive under stress conditions.