Small regulatory RNAs are mediators of the Streptococcus mutans SloR regulon.

Dental caries is among the most prevalent chronic diseases worldwide. Streptococcus mutans, the chief causative agent of caries, uses a 25-kDa manganese-dependent SloR protein to coordinate the uptake of essential manganese with the transcription of its virulence attributes. Small non-coding RNAs (sRNAs) can either enhance or repress gene expression, and reports in the literature ascribe an emerging role for sRNAs in the environmental stress response. Herein, we focused our attention on 18-50 nt sRNAs as mediators of the S. mutans SloR and manganese regulons. Specifically, the results of RNA sequencing revealed 19 sRNAs in S. mutans, which were differentially transcribed in the SloR-proficient UA159 and SloR-deficient GMS584 strains, and 10 sRNAs that were differentially expressed in UA159 cells grown in the presence of low vs high manganese. We describe SmsR1532 and SmsR1785 as SloR- and manganese-responsive sRNAs that are processed from large transcripts and that bind SloR directly in their promoter regions. The predicted targets of these sRNAs include regulators of metal ion transport, growth management via a toxin-antitoxin operon, and oxidative stress tolerance. These findings support a role for sRNAs in coordinating intracellular metal ion homeostasis with virulence gene control in an important oral cariogen. IMPORTANCE Small regulatory RNAs (sRNAs) are critical mediators of environmental signaling, particularly in bacterial cells under stress, but their role in Streptococcus mutans is poorly understood. S. mutans, the principal causative agent of dental caries, uses a 25-kDa manganese-dependent protein, called SloR, to coordinate the regulated uptake of essential metal ions with the transcription of its virulence genes. In the present study, we identified and characterized sRNAs that are both SloR and manganese responsive. Taken together, this research can elucidate the details of regulatory networks that engage sRNAs in an important oral pathogen and that can enable the development of an effective anti-caries therapeutic.

Small regulatory RNAs are mediators of the Streptococcus mutans SloR regulon.

Dental caries is among the most prevalent chronic infectious diseases worldwide. Streptococcus mutans , the chief causative agent of caries, uses a 25 kDa manganese dependent SloR protein to coordinate the uptake of essential manganese with the transcription of its virulence attributes. Small non-coding RNAs (sRNAs) can either enhance or repress gene expression and reports in the literature ascribe an emerging role for sRNAs in the environmental stress response. Herein, we identify 18-50 nt sRNAs as mediators of the S. mutans SloR and manganese regulons. Specifically, the results of sRNA-seq revealed 56 sRNAs in S. mutans that were differentially transcribed in the SloR-proficient UA159 and SloR-deficient GMS584 strains, and 109 sRNAs that were differentially expressed in UA159 cells grown in the presence of low versus high manganese. We describe SmsR1532 and SmsR1785 as SloR- and/or manganese-responsive sRNAs that are processed from large transcripts, and that bind SloR directly in their promoter regions. The predicted targets of these sRNAs include regulators of metal ion transport, growth management via a toxin-antitoxin operon, and oxidative stress tolerance. These findings support a role for sRNAs in coordinating intracellular metal ion homeostasis with virulence gene control in an important oral cariogen. IMPORTANCE: Small regulatory RNAs (sRNAs) are critical mediators of environmental signaling, particularly in bacterial cells under stress, but their role in Streptococcus mutans is poorly understood. S. mutans, the principal causative agent of dental caries, uses a 25 kDa manganese-dependent protein, called SloR, to coordinate the regulated uptake of essential metal ions with the transcription of its virulence genes. In the present study, we identified and characterize sRNAs that are both SloR- and manganese-responsive. Taken together, this research can elucidate the details of regulatory networks that engage sRNAs in an important oral pathogen, and that can enable the development of an effective anti-caries therapeutic.

Regulatory involvement of the PerR and SloR metalloregulators in the Streptococcus mutans oxidative stress response.

Streptococcus mutans is a commensal of the human oral microbiome that can promote dental caries under conditions of dysbiosis. This study investigates metalloregulators and their involvement in the S. mutans oxidative stress response. Oxidative stress in the human mouth can derive from temporal increases in reactive oxygen species (ROS) after meal consumption and from endogenous bacterial ROS-producers that colonize the dentition. We hypothesize that the S. mutans PerR (SMU.593) and SloR (SMU.186) metalloregulatory proteins contribute to the regulation of oxidative stress genes and their products. Expression assays with S. mutans UA159 wild type cultures exposed to H2O2 reveal that H2O2 upregulates perR, and that PerR represses sloR transcription upon binding directly to Fur and PerR consensus sequences within the sloR operator. In addition, the results of Western blot experiments implicate the Clp proteolytic system in SloR degradation under conditions of H2O2-stress. To reveal a potential role for SloR in the H2O2-resistant phenotype of S. mutans GMS802 (a perR-deficient strain), we generated a sloR/perR double knockout mutant, GMS1386, where we observed upregulation of the tpx and dpr antioxidant genes. These results are consistent with GMS802 H2O2 resistance and with a role for PerR as a transcriptional repressor. Cumulatively, these findings support a reciprocal relationship between PerR and SloR during the S. mutans oxidative stress response and begin to elucidate the fitness strategies that evolved to foster S. mutans persistence in the transient environments of the human oral cavity.IMPORTANCEIn 2020, untreated dental caries, especially in the permanent dentition, ranked among the most prevalent infectious diseases worldwide, disproportionately impacting individuals of low socioeconomic status. Untreated caries can lead to systemic health problems and has been associated with extended school and work absences, inappropriate use of emergency departments, and an inability for military forces to deploy. Together with public health policy, research aimed at alleviating S. mutans -induced tooth decay is important because it can improve oral health (and overall health), especially in underserved populations. This research, focused on S. mutans metalloregulatory proteins and their gene targets, is significant because it can promote virulence gene control in an important oral pathogen, and contribute to the development of an anti-caries therapeutic that can reduce tooth decay.

Manganese Uptake, Mediated by SloABC and MntH, Is Essential for the Fitness of Streptococcus mutans.

Early epidemiological studies implicated manganese (Mn) as a possible caries-promoting agent, while laboratory studies have indicated that manganese stimulates the expression of virulence-related factors in the dental pathogen Streptococcus mutans To better understand the importance of manganese homeostasis to S. mutans pathophysiology, we first used RNA sequencing to obtain the global transcriptional profile of S. mutans UA159 grown under Mn-restricted conditions. Among the most highly expressed genes were those of the entire sloABC operon, encoding a dual iron/manganese transporter, and an uncharacterized gene, here mntH, that codes for a protein bearing strong similarity to Nramp-type transporters. While inactivation of sloC, which encodes the lipoprotein receptor of the SloABC system, or of mntH alone had no major consequence for the overall fitness of S. mutans, simultaneous inactivation of sloC and mntH (ΔsloC ΔmntH) impaired growth and survival under Mn-restricted conditions, including in human saliva or in the presence of calprotectin. Further, disruption of Mn transport resulted in diminished stress tolerance and reduced biofilm formation in the presence of sucrose. These phenotypes were markedly improved when cells were provided with excess Mn. Metal quantifications revealed that the single mutant strains contained intracellular levels of Mn similar to those seen with the parent strain, whereas Mn was nearly undetectable in the ΔsloC ΔmntH strain. Collectively, these results reveal that SloABC and MntH work independently and cooperatively to promote cell growth under Mn-restricted conditions and that maintenance of Mn homeostasis is essential for the expression of major virulence attributes in S. mutansIMPORTANCE As transition biometals such as manganese (Mn) are essential for all forms of life, the ability to scavenge biometals in the metal-restricted host environment is an important trait of successful cariogenic pathobionts. Here, we showed that the caries pathogen Streptococcus mutans utilizes two Mn transport systems, namely, SloABC and MntH, to acquire Mn from the environment and that the ability to maintain the cellular levels of Mn is important for the manifestation of characteristics that associate S. mutans with dental caries. Our results indicate that the development of strategies to deprive S. mutans of Mn hold promise in the combat against this important bacterial pathogen.

In vitro manganese-dependent cross-talk between Streptococcus mutans VicK and GcrR: implications for overlapping stress response pathways.

Streptococcus mutans, a major acidogenic component of the dental plaque biofilm, has a key role in caries etiology. Previously, we demonstrated that the VicRK two-component signal transduction system modulates biofilm formation, oxidative stress and acid tolerance responses in S. mutans. Using in vitro phosphorylation assays, here we demonstrate for the first time, that in addition to activating its cognate response regulator protein, the sensor kinase, VicK can transphosphorylate a non-cognate stress regulatory response regulator, GcrR, in the presence of manganese. Manganese is an important micronutrient that has been previously correlated with caries incidence, and which serves as an effector of SloR-mediated metalloregulation in S. mutans. Our findings supporting regulatory effects of manganese on the VicRK, GcrR and SloR, and the cross-regulatory networks formed by these components are more complex than previously appreciated. Using DNaseI footprinting we observed overlapping DNA binding specificities for VicR and GcrR in native promoters, consistent with these proteins being part of the same transcriptional regulon. Our results also support a role for SloR as a positive regulator of the vicRK two component signaling system, since its transcription was drastically reduced in a SloR-deficient mutant. These findings demonstrate the regulatory complexities observed with the S. mutans manganese-dependent response, which involves cross-talk between non-cognate signal transduction systems (VicRK and GcrR) to modulate stress response pathways.

Characterization of the functional domains of the SloR metalloregulatory protein in Streptococcus mutans.

Streptococcus mutans is a commensal member of the healthy plaque biofilm and the primary causative agent of dental caries. The present study is an investigation of SloR, a 25-kDa metalloregulatory protein that modulates genes responsible for S. mutans-induced cariogenesis. Previous studies of SloR homologues in other bacterial pathogens have identified three domains critical to repressor functionality: an N-terminal DNA-binding domain, a central dimerization domain, and a C-terminal FeoA (previously SH3-like) domain. We used site-directed mutagenesis to identify critical amino acid residues within each of these domains of the SloR protein. Select residues were targeted for mutagenesis, and nonconservative amino acid substitutions were introduced by overlap extension PCR. Furthermore, three C-terminally truncated SloR variants were generated using conventional PCR. The repressor functionality and DNA-binding ability of each variant was assessed using CAT reporter gene assays, real-time semiquantitative reverse transcriptase (qRT)-PCR, and electrophoretic mobility shift assays. We identified 12 residues within SloR that cause significant derepression of sloABC promoter activity (P < 0.05) compared to the results for wild-type SloR. Derepression was particularly noteworthy in metal ion-binding site 1 mutants, consistent with the site's importance in gene repression by SloR. In addition, a hyperactive SloR(E169A/Q170A) mutant was identified as having significantly heightened repression of sloABC promoter activity, and experiments with C-terminal deletion mutants support involvement of the FeoA domain in SloR-mediated gene repression. Given these results, we describe the functional domains of the S. mutans SloR protein and propose that the hyperactive mutant could serve as a target for rational drug design aimed at repressing SloR-mediated virulence gene expression.

Genome-wide characterization of the SloR metalloregulome in Streptococcus mutans.

Streptococcus mutans is the primary causative agent of human dental caries, a ubiquitous infectious disease for which effective treatment strategies remain elusive. We investigated a 25-kDa SloR metalloregulatory protein in this oral pathogen, along with its target genes that contribute to cariogenesis. Previous studies have demonstrated manganese- and SloR-dependent repression of the sloABCR metal ion transport operon in S. mutans. In the present study, we demonstrate that S. mutans coordinates this repression with that of certain virulence attributes. Specifically, we noted virulence gene repression in a manganese-containing medium when SloR binds to promoter-proximal sequence palindromes on the S. mutans chromosome. We applied a genome-wide approach to elucidate the sequences to which SloR binds and to reveal additional "class I" genes that are subject to SloR- and manganese-dependent repression. These analyses identified 204 S. mutans genes that are preceded by one or more conserved palindromic SloR recognition elements (SREs). We cross-referenced these genes with those that we had identified previously as SloR and/or manganese modulated in microarray and real-time quantitative reverse transcription-PCR (qRT-PCR) experiments. From this analysis, we identified a number of S. mutans virulence genes that are subject to transcriptional upregulation by SloR and noted that such "class II"-type regulation is dependent on direct SloR binding to promoter-distal SREs. These observations are consistent with a bifunctional role for the SloR metalloregulator and implicate it as a target for the development of therapies aimed at alleviating S. mutans-induced caries formation.

SloR modulation of the Streptococcus mutans acid tolerance response involves the GcrR response regulator as an essential intermediary.

Streptococcus mutans, the primary causative agent of human dental caries, grows as a biofilm on the tooth surface, where it metabolizes dietary carbohydrates and generates acid byproducts that demineralize tooth enamel. A drop in plaque pH stimulates an adaptive acid-tolerance response (ATR) in this oral pathogen that allows it to survive acid challenge at pHs as low as 3.0. In the present study, we describe the growth of an S. mutans mutant, GMS901, that harbours an insertion-deletion mutation in gcrR, a gene that encodes a transcriptional regulatory protein. The mutant is acid-sensitive and significantly compromised in its ATR relative to the UA159 wild-type progenitor strain. Consistent with these findings are the results of real-time quantitative RT-PCR (qRT-PCR) experiments that support the GcrR-regulated expression of known ATR genes, including atpA/E and ffh. Although we observed gcrR transcription that was not responsive to acidic pH, we did note a significant increase in gcrR expression when S. mutans cells were grown in a manganese-restricted medium. Interestingly, the results of gel mobility shift assays indicate that the S. mutans SloR metalloregulatory protein is a potential regulator of gcrR by virtue of its manganese-dependent binding to the gcrR promoter region, and expression studies support the hypothesis that sloR transcription is responsive to manganese deprivation and acidic pH. Taking these results together, we propose that SloR-Mn modulates S. mutans gcrR expression as part of a general stress response, and that GcrR acts downstream of SloR to control the ATR.

The Streptococcus mutans vicX gene product modulates gtfB/C expression, biofilm formation, genetic competence, and oxidative stress tolerance.

Streptococcus mutans is considered one of the primary etiologic agents of dental caries. Previously, we characterized the VicRK two-component signal transduction system, which regulates multiple virulence factors of S. mutans. In this study, we focused on the vicX gene of the vicRKX tricistronic operon. To characterize vicX, we constructed a nonpolar deletion mutation in the vicX coding region in S. mutans UA159. The growth kinetics of the mutant (designated SmuvicX) showed that the doubling time was longer and that there was considerable sensitivity to paraquat-induced oxidative stress. Supplementing a culture of the wild-type UA159 strain with paraquat significantly increased the expression of vicX (P < 0.05, as determined by analysis of variance [ANOVA]), confirming the role of this gene in oxidative stress tolerance in S. mutans. Examination of mutant biofilms revealed architecturally altered cell clusters that were seemingly denser than the wild-type cell clusters. Interestingly, vicX-deficient cells grown in a glucose-supplemented medium exhibited significantly increased glucosyltransferase B/C (gtfB/C) expression compared with the expression in the wild type (P < 0.05, as determined by ANOVA). Moreover, a sucrose-dependent adhesion assay performed using an S. mutans GS5-derived vicX null mutant demonstrated that the adhesiveness of this mutant was enhanced compared with that of the parent strain and isogenic mutants of the parent strain lacking gtfB and/or gtfC. Also, disruption of vicX reduced the genetic transformability of the mutant approximately 10-fold compared with that of the parent strain (P < 0.05, as determined by ANOVA). Collectively, these findings provide insight into important phenotypes controlled by the vicX gene product that can impact S. mutans pathogenicity.

A VicRK signal transduction system in Streptococcus mutans affects gtfBCD, gbpB, and ftf expression, biofilm formation, and genetic competence development.

Bacteria exposed to transient host environments can elicit adaptive responses by triggering the differential expression of genes via two-component signal transduction systems. This study describes the vicRK signal transduction system in Streptococcus mutans. A vicK (putative histidine kinase) deletion mutant (SmuvicK) was isolated. However, a vicR (putative response regulator) null mutation was apparently lethal, since the only transformants isolated after attempted mutagenesis overexpressed all three genes in the vicRKX operon (Smuvic+). Compared with the wild-type UA159 strain, both mutants formed aberrant biofilms. Moreover, the vicK mutant biofilm formed in sucrose-supplemented medium was easily detachable relative to that of the parent. The rate of total dextran formation by this mutant was remarkably reduced compared to the wild type, whereas it was increased in Smuvic+. Based on real-time PCR, Smuvic+ showed increased gtfBCD, gbpB, and ftf expression, while a recombinant VicR fusion protein was shown to bind the promoter regions of the gtfB, gtfC, and ftf genes. Also, transformation efficiency in the presence or absence of the S. mutans competence-stimulating peptide was altered for the vic mutants. In vivo studies conducted using SmuvicK in a specific-pathogen-free rat model resulted in significantly increased smooth-surface dental plaque (Pearson-Filon statistic [PF], <0.001). While the absence of vicK did not alter the incidence of caries, a significant reduction in SmuvicK CFU counts was observed in plaque samples relative to that of the parent (PF, <0.001). Taken together, these findings support involvement of the vicRK signal transduction system in regulating several important physiological processes in S. mutans.