Abstract

Mutational analysis revealed that members of the Clp system, specifically the ClpL chaperone and the ClpXP proteolytic complex, modulate the expression of important virulence attributes of Streptococcus mutans. Compared to its parent, the DeltaclpL strain displayed an enhanced capacity to form biofilms in the presence of sucrose, had reduced viability, and was more sensitive to acid killing. The DeltaclpP and DeltaclpX strains displayed several phenotypes in common: slow growth, tendency to aggregate in culture, reduced autolysis, and reduced ability to grow under stress, including acidic pH. Unexpectedly, the DeltaclpP and DeltaclpX mutants were more resistant to acid killing and demonstrated enhanced viability in long-term survival assays. Biofilm formation by the DeltaclpP and DeltaclpX strains was impaired when grown in glucose but enhanced in sucrose. In an animal study, the average number of S. mutans colonies recovered from the teeth of rats infected with the DeltaclpP or DeltaclpX strain was slightly lower than that of the parent strain. In Bacillus subtilis, the accumulation of the Spx global regulator, a substrate of ClpXP, has accounted for the DeltaclpXP phenotypes. Searching the S. mutans genome, we identified two putative spx genes, designated spxA and spxB. The inactivation of either of these genes bypassed phenotypes of the clpP and clpX mutants. Western blotting demonstrated that Spx accumulates in the DeltaclpP and DeltaclpX strains. Our results reveal that the proteolysis of ClpL and ClpXP plays a role in the expression of key virulence traits of S. mutans and indicates that the underlying mechanisms by which ClpXP affect virulence traits are associated with the accumulation of two Spx orthologues.