Human saliva modifies growth, biofilm architecture, and competitive behaviors of oral streptococci.

The bacteria within supragingival biofilms participate in complex exchanges with other microbes inhabiting the same niche. One example is the mutans group streptococci (Streptococcus mutans), implicated in the development of tooth decay, and other health-associated commensal streptococci species. Previously, our group transcriptomically characterized intermicrobial interactions between S. mutans and several species of oral bacteria. However, these experiments were carried out in a medium without human saliva. To better mimic their natural environment, we first evaluated how inclusion of saliva affected growth and biofilm formation of eight Streptococcus species individually and found saliva to positively benefit growth rates while negatively influencing biofilm biomass accumulation and altering spatial arrangement. These results carried over during evaluation of 29 saliva-derived isolates of various species. Surprisingly, we also found that addition of saliva increased the competitive behaviors of S. mutans in coculture competitions against commensal streptococci that led to increases in biofilm microcolony volumes. Through transcriptomically characterizing mono- and cocultures of S. mutans and Streptococcus oralis with and without saliva, we determined that each species developed a nutritional niche under mixed-species growth, with S. mutans upregulating carbohydrate uptake and utilization pathways while S. oralis upregulated genome features related to peptide uptake and glycan foraging. S. mutans also upregulated genes involved in oxidative stress tolerance, particularly manganese uptake, which we could artificially manipulate by supplementing in manganese leading to an advantage over its opponent. Our report highlights observable changes in microbial behaviors through leveraging environmental- and host-supplied resources over their competitors. IMPORTANCE: Dental caries (tooth decay) is the most prevalent disease for both children and adults nationwide. Caries are initiated from demineralization of the enamel due to organic acid production through the metabolic activity of oral bacteria growing in biofilm communities attached to the tooth's surface. Mutans group streptococci are closely associated with caries development and initiation of the cariogenic cycle, which decreases the amount of acid-sensitive, health-associated commensal bacteria while selecting for aciduric and acidogenic species that then further drives the disease process. Defining the exchanges that occur between mutans group streptococci and oral commensals in a condition that closely mimics their natural environment is of critical need toward identifying factors that can influence odontopathogen establishment, persistence, and outgrowth. The goal of our research is to develop strategies, potentially through manipulation of microbial interactions characterized here, that prevent the emergence of mutans group streptococci while keeping the protective flora intact.

Human Saliva Modifies Growth, Biofilm Architecture and Competitive Behaviors of Oral Streptococci.

The bacteria within supragingival biofilms participate in complex exchanges with other microbes inhabiting the same niche. One example are the mutans group streptococci (Streptococcus mutans), implicated in the development of tooth decay, and other health-associated commensal streptococci species. Previously, our group transcriptomically characterized intermicrobial interactions between S. mutans and several species of oral bacteria. However, these experiments were carried out in a medium that was absent of human saliva. To better mimic their natural environment, we first evaluated how inclusion of saliva affected growth and biofilm formation of eight streptococci species individually, and found saliva to positively benefit growth rates while negatively influencing biomass accumulation and altering spatial arrangement. These results carried over during evaluation of 29 saliva-derived isolates of various species. Surprisingly, we also found that addition of saliva increased the competitive behaviors of S. mutans in coculture competitions against commensal streptococci that led to increases in biofilm microcolony volumes. Through transcriptomically characterizing mono- and cocultures of S. mutans and Streptococcus oralis with and without saliva, we determined that each species developed a nutritional niche under mixed-species growth, with S. mutans upregulating carbohydrate uptake and utilization pathways while S. oralis upregulated genome features related to peptide uptake and glycan foraging. S. mutans also upregulated genes involved in oxidative stress tolerance, particularly manganese uptake, which we could artificially manipulate by supplementing in manganese to give it an advantage over its opponent. Our report highlights observable changes in microbial behaviors via leveraging environmental- and host-supplied resources over their competitors.

Complete Genome Sequence of Streptococcus oralis 34.

Streptococcus oralis is an early colonizer and one of the most abundant species found in the human oral cavity. We report the complete genome sequence of S. oralis 34 (1,920,884 bp; GC content, 41.3%), commonly used in many oral microbiology studies exploring bacterial attachment and interaction(s) within mixed-species model systems.

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.

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.

Spatial Correlations and Distribution of Competence Gene Expression in Biofilms of Streptococcus mutans.

Streptococcus mutans is an important pathogen in the human oral biofilm. It expresses virulent behaviors that are linked to its genetic competence regulon, which is controlled by comX. Expression of comX is modulated by two diffusible signaling peptides, denoted CSP and XIP, and by other environmental cues such as pH and oxidative stress. The sensitivity of S. mutans competence to environmental inputs that may vary on microscopic length scales raises the question of whether the biofilm environment creates microniches where competence and related phenotypes are concentrated, leading to spatial clustering of S. mutans virulence behaviors. We have used two-photon microscopy to characterize the spatial distribution of comX expression among individual S. mutans cells in biofilms. By analyzing correlations in comX activity, we test for spatial clustering that may suggest localized competence microenvironments. Our data indicate that both competence-signaling peptides diffuse efficiently through the biofilm. XIP elicits a population-wide response. CSP triggers a Poisson-like, spatially random comX response from a subpopulation of cells that is homogeneously dispersed. Our data indicate that competence microenvironments if they exist are small enough that the phenotypes of individual cells are not clustered or correlated to any greater extent than occurs in planktonic cultures.

Expanding the Vocabulary of Peptide Signals in Streptococcus mutans.

Streptococci, including the dental pathogen Streptococcus mutans, undergo cell-to-cell signaling that is mediated by small peptides to control critical physiological functions such as adaptation to the environment, control of subpopulation behaviors and regulation of virulence factors. One such model pathway is the regulation of genetic competence, controlled by the ComRS signaling system and the peptide XIP. However, recent research in the characterization of this pathway has uncovered novel operons and peptides that are intertwined into its regulation. These discoveries, such as cell lysis playing a critical role in XIP release and importance of bacterial self-sensing during the signaling process, have caused us to reevaluate previous paradigms and shift our views on the true purpose of these signaling systems. The finding of new peptides such as the ComRS inhibitor XrpA and the peptides of the RcrRPQ operon also suggests there may be more peptides hidden in the genomes of streptococci that could play critical roles in the physiology of these organisms. In this review, we summarize the recent findings in S. mutans regarding the integration of other circuits into the ComRS signaling pathway, the true mode of XIP export, and how the RcrRPQ operon controls competence activation. We also look at how new technologies can be used to re-annotate the genome to find new open reading frames that encode peptide signals. Together, this summary of research will allow us to reconsider how we perceive these systems to behave and lead us to expand our vocabulary of peptide signals within the genus Streptococcus.

Spontaneously Arising Streptococcus mutans Variants with Reduced Susceptibility to Chlorhexidine Display Genetic Defects and Diminished Fitness.

Chlorhexidine (CHX) has been used to control dental caries caused by acid-tolerant bacteria such as Streptococcus mutans since the 1970s. Repeat CHX exposure for other bacterial species results in the development of variants with reduced susceptibility that also become more resistant to other antimicrobials. It has not been tested if such variants arise when streptococci are exposed to CHX. Here, we passaged S. mutans in increasing concentrations of CHX and isolated spontaneously arising reduced susceptibility variants (RSVs) from separate lineages that have MICs that are up to 3-fold greater than the parental strain. The RSVs have increased growth rates at neutral pH and under acidic conditions in the presence of CHX but accumulate less biomass in biofilms. RSVs display higher MICs for daptomycin and clindamycin but increased sensitivity to dental-relevant antimicrobials triclosan and sodium fluoride. Plate-based assays for competition with health-associated oral streptococci revealed decreased bacteriocin production by the RSVs, increased sensitivity to hydrogen peroxide, and diminished competitive fitness in a human-derived ex vivo biofilm consortium. Whole-genome sequencing identified common single nucleotide polymorphisms (SNPs) within a diacylglycerol kinase homolog and a glycolipid synthesis enzyme, which could alter the accumulation of lipoteichoic acids and other envelope constituents, as well as a variety of mutations in other genes. Collectively, these findings confirm that S. mutans and likely other streptococci can develop tolerance to CHX but that increased tolerance comes at a fitness cost, such that CHX-induced variants that spontaneously arise in the human oral cavity may not persist.

Intracellular Signaling by the comRS System in Streptococcus mutans Genetic Competence.

Entry into genetic competence in streptococci is controlled by ComX, an alternative sigma factor for genes that enable the import of exogenous DNA. In Streptococcus mutans, the immediate activator of comX is the ComRS quorum system. ComS is the precursor of XIP, a seven-residue peptide that is imported into the cell and interacts with the cytosolic receptor ComR to form a transcriptional activator for both comX and comS Although intercellular quorum signaling by ComRS has been demonstrated, observations of bimodal expression of comX suggest that comRS may also function as an intracellular feedback loop, activating comX without export or detection of extracellular XIP. Here we used microfluidic and single-cell methods to test whether ComRS induction of comX requires extracellular XIP or ComS. We found that individual comS-overexpressing cells activate their own comX, independently of the rate at which their growth medium is replaced. However, in the absence of lysis they do not activate comS-deficient mutants growing in coculture. We also found that induction of comR and comS genes introduced into Escherichia coli cells leads to activation of a comX reporter. Therefore, ComRS control of comX does not require either the import or extracellular accumulation of ComS or XIP or specific processing of ComS to XIP. We also found that endogenously and exogenously produced ComS and XIP have inequivalent effects on comX activation. These data are fully consistent with identification of intracellular positive feedback in comS transcription as the origin of bimodal comX expression in S. mutansIMPORTANCE The ComRS system can function as a quorum sensing trigger for genetic competence in S. mutans The signal peptide XIP, which is derived from the precursor ComS, enters the cell and interacts with the Rgg-type cytosolic receptor ComR to activate comX, which encodes the alternative sigma factor for the late competence genes. Previous studies have demonstrated intercellular signaling via ComRS, although release of the ComS or XIP peptide to the extracellular medium appears to require lysis of the producing cells. Here we tested the complementary hypothesis that ComRS can drive comX through a purely intracellular mechanism that does not depend on extracellular accumulation or import of ComS or XIP. By combining single-cell, coculture, and microfluidic approaches, we demonstrated that endogenously produced ComS can enable ComRS to activate comX without requiring processing, export, or import. These data provide insight into intracellular mechanisms that generate noise and heterogeneity in S. mutans competence.

Competence inhibition by the XrpA peptide encoded within the comX gene of Streptococcus mutans.

Streptococcus mutans displays complex regulation of natural genetic competence. Competence development in S. mutans is controlled by a peptide derived from ComS (XIP); which along with the cytosolic regulator ComR controls the expression of the alternative sigma factor comX, the master regulator of competence development. Recently, a gene embedded within the coding region of comX was discovered and designated xrpA (comX regulatory peptide A). XrpA was found to be an antagonist of ComX, but the mechanism was not established. In this study, we reveal through both genomic and proteomic techniques that XrpA is the first described negative regulator of ComRS systems in streptococci. Transcriptomic and promoter activity assays in the ΔxrpA strain revealed an up-regulation of genes controlled by both the ComR- and ComX-regulons. An in vivo protein crosslinking and in vitro fluorescent polarization assays confirmed that the N-terminal region of XrpA were found to be sufficient in inhibiting ComR-XIP complex binding to ECom-box located within the comX promoter. This inhibitory activity was sufficient for decreases in PcomX activity, transformability and ComX accumulation. XrpA serving as a modulator of ComRS activity ultimately results in changes to subpopulation behaviors and cell fate during competence activation.

Intercellular Communication via the comX-Inducing Peptide (XIP) of Streptococcus mutans.

Gram-positive bacteria utilize exported peptides to coordinate genetic and physiological processes required for biofilm formation, stress responses, and ecological competitiveness. One example is activation of natural genetic competence by ComR and the com X -inducing peptide (XIP) in Streptococcus mutans Although the competence pathway can be activated by the addition of synthetic XIP in defined medium, the hypothesis that XIP is able to function as an intercellular signaling molecule has not been rigorously tested. Coculture model systems were developed that included a "sender" strain that overexpressed the XIP precursor (ComS) and a "responder" strain harboring a green fluorescent protein (GFP) reporter fused to a ComR-activated gene (comX) promoter. The ability of the sender strain to provide a signal to activate GFP expression was monitored at the individual cell and population levels using (i) planktonic culture systems, (ii) cells suspended in an agarose matrix, or (iii) cells growing in biofilms. XIP was shown to be freely diffusible, and XIP signaling between the S. mutans sender and responder strains did not require cell-to-cell contact. The presence of a sucrose-derived exopolysaccharide matrix diminished the efficiency of XIP signaling in biofilms, possibly by affecting the spatial distribution of XIP senders and potential responders. Intercellular signaling was greatly impaired in a strain lacking the primary autolysin, AtlA, and was substantially greater when the sender strain underwent lysis. Collectively, these data provide evidence that S. mutans XIP can indeed function as a peptide signal between cells and highlight the importance of studying signaling with an endogenously produced peptide(s) in populations in various environments and physiologic states.IMPORTANCE The comX-inducing peptide (XIP) of Streptococcus mutans is a key regulatory element in the activation of genetic competence, which allows cells to take up extracellular DNA. XIP has been found in cell culture fluids, and the addition of synthetic XIP to physiologically receptive cells can robustly induce competence gene expression. However, there is a lack of consensus as to whether XIP can function as an intercellular communication signal. Here, we show that XIP indeed signals between cells in S. mutans, but that cell lysis may be a critical factor, as opposed to a dedicated secretion/processing system, in allowing for release of XIP into the environment. The results have important implications in the context of the ecology, virulence, and evolution of a ubiquitous human pathogen and related organisms.

An Essential Role for (p)ppGpp in the Integration of Stress Tolerance, Peptide Signaling, and Competence Development in Streptococcus mutans.

The microbes that inhabit the human oral cavity are subjected to constant fluctuations in their environment. To overcome these challenges and gain a competitive advantage, oral streptococci employ numerous adaptive strategies, many of which appear to be intertwined with the development of genetic competence. Here, we demonstrate that the regulatory circuits that control development of competence in Streptococcus mutans, a primary etiological agent of human dental caries, are integrated with key stress tolerance pathways by the molecular alarmone (p)ppGpp. We first observed that the growth of a strain that does not produce (p)ppGpp (ΔrelAPQ, (p)ppGpp(0)) is not sensitive to growth inhibition by comX inducing peptide (XIP), unlike the wild-type strain UA159, even though XIP-dependent activation of the alternative sigma factor comX by the ComRS pathway is not impaired in the (p)ppGpp(0) strain. Overexpression of a (p)ppGpp synthase gene (relP) in the (p)ppGpp(0) mutant restored growth inhibition by XIP. We also demonstrate that exposure to micromolar concentrations of XIP elicited changes in (p)ppGpp accumulation in UA159. Loss of the RelA/SpoT homolog (RSH) enzyme, RelA, lead to higher basal levels of (p)ppGpp accumulation, but to decreased sensitivity to XIP and to decreases in comR promoter activity and ComX protein levels. By introducing single amino acid substitutions into the RelA enzyme, the hydrolase activity of the enzyme was shown to be crucial for full com gene induction and transformation by XIP. Finally, loss of relA resulted in phenotypic changes to ΔrcrR mutants, highlighted by restoration of transformation and ComX protein production in the otherwise non-transformable ΔrcrR-NP mutant. Thus, RelA activity and its influence on (p)ppGpp pools appears to modulate competence signaling and development through RcrRPQ and the peptide effectors encoded within rcrQ. Collectively, this study provides new insights into the molecular mechanisms that integrate intercellular communication with the physiological status of the cells and the regulation of key virulence-related phenotypes in S. mutans.

A unique open reading frame within the comX gene of Streptococcus mutans regulates genetic competence and oxidative stress tolerance.

Streptococcus mutans displays complex regulation of genetic competence, with ComX controlling late competence gene transcription. The rcrRPQ operon has been shown to link oxidative stress tolerance, (p)ppGpp metabolism and competence in S. mutans. Importantly, an rcrR polar (ΔrcrR-P) mutant is hyper-transformable, but an rcrR non-polar (ΔrcrR-NP) mutant cannot be transformed. Transcriptome comparisons of the rcrR mutants using RNA-Seq and quantitative real-time polymerase chain reaction revealed little expression in the 5' region of comX in ΔrcrR-NP, but high level expression in the 3' region. Northern blotting with comX probes revealed two distinct transcripts in the ΔrcrR-P and ΔrcrR-NP strains, and 5' Rapid Amplification of cDNA Ends mapped the 5' terminus of the shorter transcript to nt +140 of the comX structural gene, where a unique 69-aa open reading frame, termed XrpA, was encoded in a different reading frame than ComX. Two single-nucleotide substitution mutants (comX::T162C; comX::T210A) were introduced to disrupt XrpA without affecting the sequence of ComX. When the mutations were in the ΔrcrR-NP genetic background, ComX production and transformation were restored. Overexpression of xrpA led to impaired growth in aerobic conditions and decreased transformability. These results reveal an unprecedented mechanism for competence regulation and stress tolerance by a gene product encoded within the comX gene that appears unique to S. mutans.

Discovery of novel peptides regulating competence development in Streptococcus mutans.

A MarR-like transcriptional repressor (RcrR) and two predicted ABC efflux pumps (RcrPQ) encoded by a single operon were recently shown to be dominant regulators of stress tolerance and development of genetic competence in the oral pathogen Streptococcus mutans. Here, we focused on polar (ΔrcrR-P) and nonpolar (ΔrcrR-NP) rcrR mutants, which are hyper- and nontransformable, respectively, to dissect the mechanisms by which these mutations impact competence. We discovered two open reading frames (ORFs) in the 3' end of the rcrQ gene that encode peptides of 27 and 42 amino acids (aa) which are also dramatically upregulated in the ΔrcrR-NP strain. Deletion of, or start codon mutations in, the ORFs for the peptides in the ΔrcrR-NP background restored competence and sensitivity to competence-stimulating peptide (CSP) to levels seen in the ΔrcrR-P strain. Overexpression of the peptides adversely affected competence development. Importantly, overexpression of mutant derivatives of the ABC exporters that lacked the peptides also resulted in impaired competence. FLAG-tagged versions of the peptides could be detected in S. mutans, and FLAG tagging of the peptides impaired their function. The competence phenotypes associated with the various mutations, and with overexpression of the peptides and ABC transporters, were correlated with the levels of ComX protein in cells. Collectively, these studies revealed multiple novel mechanisms for regulation of competence development by the components of the rcrRPQ operon. Given their intimate role in competence and stress tolerance, the rcrRPQ-encoded peptides may prove to be useful targets for therapeutics to diminish the virulence of S. mutans.

Growth phase and pH influence peptide signaling for competence development in Streptococcus mutans.

The development of competence by the dental caries pathogen Streptococcus mutans is mediated primarily through the alternative sigma factor ComX (SigX), which is under the control of multiple regulatory systems and activates the expression of genes involved in DNA uptake and recombination. Here we report that the induction of competence and competence gene expression by XIP (sigX-inducing peptide) and CSP (competence-stimulating peptide) is dependent on the growth phase and that environmental pH has a potent effect on the responses to XIP. A dramatic decline in comX and comS expression was observed in mid- and late-exponential-phase cells. XIP-mediated competence development and responses to XIP were optimal around a neutral pH, although mid-exponential-phase cells remained refractory to XIP treatment, and acidified late-exponential-phase cultures were resistant to killing by high concentrations of XIP. Changes in the expression of the genes for the oligopeptide permease (opp), which appears to be responsible for the internalization of XIP, could not entirely account for the behaviors observed. Interestingly, comS and comX expression was highly induced in response to endogenously overproduced XIP or ComS in mid-exponential-phase cells. In contrast to the effects of pH on XIP, competence induction and responses to CSP in complex medium were not affected by pH, although a decreased response to CSP in cells that had exited early-exponential phase was observed. Collectively, these results indicate that competence development may be highly sensitive to microenvironments within oral biofilms and that XIP and CSP signaling in biofilms could be spatially and temporally heterogeneous.