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Identification of highly potent competence stimulating peptide-based quorum sensing activators in Streptococcus mutans through the utilization of N-methyl and reverse alanine scanning.

ABSTRACT: Quorum sensing (QS) controls the pathogenic behavior of Streptococcus mutans, a primary cause of dental caries. S. mutans uses the competence stimulating peptide (CSP) to control mutacin production, a bacteriocin utilized by S. mutans to outcompete different commensal bacteria in mixed biofilm environments. In this study, we performed an N-methyl scan of an 18-CSP-based scaffold lacking the first two amino acid residues that were shown to be dispensable, to gain important mechanistic insight as to the role of backbone amide protons in the interaction between CSP and the ComD receptor. We then utilized the reverse alanine approach to develop CSP-based analogs with enhanced activities. The two most potent analogs were found to induce bacteriocin production at sub-nanomolar concentration using an interspecies inhibition assay. Overall, our analysis revealed that the 18-CSP sequence is not optimized and can be improved by replacement of multiple positions with alanine. Our results further suggest that the hydrophobic residues in S. mutans 18-CSP are involved in both receptor binding and activation.

SUBMITTER: Bikash CR

PROVIDER: S-EPMC6379129 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Identification of highly potent competence stimulating peptide-based quorum sensing activators in Streptococcus mutans through the utilization of N-methyl and reverse alanine scanning.

Bikash Chowdhury Raihan CR Tal-Gan Yftah Y

Bioorganic & medicinal chemistry letters 20190125 6

Quorum sensing (QS) controls the pathogenic behavior of Streptococcus mutans, a primary cause of dental caries. S. mutans uses the competence stimulating peptide (CSP) to control mutacin production, a bacteriocin utilized by S. mutans to outcompete different commensal bacteria in mixed biofilm environments. In this study, we performed an N-methyl scan of an 18-CSP-based scaffold lacking the first two amino acid residues that were shown to be dispensable, to gain important mechanistic insight as ...[more]

PMID: 30711392

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Project description:Extracellular DNA (eDNA) is a biofilm component that contributes to the formation and structural stability of biofilms. Streptococcus mutans, a major cariogenic bacterium, induces eDNA-dependent biofilm formation under specific conditions. Since cell death can result in the release and accumulation of DNA, the dead cells in biofilms are a source of eDNA. However, it remains unknown how eDNA is released from dead cells and is localized within S. mutans biofilms. We focused on cell death induced by the extracellular signaling peptide called competence-stimulating peptide (CSP). We demonstrate that nucleic acid release into the extracellular environment occurs in a subpopulation of dead cells. eDNA production induced by CSP was highly dependent on the lytF gene, which encodes an autolysin. Although lytF expression was induced bimodally by CSP, lytF-expressing cells further divided into surviving cells and eDNA-producing dead cells. Moreover, we found that lytF-expressing cells were abundant near the bottom of the biofilm, even when all cells in the biofilm received the CSP signal. Dead cells and eDNA were also abundantly present near the bottom of the biofilm. The number of lytF-expressing cells in biofilms was significantly higher than that in planktonic cultures, which suggests that adhesion to the substratum surface is important for the induction of lytF expression. The deletion of lytF resulted in reduced adherence to a polystyrene surface. These results suggest that lytF expression and eDNA production induced near the bottom of the biofilm contribute to a firmly attached and structurally stable biofilm.IMPORTANCE Bacterial communities encased by self-produced extracellular polymeric substances (EPSs), known as biofilms, have a wide influence on human health and environmental problems. The importance of biofilm research has increased, as biofilms are the preferred bacterial lifestyle in nature. Furthermore, in recent years it has been noted that the contribution of phenotypic heterogeneity within biofilms requires analysis at the single-cell or subpopulation level to understand bacterial life strategies. In Streptococcus mutans, a cariogenic bacterium, extracellular DNA (eDNA) contributes to biofilm formation. However, it remains unclear how and where the cells produce eDNA within the biofilm. We focused on LytF, an autolysin that is induced by extracellular peptide signals. We used single-cell level imaging techniques to analyze lytF expression in the biofilm population. Here, we show that S. mutans generates eDNA by inducing lytF expression near the bottom of the biofilm, thereby enhancing biofilm adhesion and structural stability.

Project description:Two small quorum sensing (QS) peptides regulate competence in S. mutans in a cell density dependent manner: XIP (sigX inducing peptide) and CSP (competence stimulating peptide). Depending on the environmental conditions isogenic S. mutans cells can split into a competent and non-competent subpopulation. The origin of this population heterogeneity has not been experimentally determined and it is unknown how the two QS systems are connected. We developed a toolbox of single and dual fluorescent reporter strains and systematically knocked out key genes of the competence signaling cascade in the reporter strain backgrounds. By following signal propagation on the single cell level we discovered that the master regulator of competence, the alternative sigma factor SigX, directly controls expression of the response regulator for bacteriocin synthesis ComE. Consequently, a SigX binding motif (cin-box) was identified in the promoter region of comE. Overexpressing the genetic components involved in competence development demonstrated that ComRS represents the origin of bimodality and determines the modality of the downstream regulators SigX and ComE. Moreover these analysis showed that there is no direct regulatory link between the two QS signaling cascades. Competence is induced through a hierarchical XIP signaling cascade, which has no regulatory input from the CSP cascade. CSP exclusively regulates bacteriocin synthesis. We suggest renaming it mutacin inducing peptide (MIP). Finally, using phosphomimetic comE mutants we show that unimodal bacteriocin production is controlled posttranslationally, thus solving the puzzling observation that in complex media competence is observed in a subpopulation only, while at the same time all cells produce bacteriocins. The control of both bacteriocin synthesis and competence through the alternative sigma-factor SigX suggests that S. mutans increases its genetic repertoire via QS controlled predation on neighboring species in its natural habitat.

Project description:UnlabelledThe capacity to internalize and catabolize carbohydrates is essential for dental caries pathogens to persist and cause disease. The expression of many virulence-related attributes by Streptococcus mutans, an organism strongly associated with human dental caries, is influenced by the peptide signaling pathways that control genetic competence. Here, we demonstrate a relationship between the efficiency of competence signaling and carbohydrate source. A significant increase in the activity of the promoters for comX, comS, and comYA after exposure to competence-stimulating peptide (CSP) was observed in cells growing on fructose, maltose, sucrose, or trehalose as the primary carbohydrate source, compared to cells growing on glucose. However, only cells grown in the presence of trehalose or sucrose displayed a significant increase in transformation frequency. Notably, even low concentrations of these carbohydrates in the presence of excess glucose could enhance the expression of comX, encoding a sigma factor needed for competence, and the effects on competence were dependent on the cognate sugar:phosphotransferase permease for each carbohydrate. Using green fluorescent protein (GFP) reporter fusions, we observed that growth in fructose or trehalose resulted in a greater proportion of the population activating expression of comX and comS, encoding the precursor of comX-inducing peptide (XIP), after addition of CSP, than growth in glucose. Thus, the source of carbohydrate significantly impacts the stochastic behaviors that regulate subpopulation responses to CSP, which can induce competence in S. mutansImportanceThe signaling pathways that regulate development of genetic competence in Streptococcus mutans are intimately intertwined with the pathogenic potential of the organism, impacting biofilm formation, stress tolerance, and expression of known virulence determinants. Induction of the gene for the master regulator of competence, ComX, by competence-stimulating peptide (CSP) occurs in a subpopulation of cells. Here, we show that certain carbohydrates that are common in the human diet enhance the ability of CSP to activate transcription of comX and that a subset of these carbohydrates stimulates progression to the competent state. The cognate sugar:phosphotransferase permeases for each sugar are needed for these effects. Interestingly, single-cell analysis shows that the carbohydrates that increase com gene expression do so by enhancing the proportion of cells that respond to CSP. A mathematical model is developed to explain how carbohydrates modulate bistable behavior in the system via the ComRS pathway and ComX stability.

Dataset Information

Identification of highly potent competence stimulating peptide-based quorum sensing activators in Streptococcus mutans through the utilization of N-methyl and reverse alanine scanning.

Publications

Identification of highly potent competence stimulating peptide-based quorum sensing activators in Streptococcus mutans through the utilization of N-methyl and reverse alanine scanning.

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