Phenotypic Characterization of spx mutant Strains in Streptococcus mutans

Streptococcus mutans is the major etiologic agent of dental caries.  The ability to survive the periodic shifts in pH, oxygen tension and nutrient availability encountered in oral biofilms is a key factor in the virulence of this organism. Therefore, the elucidation of the mechanisms used by these bacteria to thrive in stressful conditions is central for a complete understanding of the pathogenesis of dental caries.  Recently, we demonstrated that the ClpXP protease plays a role in the expression of virulence attributes of S. mutans, including stress tolerance and biofilm formation.  In addition to targeting damaged proteins for degradation, the ClpXP complex regulates the stability of central regulatory proteins.  In Bacillus subtilis, inactivation of the transcriptional regulator Spx alleviated phenotypes associated with clpXP mutations. The S. mutans UA159 genome harbors two copies of spx-like genes and inactivation of either gene (herein spxA and spxB) also bypasses the physiologic effects observed in the clpXP mutants. Objectives: The interactions between ClpXP and Spx prompted us to characterize the Dspx strains in relation to stress and biofilm formation.  Methods:  The Dspx mutants were subjected to standard in vitro stress challenges and biofilm assays.  Results:  The DspxA and DspxAB strains showed impaired growth when exposed to acidic and oxidative stresses.  There were no differences in survival when these strains were exposed to a short-term acid killing, but viability of DspxA and DspxAB was substantially lower in long-term survival assays.  When compared to its parent, both DspxB and DspxAB formed significantly more biofilms in the presence of glucose.  In sucrose, the DspxB continued to show enhanced capacity to form biofilms but the DspxAB displayed the opposite trend.  Conclusion: Our results show that Spx plays a major role in the regulation of traits that are associated with the pathophysiology of S. mutans and dental caries.  Funding T32DE007-202