Structure-Activity Relationships of the Competence Stimulating Peptide in Streptococcus mutans Reveal Motifs Critical for Membrane Protease SepM Recognition and ComD Receptor Activation.
ABSTRACT: Streptococcus mutans ( S. mutans) is a Gram-positive human pathogen that is one of the major contributors to dental caries, a condition with an economic cost of over $100 billion per year in the United States. S. mutans secretes a 21-amino-acid peptide termed the competence stimulating peptide (21-CSP) to assess its population density in a process termed quorum sensing (QS) and to initiate a variety of phenotypes such as biofilm formation and bacteriocin production. 21-CSP is processed by a membrane bound protease SepM into active 18-CSP, which then binds to the ComD receptor. This study seeks to determine the molecular mechanism that ties 21-CSP:SepM recognition and 18-CSP:ComD receptor binding and to identify QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in both 21-CSP and 18-CSP and assessed the ability of the mutated analogs to modulate QS. We identified residues that are important to SepM recognition and ComD receptor binding. Our results shed light on the S. mutans competence QS pathway at the molecular level. Moreover, our structural insights of the CSP signal can be used to design QS-based anti-infective therapeutics against S. mutans.
Project description:The competence stimulating peptide (CSP) plays a key role in the regulation of pneumococcal quorum sensing (QS), a communication system that is critical to the infectivity of pneumococci. CSP functions through binding and activating a transmembrane receptor, ComD. Molecules that can modulate pneumococcal QS through intercepting CSP:ComD interaction may serve as new generation of antibacterial agents to treat pneumococcal infections. In this work, we systematically modified the N-terminus of CSP1, a region that is essential to ComD activation, to identify detailed structural features of the N-terminus that are responsible for its function. Our results revealed structural features that are optimal to achieve receptor activation and structure-activity trends that improve our understanding of CSP:ComD interaction, all of which will contribute to the design of novel pneumococcal QS modulators with higher potency and improved pharmacological properties.
Project description:Streptococcus mutans, a causative agent of dental caries, relies on multiple quorum-sensing (QS) pathways that coordinate the expression of factors needed for colonization in the oral cavity. S. mutans uses small peptides as QS signaling molecules that typically are secreted into the outside milieu. Competence-stimulating peptide (CSP) is one such QS signaling molecule that functions through the ComDE two-component signal transduction pathway. CSP is secreted through NlmTE, a dedicated ABC transporter that cleaves off the N-terminal leader peptide to generate a mature peptide that is 21 residues long (CSP-21). We recently identified a surface-localized protease, SepM, which further cleaves the CSP-21 peptide at the C-terminal end and removes the last 3 residues to generate CSP-18. CSP-18 is the active QS molecule that interacts with the ComD sensor kinase to activate the QS pathway. In this study, we show that SepM specifically cleaves CSP-21 between the Ala18 and Leu19 residues. We also show that SepM recognizes only Ala at position 18 and Leu at position 19, although some CSP-18 variants with a substitution at position 18 can function equally as well as the QS peptide. Furthermore, we demonstrate that SepM homologs from other streptococci are capable of processing CSP-21 to generate functional CSP-18.SepM is a membrane-associated streptococcal protease that processes competence-stimulating peptide (CSP) to generate an active quorum-sensing molecule in S. mutans. SepM belongs to the S16 family of serine proteases, and in this study, we found that SepM behaves as an endopeptidase. SepM displays strict substrate specificity and cleaves the peptide bond between the Ala and Leu residues. This is the first report of an endopeptidase that specifically cleaves these two residues.
Project description: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.
Project description:Quorum sensing activation by signal pheromone (CSP) in Streptococcus mutans depends on the membrane-associated receptor ComD, which senses the signal and triggers the signaling cascade for bacteriocin production and other cell density-dependent activities. However, the mechanism of the signal recognition via the ComD receptor in this species is nearly unexplored. Here, we show that the membrane domain of the ComD protein forms six transmembrane segments with three extracellular loops, loopA, loopB and loopC. By structural and functional analyses of these extracellular loops, we demonstrate that both loopC and loopB are required for CSP recognition, while loopA plays little role in CSP detection. A deletion or substitution mutation of four residues NVIP in loopC abolishes CSP recognition for quorum sensing activities. We conclude that both loopC and loopB are required for forming the receptor and residues NVIP of loopC are essential for CSP recognition and quorum sensing activation in S. mutans.
Project description:Streptococcus pneumoniae is a highly recombinogenic human pathogen that utilizes the competence stimulating peptide (CSP)-based quorum sensing (QS) circuitry to acquire antibiotic resistance genes from the environment and initiate its attack on the human host. Modulation of QS in this bacterium, either inhibition or activation, can therefore be used to attenuate S. pneumoniae infectivity and slow down pneumococcal resistance development. In this study, we set to determine the molecular mechanism that drives CSP:receptor binding and identify CSP-based QS modulators with distinct activity profiles. To this end, we conducted systematic replacement of the amino acid residues in the two major CSP signals (CSP1 and CSP2) and assessed the ability of the mutated analogs to modulate QS against both cognate and noncognate ComD receptors. We then evaluated the overall 3D structures of these analogs using circular dichroism (CD) to correlate between the structure and function of these peptides. Our CD analysis revealed a strong correlation between ?-helicity and bioactivity for both specificity groups (CSP1 and CSP2). Furthermore, we identified the first pan-group QS activator and the most potent group-II QS inhibitor to date. These chemical probes can be used to study the role of QS in S. pneumoniae and as scaffolds for the design of QS-based anti-infective therapeutics against S. pneumoniae infections.
Project description:Competence-stimulating peptide (CSP) and ComD of the streptococcal species are a pheromone and its receptor, respectively, involved in the regulation of competence for natural genetic transformation. We show here that these molecules have undergone positive selection. This study is the first report of positive selection due to competition among bacterial populations.
Project description:Quorum sensing (QS) is a cell-cell communication mechanism that enables bacteria to assess their population density and alter their behavior upon reaching high cell number. Many bacterial pathogens utilize QS to initiate an attack on their host, thus QS has attracted significant attention as a potential antivirulence alternative to traditional antibiotics. Streptococcus pneumoniae, a notorious human pathogen responsible for a variety of acute and chronic infections, utilizes the competence regulon and its associated signaling peptide, the competence stimulating peptide (CSP), to acquire antibiotic resistance and establish an infection. In this work, we sought to define the binding pockets within the ComD1 receptor used for binding the hydrophobic side-chain residues in CSP1 through the introduction of highly-conservative point mutations within the peptide. Optimization of these binding interactions could lead to the development of highly potent CSP-based QS modulators while the inclusion of non-natural amino acids within the CSP sequence would confer resistance to protease degradation, a requirement for drug candidates.
Project description:Streptococcus pneumoniae is an important pathogen that utilizes quorum sensing (QS) to regulate genetic transformation, virulence, and biofilm formation. The competence-stimulating peptide (CSP) is a 17-amino acid signal peptide that is used by S. pneumoniae to trigger QS. S. pneumoniae strains can be divided into two main specificity groups based on the CSP signal they produce (CSP1 or CSP2) and their compatible receptors (ComD1 or ComD2, respectively). Modulation of QS in S. pneumoniae can be achieved by targeting the CSP:ComD interaction using synthetic CSP analogues. However, to rationally design CSP-based QS modulators with enhanced activities, an in-depth understanding of the structural features that are required for receptor binding is needed. Herein, we report a comprehensive in-solution three-dimensional structural characterization of eight CSP1 and CSP2 analogues with varied biological activities using nuclear magnetic resonance spectroscopy. Analysis of these structures revealed two distinct hydrophobic patches required for effective ComD1 and ComD2 binding.
Project description:Streptococcus mutans is a bacterium that has evolved to be dependent upon a biofilm "lifestyle" for survival and persistence in its natural ecosystem, dental plaque. We initiated this study to identify the genes involved in the development of genetic competence in S. mutans and to assay the natural genetic transformability of biofilm-grown cells. Using genomic analyses, we identified a quorum-sensing peptide pheromone signaling system similar to those previously found in other streptococci. The genetic locus of this system comprises three genes, comC, comD, and comE, that encode a precursor to the peptide competence factor, a histidine kinase, and a response regulator, respectively. We deduced the sequence of comC and its active pheromone product and chemically synthesized the corresponding 21-amino-acid competence-stimulating peptide (CSP). Addition of CSP to noncompetent cells facilitated increased transformation frequencies, with typically 1% of the total cell population transformed. To further confirm the roles of these genes in genetic competence, we inactivated them by insertion-duplication mutagenesis or allelic replacement followed by assays of transformation efficiency. We also demonstrated that biofilm-grown S. mutans cells were transformed at a rate 10- to 600-fold higher than planktonic S. mutans cells. Donor DNA included a suicide plasmid, S. mutans chromosomal DNA harboring a heterologous erythromycin resistance gene, and a replicative plasmid. The cells were optimally transformed during the formation of 8- to 16-h-old biofilms primarily consisting of microcolonies on solid surfaces. We also found that dead cells in the biofilms could act as donors of a chromosomally encoded antibiotic resistance determinant. This work demonstrated that a peptide pheromone system controls genetic competence in S. mutans and that the system functions optimally when the cells are living in actively growing biofilms.
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.