dStreptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived ␤1,3-glucans contribute to the EPS matrix structure, while fungal mannan and ␤-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.

No abstract

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 Culturing methods are the primary approach for microbiological analysis of plaque-biofilms in rodent models of dental caries. In this study, we developed strategies for isolation of DNA and RNA from in vivo formed plaque-biofilms to analyze the viable bacterial population and gene expression. Plaque-biofilm samples from rats were treated with propidium monoazide to isolate DNA from viable cells, and the purified DNA was used to quantify total bacteria and S. mutans population via qPCR and specific primers; the same samples were also analyzed by colony forming unit (CFU) counting. In parallel, RNA was isolated from plaque-biofilm samples (from same animals) and used for transcriptional analyses via RT-qPCR. The viable population of both S. mutans and total bacteria assessed by qPCR were positively correlated with the CFU data (P<0.001; r>0.8). However, the qPCR data showed higher bacterial cell counts, particularly for total bacteria (vs. CFU). Moreover, S. mutans proportion in the plaque-biofilm determined by qPCR analysis showed strong correlation with incidence of smooth-surface caries (P=0.0022, r=0.71). The purified RNAs presented high RNA integrity numbers (>7), which allowed measurement of the expression of genes that are critical for S. mutans virulence (e.g. gtfB and gtfC). Our data show that the viable microbial population and the gene expression can be analyzed simultaneously, providing a global assessment of the infectious aspect of the disease dental caries. Our approach could enhance the value of the current rodent model in further understanding the pathophysiology of this disease and facilitating the exploration of novel anti-caries therapies.