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 (⌬spxA, ⌬spxB, and ⌬spxA ⌬spxB) revealed that Spx, indeed, participates in the regulation of processes associated with S. mutans pathogenesis. The ⌬spxA strain displayed impaired ability to grow under acidic and oxidative stress conditions and had diminished long-term viability at low pH. Although the ⌬spxB strain did not show any inherent stress-sensitive phenotype, the phenotypes observed in ⌬spxA were more pronounced in the ⌬spxA ⌬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.

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 ⌬clpL strain displayed an enhanced capacity to form biofilms in the presence of sucrose, had reduced viability, and was more sensitive to acid killing. The ⌬clpP and ⌬clpX 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 ⌬clpP and ⌬clpX mutants were more resistant to acid killing and demonstrated enhanced viability in long-term survival assays. Biofilm formation by the ⌬clpP and ⌬clpX 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 ⌬clpP or ⌬clpX 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 ⌬clpXP 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 ⌬clpP and ⌬clpX 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.

c NADH oxidase (Nox, encoded by nox) is a flavin-containing enzyme used by the oral pathogen Streptococcus mutans to reduce diatomic oxygen to water while oxidizing NADH to NAD ؉ . The critical nature of Nox is 2-fold: it serves to regenerate NAD ؉ , a carbon cycle metabolite, and to reduce intracellular oxygen, preventing formation of destructive reactive oxygen species (ROS). As oxygen and NAD؉ have been shown to modulate the activity of the global transcription factors Spx and Rex, respectively, Nox is potentially poised at a critical junction of two stress regulons. In this study, microarray data showed that either addition of oxygen or loss of nox resulted in altered expression of genes involved in energy metabolism and transport and the upregulation of genes encoding ROS-metabolizing enzymes. Loss of nox also resulted in upregulation of several genes encoding transcription factors and signaling molecules, including the redox-sensing regulator gene rex. Characterization of the nox promoter revealed that nox was regulated by oxygen, through SpxA, and by Rex. These data suggest a regulatory loop in which the roles of nox in reduction of oxygen and regeneration of NAD ؉ affect the activity levels of Spx and Rex, respectively, and their regulons, which control several genes, including nox, crucial to growth of S. mutans under conditions of oxidative stress.

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 H 2 O 2 , 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 (Spx G52R ) resulted in strains that phenocopied the ⌬spx strains. Moreover, addition of purified SpxA1 G52R completely failed to activate transcription of ahpC, sodA, and tpx, further confirming that the G52 residue is critical for Spx functionality. IMPORTANCEStreptococcus 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.T he oral cavity is colonized by hundreds of bacterial species, some of which contribute to overall oral health and others that are associated with disease, such as dental caries and periodontitis. Among the pathogenic oral bacteria, clinical evidence paired with in vitro and in vivo studies strongly associates Streptococcus mutans with dental caries onset and development (1, 2). For all organisms that inhabit the oral cavity, oxidative stresses are relevant threats for which defense mechanisms must be in place. Aside from the presence of hydrogen peroxide (H 2 O 2 ) in oral care products, members of the mitis group of streptococci, which cohabit the dental plaque along with S. mutans, are net producers of H 2 O 2 (3-5). Notably, there is an inverse correlation between the proportion of S. mutans and of members of the mitis group in health and disease, with high numbers of S. mutans organisms associated with disease and a high proportion of mitis streptococci associated with oral health (6, 7). Ultimately, the breakdown of H 2 O 2 into other variants of reactive oxygen species (ROS) can disturb the integrity of DNA and proteins and thereby pose a significant threat to...

SUMMARY In oral biofilms, the major environmental challenges encountered by Streptococcus mutans are acid and oxidative stresses. Previously, we showed that the transcriptional regulators SpxA1 and SpxA2 are involved in general stress survival of S. mutans with SpxA1 playing a primary role in activation of antioxidant and detoxification strategies whereas SpxA2 serves as a back up activator of oxidative stress genes. We have also found that spxA1 mutant strains (ΔspxA1 and ΔspxA1ΔspxA2) are outcompeted by peroxigenic oral streptococci in vitro and have impaired abilities to colonize the teeth of rats fed a highly cariogenic diet. Here, we show that the Spx proteins can also exert regulatory roles in the expression of additional virulence attributes of S. mutans. Competence activation is significantly impaired in Δspx strains and the production of mutacin IV and V is virtually abolished in ΔspxA1 strains. Unexpectedly, the ΔspxA2 strain showed increased production of glucans from sucrose, without affecting the total amount of bacteria within biofilms when compared to the parent strain. By using the rat caries model, we showed that the capacity of the ΔspxA1 and ΔspxA2 strains to cause caries on smooth tooth surfaces is significantly impaired. The ΔspxA2 strain also formed fewer lesions on sulcal surfaces. This report reveals that global regulation via Spx contributes to the cariogenic potential of S. mutans and highlights the essentiality of animal models in the characterization of bacterial traits implicated in virulence.