Conclusions
Quorum-sensing signaling systems involving the interaction between a signaling peptide and its cognate histidine kinase receptor are widely distributed in Gram-positive bacteria. Structure–activity analysis of signaling peptides and their analogs may provide clues for understanding the specificity of peptide–receptor interactions in these bacteria. In this report, we have used the S. mutans CSP as an example to describe the methods for structure–activity analysis of quorum-sensing signaling peptides by combining activity assays with CD and NMR spectroscopy. By analyzing these peptides, we have identified several signaling peptide agonists that activate quorum-sensing cascade for induction of bacteriocin production and genetic competence in S. mutans. The methods described herein may provide a platform for analyzing structure–activity relationships of signaling peptides from many other naturally transformable streptococci.
Acknowledgments We thank Dr. John Walter and Ian Burton, Institute for Marine Biosciences, National Research Council of Canada, for their assistance in, and access to, an NMR spectrometer, and Dr. Alison Thompson, Department of Chemistry of Dalhousie University, for access to a CD instrument. We also thank Dr. Howard Kuramitsu of the State University of New York, Dr. van der Ploeg of the University of Zurich, and Dr. Dennis Cvitkovitch of the University of Toronto for their bacterial strains and plasmids. This work was supported by Discovery Grant RGPIN 311682-07 from the Natural Sciences and Engineering Research of Canada (NSERC). YH Li is a recipient of the Nova Scotia-CIHR Regional Partnership New Investigator Award. RTS was a recipient of an Izaak Walton Killam Memorial Post-Doctoral Fellowship from the Killam Trust Foundation and a Cancer Research Training Program fellowship. The authors declare that they have no competing financial interest.
Appendix
Protocol 1. Structural Determination of CSPs by CD Spectroscopy
| 1. | Prepare 2–5 mL of a stock solution (A) to the appropriate concentrations with non-chiral, sodium-free buffers, solvents, and additives. |
| 2. | Dissolve lyophilized peptide in 1 mL of stock solution A at a concentration of 500 µM (stock solution B). For structural determination, the stock solutions A and B must have identical concentrations of buffers, solvents, and additives. |
| 3. | Dilute stock solution B to make the peptide concentration of 100 µM. Note: The quality of a CD spectrum is dependent on the peptide concentration. Low peptide concentrations result in poor S/N, whereas high concentrations saturate the detector leading to artifacts. |
| 4. | Place the peptide solution in a cuvette and ensure the beam path of the cuvette is clean. Record and average the CD spectra in the range of 180 to 250 nm until a smooth CD spectrum is obtained (e.g., 16 scans with a 1.0-nm increment). |
| 5. | Further dilute the peptide solution with A to 50 µM, and record the CD spectra in a similar manner. Adjust the peptide concentration and recording CD spectra so that the receiver is not saturated, but there is enough signal for a spectrum. |
| 6. | Record the CD spectrum of solution A using identical spectrometer conditions. |
| 7. | Subtract the CD spectra of solution A from that obtained for the peptide solution by using CD software. |
| 8. | Analyze the CD spectra to determine the secondary structural features. There are several CD analysis programs and web-based analysis interfaces such as CD analysis package DICHROWEB (www.cryst.bbk.ac.uk/cdweb/html). Typically, the software is capable of multiple analysis algorithms. |
Protocol 2. Structural Determination of CSPs by 1H NMR Spectroscopy
| 1. | Dissolve purified peptides to a concentration of >1 mM in appropriate deuterated buffers, solvents, and additives. |
| 2. | Record 1H-1H COSY, TOCSY, NOESY, DOSY spectra with appropriate parameters. |
| 3. | Typically, NMR spectra are processed using sin-bell squared apodization functions to “sharpen” the signals. Process the spectra with the appropriate functions and phase corrections to produce a defined spectrum. |
| 4. | Identify as many individual amino acids as possible by correlating protons though bonds using COSY and TOCSY spectra. |
| 5. | Determine sequence-specific assignments by connecting HN i to Hα i − 1 and/or HN i to HN i ± 1 protons with the NOESY spectra. Determine longer range, non-sequential 1H-1H correlations from NOESY spectra. |
| 6. | Once a number of sequence specific residues have been assigned, integrate the appropriate NOESY cross-peaks and estimate the corresponding distance. |
| 7. | If possible, determine the 3JHN Hα coupling values from the COSY spectra. It may be necessary to fit the line shape to determine an accurate coupling value. With the 3JHN Hα coupling values, estimate the dihedral angles (if 3JHN Hα coupling value is <6.0 Hz, α-helix; 3JHN Hα values between 6.0 and 8.0 Hz, random coil; and 3JHN Hα values >8.0 Hz, β-sheet structures). |
| 8. | Using a simulated annealing molecular dynamics program, determine a preliminary family of non-violating structures from determined distances and dihedral angles. |
| 9. | Based on the preliminary structure, examine the NOESY spectrum and assign any ambiguous peaks. Then, recalculate structures in an iterative fashion. |
Protocol 3. Signaling Peptide-Dependent Competence Assay
| 1. | Inoculate a single colony of SMdC mutant into 2 mL broth of Todd–Hewitt yeast extract (THYE) (BBL®; Becton Dickinson, MD, USA) supplemented with 500 µg/mL of spectinomycin. The culture is incubated at 37°C overnight. |
| 2. | Transfer the overnight culture into 2 mL of pre-warmed, fresh THYE broth in 1:20 dilution. Each culture is incubated for 2–3 h to reach to the early mid-log phase (OD600 ≈ 0.2–0.3). |
| 3. | The culture is divided into two. One is added with a synthetic peptide at a final concentration of 50 nM, while another (negative control) is added with an equal volume of distilled water. |
| 4. | The cultures are incubated at 37°C for 15 min before added with a transforming DNA (plasmid pVA-gtfA) at the final concentration of 1 µg/mL. The cultures are incubated at 37°C for additional 2 h. |
| 5. | A 100-µL cell suspension from each culture is spread on a THYE agar plate plus erythromycin (10 µg/mL) after gentle vortexing. |
| 6. | An aliquot of the cell suspension is taken, diluted, and inoculated on THYE agar plates with no antibiotic to determine total viable cell counts. |
| 7. | All the plates are incubated at 37°C for 2 days before assessment of transformation frequency, which is expressed as percentages of transformants (erythromycin-resistant colonies) against total recipient cells per millimeter cell suspension. |
| 8. | An aliquot of the cell suspension prior to addition of the transforming DNA is also taken and inoculated on THYE plates supplemented with the same antibiotic to determine spontaneous mutation. |
Protocol 4. lacZ Reporter Assay for Signaling Peptide–Receptor Activation
| 1. | Inoculate a single colony of each of the constructed lacZ transcriptional reporter strains into 2 mL THYE broth for overnight culture. |
| 2. | Transfer the overnight culture into 40 mL of pre-warmed, fresh THYE broth in 1:20 dilution. Each culture is incubated for 2–3 h to reach the early mid-log phase (OD600 ≈ 0.2–0.3). |
| 3. | The culture is then divided into two. One is added with a synthetic peptide at a final concentration of 50 nM, while another (negative control) is added with an equal volume of distilled water. |
| 4. | Aliquots of samples are taken from each culture at time points of T 0, T 15, T 30, T 60, and T 120 following the addition of a test peptide. |
| 5. | The cell suspensions are immediately centrifuged at 10,000×g at 4°C for 10 min and the pellet is re-suspended into chilled 50 mM Tris–HCl buffer (pH 7.5) containing 0.27% (v/v) β-mercaptoethanol in a total volume of 1 mL in a Fastprep tube. |
| 6. | Cells are permeabilized by a Fastprep instrument at a setting of 6.0 for 30 s after addition of 50 µL chloroform and 20 µL of 0.1% sodium dodecyl sulfate (SDS). The samples are immediately centrifuged at 10,000×g at 4°C for 5 min. |
| 7. | An aliquot (100 µL) of the supernatant from each sample is added in triplicate onto a 96-well microtiter plate. Then, 50 µL of O-nitrophenyl-d-galactopyranoside is added into each well at a final concentration of 80 µM. |
| 8. | The reactions are incubated for 1 h, stopped by adding 50 µL of 1 M Na2CO3 into each well, and then quantified by a multi-detection micro-plate reader (Synergy) at 420 nm. The reading results are saved for calculation of specific β-gal activity. |
| 9. | An aliquot (100 µL) of the supernatants is also transferred into a microtiter plate for determination of the concentration of proteins using Bio-Rad protein assay (Bio-Rad). |
| 10. | Specific β-gal activity of each sample is calculated from triplicate samples from two independent experiments and the data are then normalized against negative or , background controls. Specific β-gal activity is expressed as Miller units (A 420 per min−1 mL−1 mg−1 protein). |
| 11. | The data from specific β-gal activity are then plotted as percentages of maximal activation versus log peptide concentrations. The half maximal activation concentration (AC50) is determined from Sigmoidal dose–response curves using Prism 4 (Graphpad). |