virulência sistêmica de Streptococcus mutans Analysis of factors regulated by CovR and VicRK involved in the susceptibility to opsonization by the complement system and systemic virulence in Streptococcus mutans Piracicaba 2017

Streptococcus mutans (SM) is a common bacterial species of the oral cavity of humans involved in the pathogenesis of dental caries, which can promote systemic infections once reaching and persisting in the bloodstream (Nomura et al., 2006). To persist in the host niches, SM applies two-component transcriptional regulatory systems (TCS). The TCS CovR and VicRK of SM regulate virulence genes associated with SM-binding to extracellular polysaccharides (EPS) produced from sucrose (gpbB, gbpC and epsC) and /or involved in the interaction of the cell wall with the biofilm matrix (wapE, lysM, 2146c, smaA). Inactivation of this TCS promotes resistance to phagocytosis by PMN when in the presence of blood (Negrini et al., 2012), indicating that these systems regulate genes for evasion to blood opsonins, such as the complement system. The aim of this study was to identify the molecular mechanisms through CovR and VicRK modulate the susceptibility of S. mutans to complement mediated immunity and survival in the bloodstream. The results obtained were included in three scientific articles which are presented as chapters. In chapter I, we characterize the profile of SM strains susceptibilities to the complement. Flow cytometry and RT-PCR analysis revealed that strains isolated from systemic infections show lower susceptibility to complement deposition and reduced expression of covR compared to oral isolates. Inactivation of covR in SM UA159 increased resistance to complement mediated opsonophagocytosis by PMN in a way dependent on binding to EPS, ex vivo survival in human blood and in the bloodstream of rats. Blood isolates showed significantly higher expression of CovRrepressed genes (gbpC and epsC) and, consistently, increased binding to EPS. Consistently, inactivation of gbpC or epsC reduced binding to EPS and increased susceptibility to complement. In Chapter II, we show that VicRK regulates SM susceptibility to complement-mediated immunity, in a EPS-independent way. Inactivation of vicK increased resistance to complement opsonization. VicRK was shown to directly or indirectly repress the expression of Smu.399 and PepO complement proteases and the enolase and surface glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins implicated in systemic virulence in other streptococci. Finally, in Chapter III, we established that CovR and VicRK directly regulate the Cnm protein, a which is important for systemic virulence in a sub-set of strains harboring cnm gene. Transcriptional analyses and assessment of interaction of recombinant proteins rCovR and rVicR with the cnm promoter region established that CovR acts as an inducer of cnm, whereas vicR acts as a repressor. Cnm-mediated virulence phenotypes were significantly associated with lower and higher cnm expression in the covR and vicK mutants, respectively. Thus, we showed that the TCSs CovR and VicRK regulate several systemic virulence factors dependent and independent of the interaction with EPS, involved in evasion to complement-mediated immunity and survival in blood. These results further confirm the importance of SM in systemic infections and contribute to the definition of therapeutic targets for controlling these infections.