Paenipeptin C′ (C8-pat) preparation
Paenipeptin C′ was synthesized by solid-phase peptide synthesis (SPPS) and purified by HPLC to homogeneity (> 95% purity) through custom peptide service (Genscript Inc., Piscataway, NJ). The accurate mass of paenipeptin C′ was determined using liquid chromatography mass spectrometer (Agilent 6210 Time-of-Flight, Agilent Technologies, Santa Clara, CA). The theoretical mass of paenipeptin C′ [M + H]+ ion is 1116.7510 m/z, which is consistent with the measured mass of 1116.7518 m/z.
Bacterial strains, growth media, and culture conditions
P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 were obtained from American Type Culture Collection (ATCC, Manassas, VA). Both bacterial strains were cultured in tryptic soy broth (TSB; Becton Dickinson, Sparks, MD) or tryptic soy agar (TSA) at 37 °C aerobically.
Time-kill kinetic assays
Time-kill kinetic assays were used to determine the bactericidal effect of paenipeptin C′ against P. aeruginosa ATCC 27853 and S. aureus ATCC 29213 as described previously . An overnight bacterial culture of P. aeruginosa or S. aureus was diluted (1/100 dilution) into tryptic soy broth. One mL of the diluted P. aeruginosa cells (approximately 106 CFU/mL) was mixed with an equal volume of paenipeptin C' solution in TSB at a final concentration of 8, 16, or 32 μg/mL. Similarly, paenipeptin C' was added to the diluted S. aureus cell suspension (approximately 106 CFU/mL) at a final concentration of 16, 32, or 64 μg/mL. The mixtures were incubated at 37 °C and the number of surviving cells was determined by spread-plating on tryptic soy agar at 0, 2, 4, 6, and 24 h. The colonies on agar plates were counted after 24 h of incubation at 37 °C. The detection limit of quantification in this assay was 10 CFU/mL.
Effect of lipopolysaccharides (LPS) and lipoteichoic acid (LTA) on antimicrobial activity
Purified LPS from the outer membrane of E. coli (Sigma, St. Louis, MO) or LTA from S. aureus (Sigma) was used to measure the possible interaction between paenipeptin C' and LPS or LTA according to previous studies [14, 15]. A stock solution of LPS or LTA was prepared in sterilized water, at 1 mg/mL, and kept at − 20 °C. LPS was added to the P. aeruginosa ATCC 27853 cell suspension (106 CFU/mL) at a final concentration of 0, 10, 25, or 100 μg/mL. Then paenipeptin C′ was added to a final concentration of 16 μg/mL. The mixtures were incubated at 37 °C for 1 h. The number of surviving cells after treatment was determined by spread-plating on tryptic soy agar. Polymyxin B which binds to LPS was used at 2 μg/mL as a positive control. Similarly, the effect of LTA at various concentrations (0, 10, 25, or 100 μg/mL) on the antibacterial activity of paenipeptin C′ at 32 μg/mL was tested against a Gram-positive bacterium, S. aureus ATCC 29213. The inoculum size, incubation conditions, and the methods for surviving cell determination were similar to the procedures as described above in the LPS binding experiment. Nisin, a cationic lantibiotic antimicrobial peptide with known binding ability to LTA , was used at 16 μg/mL as a positive control.
Membrane potential depolarization
Cytoplasmic membrane depolarization after paenipeptin C′ treatment was measured using a fluorescent probe DiSC3(5) as described by Huang and Yousef . An overnight bacterial culture of P. aeruginosa ATCC 27853 or S. aureus ATCC 29213 was diluted 100 times into tryptic soy broth. The diluted cells were incubated at 37 °C with agitation at 200 rpm for approximate 5 h. The resulting cells at early exponential phase were centrifuged at 3660×g at 4 °C for 10 min and washed two times with 5 mM HEPES buffer (Sigma) containing 5 mM glucose (buffer A, pH 7.2). The washed S. aureus cells were resuspended in buffer A to an OD600nm of 0.05. Conversely, P. aeruginosa cells were resuspended to an OD600nm of 0.05 in a solution composed of buffer A and 0.2 mM EDTA (buffer B); the chelating agent EDTA in buffer B can facilitate the uptake of the DiSC3(5) probe into cells of Gram-negative bacteria. Then the cell suspension (20 mL) was mixed with 10 μL of 100 μM fluorescent probe DiSC3(5). The mixture was incubated for 15 min at room temperature in dark to allow the uptake of the DiSC3(5) probe. After incubation, KCl was added to both cell suspensions at a final concentration of 100 mM. Aliquots (90 μL) of the cell suspension were transferred to wells of a black NBS microplate (Corning Inc., Corning, NY), followed by adding paenipeptin C' at various final concentrations (0–128 μg/mL). A change in fluorescence due to membrane depolarization was recorded using a Cell Imaging Multimode Reader (Cytation 3, BioTek, Winooski, VT) at excitation and emission wavelengths of 493 nm and 516 nm, respectively.
Intracellular potassium release assay
Potassium ions released from paenipeptin C'-treated bacterial cells were determined using a K+-sensitive fluorescence probe (PBFI; Invitrogen) . Bacterial cells of P. aeruginosa ATCC 27853 or S. aureus ATCC 29213 were prepared, washed and resuspended in HEPES buffer with 5 mM glucose (buffer A, pH 7.2) as described earlier, and the cell density was adjusted to an OD600nm of 0.3. The potassium-sensitive probe, PBFI, was added to the cell suspensions at a final concentration of 2 μM. Ninety μL of the cell suspension in buffer A were added to wells of a black NBS microplate, followed by adding paenipeptin C′ at various final concentrations (0–128 μg/mL). The fluorescence reading was monitored by a Cell Imaging Multimode Reader (Cytation 3, BioTek) at an excitation wavelength of 346 nm and an emission wavelength of 505 nm.
Transmission electron microscopy (TEM)
An overnight bacterial culture of P. aeruginosa ATCC 27853 or S. aureus ATCC 29213 was 1/10 diluted in sterile saline and treated by paenipeptin C′ at 16 μg/mL or 32 μg/mL, respectively, at 37 °C for 2 h. The treated cells were washed using Hank’s Balanced Salt Solution (HBSS) and pelleted by centrifugation at 3660×g at 4 °C for 10 min. The washed cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 20 min. After washing in cacodylate buffer, cells were post-fixed for 30 min in 1% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) with 0.8% potassium hexacyanoferrate (III) (Sigma) in cacodylate buffer. The cells were treated with 1% tannic acid in molecular grade water before dehydrating in a graded ethanol series followed by propylene oxide. The dehydrated cells were embedded in araldite/Embed 812 resins (Electron Microscopy Sciences) and cured for 48 h at 60 °C. Thin sections (50 nm) were collected on 150 mesh copper grids and post-stained with uranyl acetate and lead citrate for imaging at 80 kV with a Technai F20 TEM (Fei, Hillsboro, OR).
For LPS/LTA binding experiments, the bacterial population counts were analyzed and compared. For membrane potential depolarization and potassium release assays, the changes of fluorescence before and after adding paenipeptin C' were recorded and compared. All experiments were conducted at least three times independently. The above data were analyzed using Analysis of Variance (ANOVA) and Tukey’s honest significant difference (HSD) in SPSS Statistics (version 24; SPSS Inc., Chicago, IL).