Many species of streptococci secrete and use a competence-stimulating peptide (CSP) to initiate quorum sensing for induction of genetic competence, bacteriocin production, and other activities. These signaling molecules are small, unmodified peptides that induce powerful strain-specific activity at nano-molar concentrations. This feature has provided an excellent opportunity to explore their structure–function relationships. However, CSP variants have also been identified in many species, and each specifically activates its cognate receptor. How such minor changes dramatically affect the specificity of these peptides remains unclear. Structure–activity analysis of these peptides may provide clues for understanding the specificity of signaling peptide–receptor interactions. Here, we use the Streptococcus mutans CSP as an example to describe methods of analyzing its structure–activity relationship. The methods described here may provide a platform for studying quorum-sensing signaling peptides of other naturally transformable streptococci.
Key Words: Quorum sensing - Signaling peptides - Structure–activity analysis - Circular dichroism and nuclear magnetic resonance spectroscopy - Streptococcus mutans
Introduction
Natural genetic transformation is a process by which bacteria are able to take up and integrate exogenous free DNA from their environment (1, 2). This process enables the recipient organisms to acquire novel genes, thereby promoting the emergence of antibiotic resistance, genetic variation, and rapid evolution of virulence factors (1–4). Many members of the genus Streptococcus are naturally transformable and each depends on a signaling peptide-mediated quorum-sensing system for induction of genetic competence (3–5). Activation of quorum sensing for genetic competence and other coordinated activities in these species requires at least six gene products encoded by comCDE, comAB, and comX (Fig. 1 ). The comC gene encodes a competence-stimulating peptide (CSP) precursor, which is cleaved and exported through a peptide-specific ABC transporter encoded by comAB, releasing CSP into a extracellular environment (2–6). The comDE encodes a two-component system consisting of a histidine kinase sensor protein (ComD) and its cognate response regulator (ComE) that specifically senses and responds to CSP. At a critical concentration, the CSP activates autophosphorylation of the ComD of neighboring cells. The phosphate group is then transferred to the ComE, which in turn activates its target genes, including comX that encodes a competence-specific sigma factor recognizing a consensus sequence (com-box) at the promoter regions of late competence genes, triggering the signaling cascade for genetic competence (2–5). The quorum-sensing system in Streptococcus mutans represents a unique regulatory mechanism, since this system regulates both bacteriocin production and genetic competence (2, 6, 7). In contrast to S. mutans, Streptococcus pneumoniae requires two separate signaling systems, the ComCDE and BlpRH, to regulate genetic competence and bacteriocin production (8).
Fig. 1 A schematic diagram describing the quorum-sensing system and its controlled cooperative activities in Streptococcus mutans. The signaling peptide or CSP induces quorum-sensing cascade when reaching to a critical concentration. This in turn activates the production of numerous bacteriocins and genetic competence, resulting in killing of other species, DNA release, and gene exchange.
Many CSPs from naturally transformable streptococci have been identified and their activity for genetic competence has been validated (2–4). These signaling molecules are small, unmodified peptides, ranging from 14 to 25 residues in length, and induce powerful strain-specific activity at nano-molar concentrations. Some CSPs have been chemically synthesized and used as a tool to induce genetic competence during molecular cloning. Recently, the structure–activity relationships of CSPs from both S. pneumoniae and S. mutans have been analyzed (9, 10). The CSPs from these organisms have been found to adopt an amphipathic, α-helical conformation with a defined hydrophobic face that contributes to the CSP specificity. Furthermore, the CSP from S. mutans reveals two functional domains (10). The C-terminal structural motif consisting of a sequence of polar hydrophobic charged residues is crucial for activating the signal transduction pathway, while the core α-helical structure extending from residue 5 through to the end of the peptide is required for receptor binding. Sequence alignment of CSPs from various streptococci shows that almost all CSPs have such a motif at the C-termini (10), suggesting that these CSPs likely have similar functional domains. However, CSP variants have been identified in many species and each induces competence in a highly strain-specific manner. Thus, the CSP variants may provide an excellent model to study peptide–receptor interactions in these bacteria. Here, we extend our technical information and describe methods and protocols for structure–activity analysis of CSP from S. mutans by combining activity assays with nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopies (see Appendix). The methods described herein may provide a platform and rationale for analyzing CSPs from other naturally transformable streptococci.
Materials and Methods
Chemical Synthesis of Signaling Peptides
Efficient structural characterization of signaling peptides by CD and NMR requires purified (≥95% purity) and soluble peptides. In our study, all the peptides were commercially synthesized and purified by reversed-phase high-pressure liquid chromatography. Their identity was confirmed by a mass spectrometry (10). The peptides were lyophilized and stored at –20°C until use. For CD and NMR studies, each peptide was dissolved in an appropriate solvent. For activity assays, each peptide was freshly dissolved in sterile distilled water at a concentration of 1.0 mM (stock solution), which was further diluted as required.
Sample Preparation
The first step was to optimize sample conditions by adjusting pH, ionic strength, and temperature to mimic physiological conditions with the constraint in which the conditions were compatible with data acquisition (11). For CD spectroscopy, peptide concentrations were typically on the order of 50 µM with 200 µL of solution (12). Buffers, co-solvents, and additives (e.g., detergent molecules) were non-chiral, sodium-free, and non-absorbing within the wavelength range (typically 250–180 nm). For NMR studies, buffers, co-solvents, and additives were hydrogen-free or deuterated. Samples could be dissolved in as little as 10 µL, but typically, spectrometers were designed for sample volumes of at least 500 µL. Due to the inherently low sensitivity, concentrations of peptides ranged from 1.0 to 10 mM (13).
In our study, synthetic peptides CSP (UA159sp) and TPC3 for CD and NMR were dissolved in 95/5/0%, 70/0/30%, 30/0/70%, or 0/0/100% aqueous buffer/D2O/TFE-d 2, or 95/5 aqueous buffer/D2O with 300 mM DPC-d 38 (98 atom %D), where TFE refers to trifluoroethanol and DPC-d 38 refers to perdeuterated dodecylphosphocholine (10, 13). The aqueous buffer used for sample preparation contained 50 mM K2HPO4/KH2PO4 at pH 7.0. The concentration of peptides for solutions containing 100% TFE-d 2 or DPC-d38 was 2 and 5 mM. For diffusion studies, we analyzed the solutions containing peptides and DPC-d 38 by lyophilizing and dissolving them in an equivalent volume of D2O (99.9 atom %D) followed by transferring into a 5 mm OD, D2O magnetic susceptibility-matched NMR tube (BMS-3; Shigemi, Tokyo). The sample height of 1.2 cm ensured that the entire sample was within the radio frequency coils, which was essential for artifact-free and accurate diffusion measurements.
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