Data Availability Statement.

With the exception of the NMR structural coordinates, which are publicly available at the Biological Magnetic Resonance Data Bank (BMRB) structural databank, all data and protocols reported are contained in the manuscript and SI Appendix.

Chemical Reagents and Instrumentation.

All chemical reagents and solvents were used as previously described (25). Briefly, chemical reagents and solvents were purchased from Sigma-Aldrich and used without further purification, water (18 MΩ) was purified using a Millipore Analyzer Feed System, and solid-phase resins were purchased from Advanced ChemTech and Chem-Impex International.

Reversed-phase high-performance liquid chromatography (RP-HPLC) and MS were performed as previously described (25). Briefly, RP-HPLC was performed using a Shimadzu system equipped with a CBM-20A communications bus module, two LC-20AT pumps, an SIL-20A auto sampler, an SPD-20A ultraviolet/visible (VIS) detector, a CTO-20A column oven, and an FRC-10A fraction collector. Matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF MS) data were obtained on a Bruker Microflex spectrometer equipped with a 60-Hz nitrogen laser and a reflectron. In reflectron positive ion mode, the acceleration voltage on Ion Source 1 was 19.01 kV. Exact mass data were obtained on an Agilent Technologies 6230 TOF LC/MS spectrometer. The samples were sprayed with a capillary voltage of 3,500 V, and the electrospray ionization source parameters were as follows: gas temperature of 325 °C at a drying gas flow rate of 8 L/min at a pressure of 35 psi.

Solid-Phase Peptide Synthesis.

Solid-phase peptide synthesis (SPPS) was performed as previously described (25). Briefly, the CSP1 analogs were synthesized using standard Fmoc-based SPPS procedures on 4-benzyloxybenzyl alcohol (Wang) resin. Preloaded Fmoc-l-Lys(Boc) Wang resin (0.343 mmol/g) was used for peptides that required a lysine at the C terminus, and preloaded Fmoc-l-Arg(Pbf) Wang resin (0.305 mmol/g) was used for peptides that required an arginine at the C terminus. Amino acids that have alloc- or allyl-protected amine or carboxyl group on the side chain were added in the selected positions for future cyclization (see SI Appendix for the full procedure). Upon completing the construction of the entire peptide sequence, the Fmoc protecting group was kept on the N-terminal amine, while the alloc- and allyl-protected side chains were deprotected with the following protocol. The resin was washed three times with dichloromethane (DCM) for 1 min before it was dried. Then the air in the reaction vessel was replaced with argon. Approximately 5 mL of dry DCM was added to a 15-mL polypropylene centrifuge tube, and the tube was sparged with argon for 3 min. Ten equivalents (equiv.) (relative to the resin loading) of phenyl silane (which is acting as a scavenger) was added to the centrifuge tube, and the tube was sparged with argon for an additional 2 to 3 min. Then 0.5 equiv. of tetrakis(triphenylphosphine)palladium(0) was added to the centrifuge tube, and the tube was sparged with argon for about 4 min. The resulting solution in the centrifuge tube was then added to the resin, and the reaction vessel was covered with aluminum foil and properly sealed. Then, the reaction vessel was placed on a shaker at 200 rpm for 2 h. The vessel was drained, and the resin was washed four times with 0.5% sodium ditethyldithiocarbamate trihydrate in DMF for 2 min with shaking, followed by four washes with DMF for 1 min with shaking. A solution of [ethyl cyano(hydroxyimino)acetato]-tri(1-pyrrolidinyl)-phosphonium hexafluorophosphate (Pyoxim; 1.25 equiv. relative to the resin loading) and N,N-Diisopropylethylamine (2.5 equiv. relative to the resin loading) in DMF was then added to the resin to couple the deprotected free amine and carboxyl group to form the macrocycle ring. The reaction vessel was placed on a shaker at 200 rpm for 3 h. The vessel was drained, the solution was replenished, and the reaction was allowed to proceed for 14 h, followed by three washes with DMF. The Fmoc protecting group at the N terminus was removed, and the peptide was cleaved from the resin, along with the side-chain protecting groups (see SI Appendix for the full procedure).

Peptide Purification.

Crude peptides were purified with RP-HPLC as previously described (25). Briefly, a semipreparative Phenomenex Kinetex C18 column (5 µm, 10 mm × 250 mm, 110 Å) was used for preparative RP-HPLC work, while an analytical Phenomenex Kinetex C18 column (5 µm, 4.6 mm × 250 mm, 110 Å) was used for analytical RP-HPLC work. Standard RP-HPLC conditions were as follows: Flow rates = 5 mL⋅min⁻¹ for semipreparative separations and 1 mL⋅min⁻¹ for analytical separations; mobile phase A = 18 MΩ water + 0.1% trifluoro acetic acid (TFA); mobile phase B = acetonitrile (ACN) + 0.1% TFA. Purities were determined by integration of peaks with UV detection at 220 nm. Preparative HPLC methods were used to separate the crude peptide mixture into different chemical components using a linear gradient (first prep 5% B → 45% B over 40 min, and second prep 20% B → 30% B over 30 min). Then, an analytical HPLC method was used to quantify the purity of the desired product using a linear gradient (5% B → 95% B over 27 min). Only peptide fractions that were purified to homogeneity (>95%) were used for the biological assays. High-resolution MS was used to validate the presence of synthesized peptides. The observed mass-to-charge (m/z) ratio of the peptide was compared to the expected m/z ratio for each peptide (SI Appendix, Table S1).

Biological Reagents and Strain Information.

All standard biological reagents were used as previously described (25). Briefly, standard biological reagents were purchased from Sigma-Aldrich and used according to enclosed instructions. Donor horse serum (defibrinated) was purchased from Sigma-Aldrich and stored at 4 °C until use in bacterial growth conditions. To examine the ability of the synthesized CSP analogs to modulate the ComD receptors, and thus the competence regulon in S. pneumoniae, beta-galactosidase assays were performed using D39pcomX::lacZ (group I) and TIGR4pcomX::lacZ (group II) reporter strains, while phenotypic assays were performed using D39 (group I) and TIGR4 (group II) strains.

Bacterial Growth Conditions.

Bacteria were grown as previously described (25). Briefly, frozen stocks were created from 1.5-mL aliquots of pneumococcal bacteria (0.2 optical density [OD] 600_nm) in Todd Hewitt Broth (THB) supplemented with 0.5% yeast extract (THY) and 0.5 mL of glycerol, and stored at −80 °C. For experiments, frozen stocks were streaked onto a THY agar plate containing 5% serum and chloramphenicol at a final concentration of 4 µg/mL. The plate was incubated for 8 to 9 h in a CO₂ incubator (37 °C with 5% CO₂). Fresh colonies (single colony for D39pcomX::lacZ; multiple colonies for TIGR4pcomX::lacZ) were transferred to 5 mL of THY broth supplemented with chloramphenicol at a final concentration of 4 µg/mL, and the culture was incubated in a CO₂ incubator overnight (15 h). Overnight cultures were then diluted (1:50 for D39pcomX::lacZ; 1:10 for TIGR4pcomX::lacZ) with THY, and the resulting solution was incubated in a CO₂ incubator for 3 to 4 h, until the bacteria reached exponential stage (0.30 to 0.35 for D39pcomX::lacZ; 0.20 to 0.25 for TIGR4pcomX::lacZ) as determined by using a plate reader.

beta-Galactosidase Assays.

All beta-galactosidase assays were performed as previously described (25).

NMR Sample Preparation.

Peptide samples for structural NMR experiments were prepared as previously described (62). Briefly, the peptides were dissolved in 250 mM deuterated dodecyl phosphocholine (DPC-d₃₈; CDN Isotopes) in a PBS buffer solution with 10% D₂O (Cambridge Isotope Laboratories). The final concentration of the peptides was 1.9 mM. PBS buffer solution was a water solution that contained NaCl (137 mM), KCl (2.7 mM), Na₂HPO₄ (10 mM), and KH₂PO₄ (1.8 mM), and the pH was adjusted to 7.4.

NMR Spectroscopy.

All NMR spectra were recorded as previously described (62). Briefly, NMR spectra were recorded on a Bruker 900-MHz spectrometer at 298 K. Spectra were processed using NMR Pipe software. Chemical shifts were referenced to water at 4.771 parts per million (ppm). We acquired the following 2D homonuclear experiments: gradient selection correlation spectroscopy (COSY) with presaturation, total correlation spectroscopy (TOCSY) with decoupling in the presence of scalar interactions (DIPSI) spinlock, and the 3-9-19 or excitation sculpting with gradient water suppression schemes, ¹H-¹⁵N fast heteronuclear single-quantum correlation spectroscopy (HSQC), sensitivity-enhanced ¹H-¹³C HSQC with selective 180° ¹³C pulses, nuclear Overhauser effect spectroscopy (NOESY) with flip-back and Watergate water suppression and a 200-ms mixing time, and rotating frame NOESY (ROESY) with continuous wave spin lock and 3-9-19 water suppression and a 200-ms mixing time. The COSY experiments were collected with 1,024 and 2,048 complex and real data points in the direct and indirect dimensions, respectively, with four scans per data point. The TOCSY and ROESY experiments were acquired with 1,024 direct and 512 indirect complex data points, with 80-ms and 200-ms spin lock durations, and with 8 and 16 scans per data point, respectively. The ¹H-¹⁵N and ¹H-¹³C HSQC experiments were collected with 1,024 complex data points and 13-ppm spectral width in the direct dimensions and 128 complex data points with 28-ppm spectral width and 256 complex data points and 150-ppm spectral width in the indirect dimension, with 8 and 16 scans per data point, respectively. A 1-s relaxation delay was used in all experiments except TOCSY (relaxation delay of 1.2 s). Excitation sculpting with gradient water suppression was used in the ¹H 1-D experiment, and 16,000 real data points were acquired, with eight scans per data point.

Spectra Assignment and Structure Calculation.

NMR spectra were analyzed as previously described (62). Briefly, all spectra were analyzed with National Magnetic Resonance Facility at Madison (NMRFAM)-SPARKY (71). Assignment of resonances for each peptide was achieved using the standard sequential assignment methodology. The volumes of the NOE peaks were calculated by SPARKY and converted into a continuous distribution of interproton distance restraints, with a uniform 35% distance error applied to take into account spin diffusion (see SI Appendix, Table S7 for total number of NOEs divided into backbone vs. side chain for each peptide). The 2D ¹H-¹⁵N and ¹H-¹³C HSQC experiments allowed assigning all backbone (and C^β) atoms, which were subsequently used as input in the TALOS-N program to generate backbone dihedral angle restraints (ϕ/ψ) and side-chain chi1 angle restraints (see SI Appendix for complete restraint tables used for structural calculations) (72). The 3D structure calculations and refinements made use of the torsion angle molecular dynamics and the internal variable dynamics modules of Xplor-NIH (v. 2.42) (73). Backbone and heavy atom rmsd values were calculated for the 20-structure ensembles of all peptides using the entire peptide sequence (SI Appendix, Table S7). PyMOL was used for visual analysis and presentation of the peptide structures.

Stability Assay.

To study the enzymatic stability of the CSP1 analogs, the peptides were dissolved in aqueous PBS solution (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄; pH 7.4) to afford a final concentration of 0.066 mM. Trypsin and chymotrypsin (25 µg⋅mL⁻¹) stock solution (diluted from a 2.5 mg⋅mL⁻¹ trypsin solution; Gibco) was made in PBS solution. Protease solution was added to the peptide solution to afford a final concentration of 0.05 µg⋅mL⁻¹, and then the solution was incubated with shaking at 37 °C. Aliquots (500 µL) of peptide solution were taken and mixed with ACN/H₂O (1:1, 100 µL) for 0, 1, 2, 3, 4, 5, 6, and 24 h, and then analyzed immediately for peptide degradation by analytical RP-HPLC. During the analytical RP-HPLC runs, the degradation products were manually collected and analyzed by MALDI-TOF MS.

BMRB Accession ID.

BMRB accession ID codes are as follows: CSP1-cyc(K6D10), 30593; CSP1-cyc(Orn6D10), 30594; CSP1-cyc(Dab6E10), 30595; CSP1-cyc(Dap6E10), 30601; and CSP1-E1A-cyc(Dap6E10), 30690. Data is available at: http://www.bmrb.wisc.edu and include atomic coordinates, assigned chemical shifts, experimental restraints used for structure determination and refinement, and NOESY peak lists.

Hemolysis Assay.

Hemolytic assays were performed as we have published (60, 61, 68). Briefly, pneumococcal strains were cultured in THB to OD600_nm of 0.2 and then treated with 50 nM CSP1 or CSP2 with increasing concentration of dnCSPs. Untreated controls were exposed to same volume of sterile PBS. After 20 min of incubation, supernatants were collected by pelleting pneumococcal cells at 3,000 × g for 15 min, and cell-free supernatants were filtered through 0.22-µm syringe filters (Millipore). Twofold serial dilutions of the supernatants were performed with fresh THB before incubating with 10 mM dithiothreitol at room temperature for 15 min. Then, a 500-µL aliquot of each sample was mixed with 200 µL of 2% sheep red blood cells and incubated for 30 min at 37 °C. The mixture was centrifuged at 3,000 × g for 5 min. The absorbance of the cell-free supernatant was measured at OD 541_nm. The hemolytic units were reported as the highest dilution that could lyse 50% of the red blood cells.

Mouse Toxicity Studies.

CD1 mice (five per cohort) were intratracheally inoculated with 50 µg/d of native CSP (CSP1 and CSP2) or dnCSPs (CSP1-E1A, CSP2-E1Ad10, CSP1-E1A-cyc(Dap6E10)) for 1 wk. Systemic toxicity was assessed by performing blood chemistry and CBC with differential at the University of Illinois College of Veterinary Medicine Clinical Pathology Laboratory. Histopathology of major organs (lungs, hearts, livers, kidneys, and spleens) and sternum (for genotoxicity) were analyzed at the University of Illinois College of Veterinary Medicine Comparative Pathology Laboratory, as we have previously published (69). Mouse organs were fixed with 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with haemotoxylin and eosin (H&E). Proinflammatory activities in the lungs were photographed using an Olympus DP70 light microscope.

Mouse Model of Acute Pneumonia.

Mouse studies were performed as previously described (60, 61, 68, 74), according to the recommendations in Guide for the Care and Use of Laboratory Animals of the NIH (75). The protocol was approved by the Institutional Animal Care and Use Committee at the University of Illinois at Urbana–Champaign (Protocol Number 18135). For the imminent death acute pneumonia studies, 6-wk-old CD1 mice of both sexes (15 to 20 per cohort) (Charles River Laboratories, Inc.) were acclimated for 7 d in positively ventilated microisolator cages with automatic recirculating water, located in a room with laminar, high-efficiency particle accumulation-filtered air. All mice received autoclaved food, water, and bedding. Animals were anesthetized with isoflurane and intranasally administered 5 × 10⁶ CFU pneumococcal cells (in 50 μL). Two hours after infection, mice were intranasally instilled with 100 μg of dnCSPs. Control cohorts received 50 μL of sterile PBS. Mice were monitored every 4 to 12 h for 144 h. Moribund animals that displayed rough hair coat, hunched posture, distended abdomen, lethargy, or inability to eat or drink were considered dead and euthanized. The survival analyses were performed by using the Kaplan−Meier Log Rank (Mantel−Cox) survival test.

Statistical Analysis.

Statistical significance of all data was analyzed by using the GraphPad Prism 5.0 package. Data are shown as the mean ± SEM. For two groups, statistical significance was determined by the two-tailed Student’s t test. To determine the data with three or more groups, two-way ANOVA with Tukey’s multiple comparisons test was used. Mouse survival was compared using the Kaplan−Meier Log Rank (Mantel−Cox) survival test. P < 0.05 was considered statistically significant.

SI Appendix.

Full details of peptide synthesis and characterization, dose response curves for CSP analogs, tables of resonance assignments, and additional structural figures are provided in SI Appendix.