Genetic characterization of glyoxalase pathway in oral streptococci and its contribution to interbacterial competition.

Objectives: To analyze contributions to microbial ecology of Reactive Electrophile Species (RES), including methylglyoxal, generated during glycolysis. Methods: Genetic analyses were performed on the glyoxalase pathway in Streptococcus mutans (SM) and Streptococcus sanguinis (SS), followed by phenotypic assays and transcription analysis. Results: Deleting glyoxalase I (lguL) reduced RES tolerance to a far greater extent in SM than in SS, decreasing the competitiveness of SM against SS. Although SM displays a greater RES tolerance than SS, lguL-null mutants of either species showed similar tolerance; a finding consistent with the ability of methylglyoxal to induce the expression of lguL in SM, but not in SS. A novel paralogue of lguL (named gloA2) was identified in most streptococci. SM mutant ∆gloA2SM showed little change in methylglyoxal tolerance yet a significant growth defect and increased autolysis on fructose, a phenotype reversed by the addition of glutathione, or by the deletion of a fructose: phosphotransferase system (PTS) that generates fructose-1-phosphate (F-1-P). Conclusions: Fructose contributes to RES generation in a PTS-specific manner, and GloA2 may be required to degrade certain RES derived from F-1-P. This study reveals the critical roles of RES in fitness and interbacterial competition and the effects of PTS in modulating RES metabolism.

Heterogeneous lineage-specific arginine deiminase expression within dental microbiome species.

Arginine catabolism by the bacterial arginine deiminase system (ADS) has anticariogenic properties through the production of ammonia, which modulates the pH of the oral environment. Given the potential protective capacity of the ADS pathway, the exploitation of ADS-competent oral microbes through pre- or probiotic applications is a promising therapeutic target to prevent tooth decay. To date, most investigations of the ADS in the oral cavity and its relation to caries have focused on indirect measures of activity or on specific bacterial groups, yet the pervasiveness and rate of expression of the ADS operon in diverse mixed microbial communities in oral health and disease remain an open question. Here, we use a multivariate approach, combining ultra-deep metatranscriptomic sequencing with paired metataxonomic and in vitro citrulline quantification to characterize the microbial community and ADS operon expression in healthy and late-stage cavitated teeth. While ADS activity is higher in healthy teeth, we identify multiple bacterial lineages with upregulated ADS activity on cavitated teeth that are distinct from those found on healthy teeth using both reference-based mapping and de novo assembly methods. Our dual metataxonomic and metatranscriptomic approach demonstrates the importance of species abundance for gene expression data interpretation and that patterns of differential expression can be skewed by low-abundance groups. Finally, we identify several potential candidate probiotic bacterial lineages within species that may be useful therapeutic targets for the prevention of tooth decay and propose that the development of a strain-specific, mixed-microbial probiotic may be a beneficial approach given the heterogeneity of taxa identified here across health groups. IMPORTANCE: Tooth decay is the most common preventable chronic disease, affecting more than two billion people globally. The development of caries on teeth is primarily a consequence of acid production by cariogenic bacteria that inhabit the plaque microbiome. Other bacterial strains in the oral cavity may suppress or prevent tooth decay by producing ammonia as a byproduct of the arginine deiminase metabolic pathway, increasing the pH of the plaque biofilm. While the benefits of arginine metabolism on oral health have been extensively documented in specific bacterial groups, the prevalence and consistency of arginine deiminase system (ADS) activity among oral bacteria in a community context remain an open question. In the current study, we use a multi-omics approach to document the pervasiveness of the expression of the ADS operon in both health and disease to better understand the conditions in which ADS activity may prevent tooth decay.

Supragingival mycobiome of HIV-exposed-but-uninfected children reflects a stronger correlation with caries-free-associated taxa compared to HIV-infected or uninfected children.

IMPORTANCE: Globally, caries is among the most frequent chronic childhood disease, and the fungal component of the microbial community responsible is poorly studied despite evidence that fungi contribute to increased acid production exacerbating enamel demineralization. HIV infection is another global health crisis. Perinatal HIV exposure with infection are caries risk factors; however, the caries experience in the context of perinatal HIV exposure without infection is less clear. Using high-throughput amplicon sequencing, we find taxonomic differences that become pronounced during late-stage caries. Notably, we show a stronger correlation with health-associated taxa for HIV-exposed-but-uninfected children when compared to unexposed and uninfected children. This aligns with a lower incidence of caries in primary teeth at age 6 or less for exposed yet uninfected children. Ultimately, these findings could contribute to improved risk assessment, intervention, and prevention strategies such as biofilm disruption and the informed design of pro-, pre-, and synbiotic oral therapies.

Glucose Phosphotransferase System Modulates Pyruvate Metabolism, Bacterial Fitness, and Microbial Ecology in Oral Streptococci.

Spontaneous mutants with defects in the primary glucose phosphotransferase permease (manLMNO) of Streptococcus sanguinis SK36 showed enhanced fitness at low pH. Transcriptomics and metabolomics with a manL deletion mutant (SK36/manL) revealed redirection of pyruvate to production of acetate and formate, rather than lactate. These observations were consistent with measurements of decreased lactic acid accumulation and increased excretion of acetate, formate, pyruvate, and H2O2. Genes showing increased expression in SK36/manL included those encoding carbohydrate transporters, extracellular glycosidases, intracellular polysaccharide metabolism, and arginine deiminase and pathways for metabolism of acetoin, ethanolamine, ascorbate, and formate, along with genes required for membrane biosynthesis and adhesion. Streptococcus mutans UA159 persisted much better in biofilm cocultures with SK36/manL than with SK36, an effect that was further enhanced by culturing the biofilms anaerobically but dampened by adding arginine to the medium. We posited that the enhanced persistence of S. mutans with SK36/manL was in part due to excess excretion of pyruvate by the latter, as addition of pyruvate to S. mutans-S. sanguinis cocultures increased the proportions of UA159 in the biofilms. Reducing the buffer capacity or increasing the concentration of glucose benefited UA159 when cocultured with SK36, but not with SK36/manL, likely due to the altered metabolism and enhanced acid tolerance of the mutant. When manL was deleted in S. mutans or Streptococcus gordonii, the mutants presented altered fitness characteristics. Our study demonstrated that phosphotransferase system (PTS)-dependent modulation of central metabolism can profoundly affect streptococcal fitness and metabolic interactions, revealing another dimension in commensal-pathogen relationships influencing dental caries development. IMPORTANCE Dental caries is underpinned by a dysbiotic microbiome and increased acid production. As beneficial bacteria that can antagonize oral pathobionts, oral streptococci such as S. sanguinis and S. gordonii can ferment many carbohydrates, despite their relative sensitivity to low pH. We characterized the molecular basis for why mutants of glucose transporter ManLMNO of S. sanguinis showed enhanced production of hydrogen peroxide and ammonia and improved persistence under acidic conditions. A metabolic shift involving more than 300 genes required for carbohydrate transport, energy production, and envelope biogenesis was observed. Significantly, manL mutants engineered in three different oral streptococci displayed altered capacities for acid production and interspecies antagonism, highlighting the potential for targeting the glucose-PTS to modulate the pathogenicity of oral biofilms.

Optimization and Evaluation of the 30S-S11 rRNA Gene for Taxonomic Profiling of Oral Streptococci.

Dental caries is a multifactorial disease driven by interactions between the highly complex microbial biofilm community and host factors like diet, oral hygiene habits, and age. The oral streptococci are one of the most dominant members of the plaque biofilm and are implicated in disease but also in maintaining oral health. Current methods used for studying the supragingival plaque community commonly sequence portions of the16S rRNA gene, which often cannot taxonomically resolve members of the streptococcal community past the genus level due to their sequence similarity. The goal of this study was to design and evaluate a more reliable and cost-effective method to identify oral streptococci at the species level by applying a new locus, the 30S-S11 rRNA gene, for high-throughput amplicon sequencing. The study results demonstrate that the newly developed single-copy 30S-S11 gene locus resolved multiple amplicon sequence variants (ASVs) within numerous species, providing much improved taxonomic resolution over 16S rRNA V4. Moreover, the results reveal that different ASVs within a species were found to change in abundance at different stages of caries progression. These findings suggest that strains of a single species may perform distinct roles along a biochemical spectrum associated with health and disease. The improved identification of oral streptococcal species will provide a better understanding of the different ecological roles of oral streptococci and inform the design of novel oral probiotic formulations for prevention and treatment of dental caries. IMPORTANCE The microbiota associated with the initiation and progression of dental caries has yet to be fully characterized. Although much insight has been gained from 16S rRNA hypervariable region DNA sequencing, this approach has several limitations, including poor taxonomic resolution at the species level. This is particularly relevant for oral streptococci, which are abundant members of oral biofilm communities and major players in health and caries disease. Here, we develop a new method for taxonomic profiling of oral streptococci based on the 30S-S11 rRNA gene, which provides much improved resolution over 16S rRNA V4 (resolving 10 as opposed to 2 species). Importantly, 30S-S11 can resolve multiple amplicon sequence variants (ASVs) within species, providing an unprecedented insight into the ecological progression of caries. For example, our findings reveal multiple incidences of different ASVs within a species with contrasting associations with health or disease, a finding that has high relevance toward the informed design of prebiotic and probiotic therapy.

The fruB Gene of Streptococcus mutans Encodes an Endo-Levanase That Enhances Growth on Levan and Influences Global Gene Expression.

Streptococcus mutans, the primary etiologic agent of human dental caries, and a variety of oral Streptococcus and Actinomyces spp. synthesize high molecular mass homopolymers of fructose (fructans) with predominantly ß2,1- (inulins) or ß2,6-linkages (levans). The ability of S. mutans to degrade fructans contributes to the severity of dental caries. The extracellular product of fruA of S. mutans is an exo- ß-d-fructofuranosidase that releases fructose from levan and inulin. Located 70 bp downstream of fruA, fruB encodes a member of the glycoside hydrolase family 32, but the function of FruB has not been established. Growth assays performed using wild-type UA159 and fruB-deficient derivatives, with fructans as the sole carbohydrate source, showed a significant reduction in the growth rate of a fruB mutant on levan, but not on inulin. A purified, recombinant FruB protein degraded levan to release mainly fructooligosaccharides. Driven by the fruA promoter and a secondary promoter located in the 3' region of the fruA sequence, the fruB gene is inducible by fructose and especially by levan, but a stable stem-loop structure in the intergenic region likely modulates transcriptional read-through from fruA. Transcriptomic analysis of UA159 and a fruB mutant grown on 0.2% levan revealed differential expression of genes encoding ABC transporters, transcriptional regulators and genes involved in growth and stress tolerance. The ability of FruB to enhance levan metabolism and the high degree of conservation of FruB across S. mutans isolates imply a significant contribution of FruB to the fitness and virulence of this pathogen in human dental biofilms. IMPORTANCE Carbohydrate metabolism and acid production are essential for the development of dental caries. As a by-product of sucrose metabolism, formation, and degradation of fructans enhances the severity of caries by S. mutans in animal models. This study highlights a significant breakthrough in identifying FruB in S. mutans as an endolevanase that contributes to efficient utilization of levan, a specific type of fructan produced by certain commensals but not S. mutans. Transcriptomic analysis revealed that FruB-dependent levan metabolism impacted global gene regulation, including a large number of novel genes. Considering the preference for levan by both FruA and FruB, the conservation of fruAB in S. mutans might represent a competitive advantage in access to the energy storage produced by dental microbiome. This is the first report demonstrating the presence of an endolevanase in S. mutans, therefore should be of broad interest to the fields of dental caries and complex carbohydrate metabolism.

Testing of candidate probiotics to prevent dental caries induced by Streptococcus mutans in a mouse model.

AIMS: We evaluated two species of human oral commensal streptococci in protection against dental caries induced by Streptococcus mutans. METHODS AND RESULTS: Candidate probiotics, Streptococcus sp. A12, Streptococcus sanguinis BCC23 and an arginine deiminase mutant of BCC23 (∆arcADS) were tested for their ability to reduce S. mutans-induced caries in an established mouse model. Mice were colonized with a probiotic, challenged with S. mutans, then intermittently reinoculated with a probiotic strain. Oral colonization of each strain and autochthonous bacteria was assessed by quantitative polymerase chain reaction. Both BCC23 strains, but not A12, were associated with markedly reduced sulcal caries, persistently colonized mucosal and dental biofilms, and significantly lowered S. mutans counts. All three strains enhanced mucosal colonization of autochthonous bacteria. In a follow-up experiment, when S. mutans was established first, dental and mucosal colonization of S. mutans was unaltered by a subsequent challenge with either BCC23 strain. Results between BCC23 and BCC23 ∆arcADS were equivalent. CONCLUSIONS: BCC23 is a potential probiotic to treat patients at high caries risk. Its effectiveness is independent of ADS activity, but initial dental cleaning to enhance establishment in dental biofilms may be required. SIGNIFICANCE AND IMPACT OF THE STUDY: In vivo testing of candidate probiotics is highly informative, as effectiveness is not always reflected by genotype or in vitro behaviours.

Manganese transport by Streptococcus sanguinis in acidic conditions and its impact on growth in vitro and in vivo.

Streptococcus sanguinis is an oral commensal and an etiological agent of infective endocarditis. Previous studies have identified the SsaACB manganese transporter as essential for endocarditis virulence; however, the significance of SsaACB in the oral environment has never been examined. Here we report that a ΔssaACB deletion mutant of strain SK36 exhibits reduced growth and manganese uptake under acidic conditions. Further studies revealed that these deficits resulted from the decreased activity of TmpA, shown in the accompanying paper to function as a ZIP-family manganese transporter. Transcriptomic analysis of fermentor-grown cultures of SK36 WT and ΔssaACB strains identified pH-dependent changes related to carbon catabolite repression in both strains, though their magnitude was generally greater in the mutant. In strain VMC66, which possesses a MntH transporter, loss of SsaACB did not significantly alter growth or cellular manganese levels under the same conditions. Interestingly, there were only modest differences between SK36 and its ΔssaACB mutant in competition with Streptococcus mutans in vitro and in a murine oral colonization model. Our results suggest that the heterogeneity of the oral environment may provide a rationale for the variety of manganese transporters found in S. sanguinis.

Spontaneous Mutants of Streptococcus sanguinis with Defects in the Glucose-Phosphotransferase System Show Enhanced Post-Exponential-Phase Fitness.

Genetic truncations in a gene encoding a putative glucose-phosphotransferase system (PTS) protein (manL, EIIABMan) were identified in subpopulations of two separate laboratory stocks of Streptococcus sanguinis SK36; the mutants had reduced PTS activities on glucose and other monosaccharides. To understand the emergence of these mutants, we engineered deletion mutants of manL and showed that the ManL-deficient strain had improved bacterial viability in the stationary phase and was better able to inhibit the growth of the dental caries pathogen Streptococcus mutans. Transcriptional analysis and biochemical assays suggested that the manL mutant underwent reprograming of central carbon metabolism that directed pyruvate away from production of lactate, increasing production of hydrogen peroxide (H2O2) and excretion of pyruvate. Addition of pyruvate to the medium enhanced the survival of SK36 in overnight cultures. Meanwhile, elevated pyruvate levels were detected in the cultures of a small but significant percentage (∼10%) of clinical isolates of oral commensal bacteria. Furthermore, the manL mutant showed higher expression of the arginine deiminase system than the wild type, which enhanced the ability of the mutant to raise environmental pH when arginine was present. To our surprise, significant discrepancies in genome sequence were identified between strain SK36 obtained from ATCC and the sequence deposited in GenBank. As the conditions that are likely associated with the emergence of spontaneous manL mutations, i.e., excess carbohydrates and low pH, are those associated with caries development, we propose that glucose-PTS strongly influences commensal-pathogen interactions by altering the production of ammonia, pyruvate, and H2O2. IMPORTANCE A health-associated dental microbiome provides a potent defense against pathogens and diseases. Streptococcus sanguinis is an abundant member of a health-associated oral flora that antagonizes pathogens by producing hydrogen peroxide. There is a need for a better understanding of the mechanisms that allow bacteria to survive carbohydrate-rich and acidic environments associated with the development of dental caries. We report the isolation and characterization of spontaneous mutants of S. sanguinis with impairment in glucose transport. The resultant reprograming of the central metabolism in these mutants reduced the production of lactic acid and increased pyruvate accumulation; the latter enables these bacteria to better cope with hydrogen peroxide and low pH. The implications of these discoveries in the development of dental caries are discussed.

Mutanofactin promotes adhesion and biofilm formation of cariogenic Streptococcus mutans.

Cariogenic Streptococcus mutans is known as a predominant etiological agent of dental caries due to its exceptional capacity to form biofilms. From strains of S. mutans isolated from dental plaque, we discovered, in the present study, a polyketide/nonribosomal peptide biosynthetic gene cluster, muf, which directly correlates with a strong biofilm-forming capability. We then identified the muf-associated bioactive product, mutanofactin-697, which contains a new molecular scaffold, along with its biosynthetic logic. Further mode-of-action studies revealed that mutanofactin-697 binds to S. mutans cells and also extracellular DNA, increases bacterial hydrophobicity, and promotes bacterial adhesion and subsequent biofilm formation. Our findings provided an example of a microbial secondary metabolite promoting biofilm formation via a physicochemical approach, highlighting the importance of secondary metabolism in mediating critical processes related to the development of dental caries.

A single system detects and protects the beneficial oral bacterium Streptococcus sp. A12 from a spectrum of antimicrobial peptides.

The commensal bacterium Streptococcus sp. A12 has multiple properties that may promote the stability of health-associated oral biofilms, including overt antagonism of the dental caries pathogen Streptococcus mutans. A LanFEG-type ABC transporter, PcfFEG, confers tolerance to the lantibiotic nisin and enhances the ability of A12 to compete against S. mutans. Here, we investigated the regulation of pcfFEG and adjacent genes for a two-component system, pcfRK, to better understand antimicrobial peptide resistance by A12. Induction of pcfFEG-pcfRK was the primary mechanism to respond rapidly to nisin. In addition to nisin, PcfFEG conferred tolerance by A12 to a spectrum of lantibiotic and non-lantibiotic antimicrobial peptides produced by a diverse collection of S. mutans isolates. Loss of PcfFEG resulted in the altered spatio-temporal arrangement of A12 and S. mutans in a dual-species biofilm model. Deletion of PcfFEG or PcfK resulted in constitutive activation of pcfFEG and expression of pcfFEG was inhibited by small peptides in the pcfK mutant. Transcriptional profiling of pcfR or pcfK mutants combined with functional genomics revealed peculiarities in PcfK function and a novel panel of genes responsive to nisin. Collectively, the results provide fundamental insights that strengthen the foundation for the design of microbial-based therapeutics to control oral infectious diseases.

The Route of Sucrose Utilization by Streptococcus mutans Affects Intracellular Polysaccharide Metabolism.

Streptococcus mutans converts extracellular sucrose (Suc) into exopolysaccharides (EPS) by glucosyl-transferase and fructosyl-transferase enzymes and internalizes Suc for fermentation through the phosphotransferase system (PTS). Here, we examined how altering the routes for sucrose utilization impacts intracellular polysaccharide [IPS; glycogen, (glg)] metabolism during carbohydrate starvation. Strain UA159 (WT), a mutant lacking all exo-enzymes for sucrose utilization (MMZ952), and a CcpA-deficient mutant (∆ccpA) were cultured with sucrose or a combination of glucose and fructose, followed by carbohydrate starvation. At baseline (0h), and after 4 and 24h of starvation, cells were evaluated for mRNA levels of the glg operon, IPS storage, glucose-1-phosphate (G1P) concentrations, viability, and PTS activities. A pH drop assay was performed in the absence of carbohydrates at the baseline to measure acid production. We observed glg operon activation in response to starvation (p<0.05) in all strains, however, such activation was significantly delayed and reduced in magnitude when EPS synthesis was involved (p<0.05). Enhanced acidification and greater G1P concentrations were observed in the sucrose-treated group, but mostly in strains capable of producing EPS (p<0.05). Importantly, only the WT exposed to sucrose was able to synthesize IPS during starvation. Contrary to CcpA-proficient strains, IPS was progressively degraded during starvation in ∆ccpA, which also showed increased glg operon expression and greater PTS activities at baseline. Therefore, sucrose metabolism by secreted enzymes affects the capacity of S. mutans in synthesizing IPS and converting it into organic acids, without necessarily inducing greater expression of the glg operon.

Molecular mechanisms controlling fructose-specific memory and catabolite repression in lactose metabolism by Streptococcus mutans.

Lactose is an abundant dietary carbohydrate metabolized by the dental pathogen Streptococcus mutans. Lactose metabolism presents both classic diauxic behaviors and long-term memory, where the bacteria can pause for >11 h before initiating growth on lactose. Here, we explored mechanisms contributing to unusual aspects of regulation of the lac operon. The fructose-phosphate metabolites, F-1-P and F-6-P, could modulate the DNA-binding activities of the lactose repressor. Recombinant LacR proteins bound upstream of lacA and Gal-6-P induced the formation of different LacR-DNA complexes. Deletion of lacR resulted in strain-specific growth phenotypes on lactose, but also on a number of mono- and di-saccharides that involve the glucose-PTS or glucokinase in their catabolism. The phenotypes were consistent with the novel findings that loss of LacR altered glucose-PTS activity and expression of the gene for glucokinase. CcpA was also shown to affect lactose metabolism in vivo and to bind to the lacA promoter region in vitro. Collectively, our study reveals complex molecular circuits controlling lactose metabolism in S. mutans, where LacR and CcpA integrate cellular and environmental cues to regulate metabolism of a variety of carbohydrates that are critical to persistence and pathogenicity of S. mutans.

Subpopulation behaviors in lactose metabolism by Streptococcus mutans.

When Streptococcus mutans is transferred from a preferred carbohydrate (glucose or fructose) to lactose, initiation of growth can take several hours, and substantial amounts of glucose are released during growth. Here, S. mutans strains UA159 and GS-5 were examined for stochastic behaviors in transcription of the lac operon. Using a gfp reporter fusion, we demonstrated that induction of the lac operon occurs in only a fraction of the population, with prior exposure to carbohydrate source and strain influencing the magniture of the sub-population response. Lower glucokinase activity in GS-5 was associated with release of substantially more glucose than UA159 and significantly lower lac expression. Mutants unable to use lactose grew on lactose as the sole carbohydrate when strains with an intact lac operon were also present in the cultures, indicative of the potential for population cheating. Utilizing a set of engineered obligate cheating and non-cheating strains, we confirmed that cheating can sustain a heterogeneous population. Futher, obligate cheaters of GS-5 competed well with the non-cheaters and showed a high degree of competitive fitness in a human-derived consortium biofilm model. The results show that bet-hedging behaviors in carbohydrate metabolism may substantially influence the composition and pathogenic potential of oral biofilms.

Direct interactions with commensal streptococci modify intercellular communication behaviors of Streptococcus mutans.

The formation of dental caries is a complex process that ultimately leads to damage of the tooth enamel from acids produced by microbes in attached biofilms. The bacterial interactions occurring within these biofilms between cariogenic bacteria, such as the mutans streptococci, and health-associated commensal streptococci, are thought to be critical determinants of health and disease. To better understand these interactions, a Streptococcus mutans reporter strain that actively monitors cell-cell communication via peptide signaling was cocultured with different commensal streptococci. Signaling by S. mutans, normally highly active in monoculture, was completely inhibited by several species of commensals, but only when the bacteria were in direct contact with S. mutans. We identified a novel gene expression pattern that occurred in S. mutans when cultured directly with these commensals. Finally, mutant derivatives of commensals lacking previously shown antagonistic gene products displayed wild-type levels of signal inhibition in cocultures. Collectively, these results reveal a novel pathway(s) in multiple health-associated commensal streptococci that blocks peptide signaling and induces a common contact-dependent pattern of differential gene expression in S. mutans. Understanding the molecular basis for this inhibition will assist in the rational design of new risk assessments, diagnostics, and treatments for the most pervasive oral infectious diseases.

In Vivo Colonization with Candidate Oral Probiotics Attenuates Colonization and Virulence of Streptococcus mutans.

A collection of 113 Streptococcus strains from supragingival dental plaque of caries-free individuals were recently tested in vitro for direct antagonism of the dental caries pathogen Streptococcus mutans, and for their capacity for arginine catabolism via the arginine deiminase system (ADS). To advance their evaluation as potential probiotics, twelve strains of commensal oral streptococci with various antagonistic and ADS potentials were assessed in a mouse model for oral (i.e., oral mucosal pellicles and saliva) and dental colonization under four diets (healthy or high-sucrose, with or without prebiotic arginine). Colonization by autochthonous bacteria was also monitored. One strain failed to colonize, whereas oral colonization by the other eleven strains varied by 3 log units. Dental colonization was high for five strains regardless of diet, six strains increased colonization with at least one high-sucrose diet, and added dietary arginine decreased dental colonization of two strains. Streptococcus sp. A12 (high in vitro ADS activity and antagonism) and two engineered mutants lacking the ADS (ΔarcADS) or pyruvate oxidase-mediated H2O2 production (ΔspxB) were tested for competition against S. mutans UA159. A12 wild type and ΔarcADS colonized only transiently, whereas ΔspxB persisted, but without altering oral or dental colonization by S. mutans In testing four additional candidates, S. sanguinis BCC23 markedly attenuated S. mutans' oral and dental colonization, enhanced colonization of autochthonous bacteria, and decreased severity of smooth surface caries under highly cariogenic conditions. Results demonstrate the utility of the mouse model to evaluate potential probiotics, revealing little correlation between in vitro antagonism and competitiveness against S. mutans in vivo IMPORTANCE Our results demonstrate in vivo testing of potential oral probiotics can be accomplished and can yield information to facilitate the ultimate design and optimization of novel anti-caries probiotics. We show human oral commensals associated with dental health are an important source of potential probiotics that may be used to colonize patients under dietary conditions of highly varying cariogenicity. Assessment of competitiveness against dental caries pathogen Streptococcus mutans and impact on caries identified strains or genetic elements for further study. Results also uncovered strains that enhanced oral and dental colonization by autochthonous bacteria when challenged with S. mutans, suggesting cooperative interactions for future elucidation. Distinguishing a rare strain that effectively compete with S. mutans under conditions that promote caries further validates our systematic approach to more critically evaluate probiotics for use in humans.

Amino Sugars Reshape Interactions between Streptococcus mutans and Streptococcus gordonii.

Amino sugars, particularly glucosamine (GlcN) and N-acetylglucosamine (GlcNAc), are abundant carbon and nitrogen sources supplied in host secretions and in the diet to the biofilms colonizing the human oral cavity. Evidence is emerging that these amino sugars provide ecological advantages to beneficial commensals over oral pathogens and pathobionts. Here, we performed transcriptome analysis on Streptococcus mutans and Streptococcus gordonii growing in single-species or dual-species cultures with glucose, GlcN, or GlcNAc as the primary carbohydrate source. Compared to glucose, GlcN caused drastic transcriptomic shifts in each species of bacteria when it was cultured alone. Likewise, cocultivation in the presence of GlcN yielded transcriptomic profiles that were dramatically different from the single-species results from GlcN-grown cells. In contrast, GlcNAc elicited only minor changes in the transcriptome of either organism in single- and dual-species cultures. Interestingly, genes involved in pyruvate metabolism were among the most significantly affected by GlcN in both species, and these changes were consistent with measurements of pyruvate in culture supernatants. Differing from what was found in a previous report, growth of S. mutans alone with GlcN inhibited the expression of multiple operons required for mutacin production. Cocultivation with S. gordonii consistently increased the expression of two manganese transporter operons (slo and mntH) and decreased expression of mutacin genes in S. mutans Conversely, S. gordonii appeared to be less affected by the presence of S. mutans but did show increases in genes for biosynthetic processes in the cocultures. In conclusion, amino sugars profoundly alter the interactions between pathogenic and commensal streptococci by reprogramming central metabolism.IMPORTANCE Carbohydrate metabolism is central to the development of dental caries. A variety of sugars available to dental microorganisms influence the development of caries by affecting the physiology, ecology, and pathogenic potential of tooth biofilms. Using two well-characterized oral bacteria, one pathogen (Streptococcus mutans) and one commensal (Streptococcus gordonii), in an RNA deep-sequencing analysis, we studied the impact of two abundant amino sugars on bacterial gene expression and interspecies interactions. The results indicated large-scale remodeling of gene expression induced by GlcN in particular, affecting bacterial energy generation, acid production, protein synthesis, and release of antimicrobial molecules. Our study provides novel insights into how amino sugars modify bacterial behavior, information that will be valuable in the design of new technologies to detect and prevent oral infectious diseases.

Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function.

A recent genome-wide screen identified ~300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3' end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

Site-Specific Profiling of the Dental Mycobiome Reveals Strong Taxonomic Shifts during Progression of Early-Childhood Caries.

Dental caries is one of the most common diseases worldwide. Bacteria and fungi are both commensals in the oral cavity; however, most research regarding caries has focused on bacterial impacts. The oral fungal mycobiome associated with caries is not well characterized, and its role in disease is unclear. ITS1 amplicon sequencing was used to generate taxonomic profiles from site-specific supragingival plaque samples (n = 82) obtained from 33 children with different caries status. Children were either caries free (CF), caries active with enamel lesions (CAE), or caries active with dentin lesions (CA). Plaque samples were collected from caries-free surfaces (PF) and from enamel (PE) and dentin (PD) lesions. Taxonomic profiles representing the different categorizations (CF-PF, CAE-PF, CAE-PE, CA-PF, CA-PE, and CA-PD) were used to characterize the mycobiome and its change through disease progression. A total of 139 fungal species were identified. Candida albicans was the most abundant species, followed by Candida dubliniensis We found that severely progressed plaque communities (CA-PD) were significantly different from healthy plaque communities (CF-PF). A total of 32 taxa were differentially abundant across the plaque categories. C. albicans, C. dubliniensis, Nigrospora oryzae, and an unclassified Microdochium sp. were correlated with caries, whereas 12 other taxa were correlated with health. C. dubliniensis increased steadily as caries progressed, suggesting that C. dubliniensis may play an important role in caries pathogenicity. In contrast, four health-associated fungal taxa have the potential to antagonize the cariogen Streptococcus mutans via xylitol production, suggesting a possible fungal mechanism that could contribute to maintenance of dental health.IMPORTANCE Early-childhood caries is one of the most prevalent diseases in children worldwide and, while preventable, remains a global public health concern. Untreated cavities are painful and expensive and can lead to tooth loss and a lower quality of life. Caries are driven by acid production via microbial fermentation of dietary carbohydrates, resulting in enamel erosion. While caries is a well-studied disease, most research has focused on bacterial impacts, even though fungi are commensal organisms living within the plaque biofilm. There is very little known about how fungi impact caries pathogenicity. The elucidation of fungal taxa involved in caries disease progression is necessary for a more holistic view of the human oral microbiome. Data from this study will improve our understanding of how the fungal community changes as disease progresses and provide insight into the complex etiology of dental caries, which is necessary for the development of treatment plans and preventative measures.

Peptides encoded in the Streptococcus mutans RcrRPQ operon are essential for thermotolerance.

The MarR-like transcriptional regulator and two ABC transporters encoded by the rcrRPQ operon in the dental caries pathogen Streptococcus mutans have important regulatory roles related to oxidative stress tolerance, genetic competence and (p)ppGpp metabolism. A unique feature of the rcrRPQ operon, when compared to other bacteria, is the presence of two peptides, designated Pep1 and Pep2, encoded in alternative reading frames at the 3' end of rcrQ. Here, we show that the rcrRPQ operon, including Pep1 and 2, is essential for S. mutans to survive and maintain viability at elevated temperatures. No major changes in the levels of the heat shock proteins DnaK or GroEL that could account for the thermosensitivity of rcrRPQ mutants were observed. By introducing a single amino acid substitution into the comX gene that deletes an internally encoded peptide, XrpA, we found that XrpA is a contributing factor to the thermosensitive phenotype of a ΔrcrR strain. Overexpression of XrpA on a plasmid also caused a significant growth defect at 42 °C. Interestingly, loss of the gene for the RelA/SpoT homologue (RSH) enzyme, relA, restored growth of the ΔrcrR strain at 42 °C. During heat stress and when a stringent response was induced, levels of (p)ppGpp were elevated in the ΔrcrR strain. Deletion of relA in the ΔrcrR strain lowered the basal levels of (p)ppGpp to those observed in wild-type S. mutans. Thus, (p)ppGpp pools are dysregulated in ΔrcrR, which likely leads to aberrant control of transcriptional/translational processes and the thermosensitive phenotype. In summary, the genes and peptides encoded in the rcrRPQ operon are critical for thermotolerance, and in some strains these phenotypes are related to altered (p)ppGpp metabolism and increased production of the XrpA peptide.