Grace A. Spatafora

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.

Effect of an Orphan Response Regulator on Streptococcus mutans Sucrose-Dependent Adherence and Cariogenesis

ABSTRACT Streptococcus mutans is the principal acidogenic component of dental plaque that demineralizes tooth enamel, leading to dental decay. Cell-associated glucosyltransferases catalyze the sucrose-dependent synthesis of sticky glucan polymers that, together with glucan binding proteins, promote S. mutans adherence to teeth and cell aggregation. We generated an S. mutans Tn916 transposon mutant, GMS315, which is defective in sucrose-dependent adherence and significantly less cariogenic than the UA130 wild-type progenitor in germfree rats. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and N-terminal sequence analysis confirmed the absence of a 155-kDa glucosyltransferase S (Gtf-S) from GMS315 protein profiles. Mapping of the unique transposon insertion in GMS315 revealed disruption of a putative regulatory region located upstream of gcrR, a gene previously described by Sato et al. that shares significant amino acid identity with other bacterial response regulators (Y. Sato, Y. Yamamoto, and H. Kizaki, FEMS Microbiol. Lett. 186: 187-191, 2000). The gcrR regulator, which we call “tarC,” does not align with any of the 13 proposed two-component signal transduction systems derived from in silico analysis of the S. mutans genome, but rather represents one of several orphan response regulators in the genome. The results of Northern hybridization and/or real-time reverse transcription-PCR experiments reveal increased expression of both Gtf-S and glucan binding protein C (GbpC) in a tarC knockout mutant (GMS900), thereby supporting the notion that TarC acts as a negative transcriptional regulator. In addition, we noted that GMS900 has altered biofilm architecture relative to the wild type and is hypocariogenic in germfree rats. Taken collectively, these findings support a role for signal transduction in S. mutans sucrose-dependent adherence and aggregation and implicate TarC as a potential target for controlling S. mutans-induced cariogenesis.

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.

The SloR/Dlg Metalloregulator Modulates Streptococcus mutans Virulence Gene Expression

Metal ion availability in the human oral cavity plays a putative role in Streptococcus mutans virulence gene expression and in appropriate formation of the plaque biofilm. In this report, we present evidence that supports such a role for the DtxR-like SloR metalloregulator (called Dlg in our previous publications) in this oral pathogen. Specifically, the results of gel mobility shift assays revealed the sloABC, sloR, comDE, ropA, sod, and spaP promoters as targets of SloR binding. We confirmed differential expression of these genes in a GMS584 SloR-deficient mutant versus the UA159 wild-type progenitor by real-time semiquantitative reverse transcriptase PCR experiments. The results of additional expression studies support a role for SloR in S. mutans control of glucosyltransferases, glucan binding proteins, and genes relevant to antibiotic resistance. Phenotypic analysis of GMS584 revealed that it forms aberrant biofilms on an abiotic surface, is compromised for genetic competence, and demonstrates heightened incorporation of iron and manganese as well as resistance to oxidative stress compared to the wild type. Taken together, these findings support a role for SloR in S. mutans adherence, biofilm formation, genetic competence, metal ion homeostasis, oxidative stress tolerance, and antibiotic gene regulation, all of which contribute to S. mutans-induced disease.

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.

Evidence that ORF3 at the Streptococcus parasanguis fimA locus encodes a thiol-specific antioxidant.

Streptococcus parasanguis is a primary colonizer of dental plaque and a major player in subacute bacterial endocarditis. In the present study, the authors report that an ORF (ORF3) located 77 bp downstream of the fimA operon on the S. parasanguis FW213 chromosome complements an Escherichia coli thiol peroxidase (tpx) mutation in glutamine synthetase (GS) protection assays and that GS is protected by the ORF3 gene product in S. parasanguis cell extracts. In addition, the putative streptococcal peroxidase (Tpx(Sp)) protects S. parasanguis from stress caused by H2O2 and is induced by oxygen, as revealed by Northern blot analysis. Taken collectively, these findings support a thiol-dependent antioxidant activity for Tpx in S. parasanguis.

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.

Growth of Streptococcus mutans in an iron-limiting medium

Microbial pathogens require iron to survive and promote disease. Reports in the literature indicate that iron can potentiate infection in the human host by triggering the expression of bacterial virulence determinants [8]. Streptococcus mutans is a prevalent microorganism in dental plaque and the principal causative agent of human dental caries. This oral pathogen may respond to the changing availability of iron in the plaque environment brought about by conditions of feast or famine. Our understanding of a role for iron in S. mutans-induced caries formation is limited, however, by the lack of an appropriate test medium. In this study we applied batch chromatography to remove iron from a chemically-defined medium [7] which we subsequently reconstituted with manganese and/or magnesium. The final metal ion concentrations in this medium were confirmed by inductively coupled argon plasma (ICAP) analysis. Importantly, the iron-depleted medium supported the growth of S. mutans only when supplemented with 0.01 to 10 μM iron, concentrations which are consistent with those found in human saliva. The practical applications of this medium are far-reaching and include the identification of iron sources, and iron-responsive genes and their products which may promote streptococcal pathogenesis. Other trace metals may be altered in this medium to promote investigations of their putative effect(s) on streptococcal growth and expression.

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 sites 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.

Interactions of the metalloregulatory protein SloR from Streptococcus mutans with its metal ion effectors and DNA binding site

UNLABELLED Streptococcus mutans is the causative agent of dental caries, a significant concern for human health, and therefore an attractive target for therapeutics development. Previous work in our laboratory has identified a homodimeric, manganese-dependent repressor protein, SloR, as an important regulator of cariogenesis and has used site-directed mutagenesis to map functions to specific regions of the protein. Here we extend those studies to better understand the structural interaction between SloR and its operator and its effector metal ions. The results of DNase I assays indicate that SloR protects a 42-bp region of DNA that overlaps the sloABC promoter on the S. mutans UA159 chromosome, while electrophoretic mobility shift and solution binding assays indicate that each of two SloR dimers binds to this region. Real-time semiquantitative reverse transcriptase PCR (real-time semi-qRT-PCR) experiments were used to determine the individual base pairs that contribute to SloR-DNA binding specificity. Solution studies indicate that Mn(2+) is better than Zn(2+) at specifically activating SloR to bind DNA, and yet the 2.8-Å resolved crystal structure of SloR bound to Zn(2+) provides insight into the means by which selective activation by Mn(2+) may be achieved and into how SloR may form specific interactions with its operator. Taken together, these experimental observations are significant because they can inform rational drug design aimed at alleviating and/or preventing S. mutans-induced caries formation. IMPORTANCE This report focuses on investigating the SloR protein as a regulator of essential metal ion transport and virulence gene expression in the oral pathogen Streptococcus mutans and on revealing the details of SloR binding to its metal ion effectors and binding to DNA that together facilitate this expression. We used molecular and biochemical approaches to characterize the interaction of SloR with Mn(2+) and with its SloR recognition element to gain a clearer picture of the regulatory networks that optimize SloR-mediated metal ion homeostasis and virulence gene expression in S. mutans. These experiments can have a significant impact on caries treatment and/or prevention by revealing the S. mutans SloR-DNA binding interface as an appropriate target for the development of novel therapeutic interventions.