Transcription of Oxidative Stress Genes Is Directly Activated by SpxA1 and, to a Lesser Extent, by SpxA2 in Streptococcus mutans.

UNLABELLED: The SpxA1 and SpxA2 (formerly SpxA and SpxB) transcriptional regulators of Streptococcus mutans are members of a highly conserved family of proteins found in Firmicutes, and they were previously shown to activate oxidative stress responses. In this study, we showed that SpxA1 exerts substantial positive regulatory influence over oxidative stress genes following exposure to H2O2, while SpxA2 appears to have a secondary regulatory role. In vitro transcription (IVT) assays using purified SpxA1 and/or SpxA2 showed that SpxA1 and, less often, SpxA2 directly activate transcription of some of the major oxidative stress genes. Addition of equimolar concentrations of SpxA1 and SpxA2 to the IVT reactions neither enhanced transcription of the tested genes nor disrupted the dominant role of SpxA1. Substitution of a conserved glycine residue (G52) present in both Spx proteins by arginine (SpxG52R) resulted in strains that phenocopied the Δspx strains. Moreover, addition of purified SpxA1G52R completely failed to activate transcription of ahpC, sodA, and tpx, further confirming that the G52 residue is critical for Spx functionality. IMPORTANCE: Streptococcus mutans is a pathogen associated with the formation of dental caries in humans. Within the oral cavity, S. mutans routinely encounters oxidative stress. Our previous data revealed that two regulatory proteins, SpxA1 and SpxA2 (formerly SpxA and SpxB), bear high homology to the Spx regulator that has been characterized as a critical activator of oxidative stress genes in Bacillus subtilis. In this report, we prove that Spx proteins of S. mutans directly activate transcription of genes involved in the oxidative stress response, though SpxA1 appears to have a more dominant role than SpxA2. Therefore, the Spx regulators play a critical role in the ability of S. mutans to thrive within the oral cavity.

Two Spx proteins modulate stress tolerance, survival, and virulence in Streptococcus mutans.

Previous work suggested that the underlying mechanisms by which the Streptococcus mutans ClpXP protease affects virulence traits are associated with accumulation of two orthologues of the Spx regulator, named SpxA and SpxB. Here, a thorough characterization of strains lacking the spx genes (Delta spxA, Delta spxB, and Delta spxA Delta spxB) revealed that Spx, indeed, participates in the regulation of processes associated with S. mutans pathogenesis. The Delta spxA strain displayed impaired ability to grow under acidic and oxidative stress conditions and had diminished long-term viability at low pH. Although the Delta spxB strain did not show any inherent stress-sensitive phenotype, the phenotypes observed in Delta spxA were more pronounced in the Delta spxA Delta spxB double mutant. By using two in vivo models, we demonstrate for the first time that Spx is required for virulence in a gram-positive pathogen. Microarrays confirmed the global regulatory role of SpxA and SpxB. In particular, SpxA was shown to positively regulate genes associated with oxidative stress, a finding supported by enzymatic assays. SpxB had a secondary role in regulation of oxidative stress genes but appeared to play a larger role in controlling processes associated with cell wall homeostasis. Given the high degree of conservation between Spx proteins of low-GC gram-positive bacteria, these results are likely to have broad implications.

Role of Clp proteins in expression of virulence properties of Streptococcus mutans.

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.

Modulation of elevated plus maze behavior after chronic exposure to the anabolic steroid 17alpha-methyltestosterone in adult mice.

Exposure to supraphysiological doses of androgens may disrupt affective components of behavior. In this study, behavior of adult C57Bl/6 male mice was studied after exposure to the anabolic androgenic steroid (AAS) 17alpha-methyltestosterone (17alpha-meT; 7.5 mg/kg) via a subcutaneous osmotic pump for 17 days. Controls received vehicle implants (0.9% NaCl + 30% cyclodextrine). On day 15, experimental animals were challenged with an ethanol (EtOH) injection (i.p.; 1 g/kg) while controls received saline injections. Five minutes after the injection, animals were tested in an automated elevated plus maze (EPM) or in automated activity chambers. In addition, injection-free animals were tested for ethanol consumption on day 16 after an overnight water deprivation period. Whereas chronic exposure to 17alpha-meT did not modulate open arm behavior, EtOH-exposed animals made more entries into the open arms than controls (P < 0.05). A significant reduction of risk assessment behaviors (rearing, flat approach behavior, and stretch attended posture) over the EPM was noted for EtOH-exposed animals whereas a reduction in stretch attended postures was observed among 17alpha-meT-exposed animals. Locomotor activity, and light-dark transitions in activity chambers remained unaltered. Exposure to AAS did not modulate EtOH consumption. Our data suggest that exposure to a supraphysiological dose of 17alpha-meT has minimal effects on exploratory-based anxiety.

Modulation of affect after chronic exposure to the anabolic steroid 17alpha-methyltestosterone in adult mice.

A battery of behavioral tasks in C57BL/6J mice was used to assess changes in affective components of behavior after systemic exposure to the anabolic-androgenic steroid (AAS) 17alpha-methyltestosterone (7.5 mg/kg). Gonadal weight in both sexes was reduced after 16 days of AAS exposure. Changes in discrete components of social behaviors were observed. No changes were recorded in the elevated plus-maze, the light-dark transition, and defensive behavior tests on exposure to 17alpha-methyltestosterone. When compared with controls, AAS-exposed females received a greater number of shocks, and AAS-exposed males displayed a shorter recovery time to consume water after a negative reinforcer in the modified Vogel conflict test. Results show that systemic exposure to a single AAS modified social behaviors, whereas minimal effects on anxiety-related behaviors were observed according to sex.