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.