Peptide synthesis
Thirty-mer peptides corresponding to the functional region helices 2-loop-3 of bovine NK-lysins (NK1: VIIHVTSKVCSKMGLWSILCNQMMKKYLNR, net charge = + 5.0; NK2A: TVIEVASKMCSKMRLLKGLCKSITKRFLRR, net charge = + 7.9; NK2B: TVIEAASKVCGKMGPLKGLCKSITKRFLRR, net charge = + 6.9; NK2C: TVIEEASKVCSKMRLLKGLCKSIMKKFLRT, net charge = + 5.9 and a scrambled version of NK2A (Sc-NK2A): MFVSLSLATIRLTGKSKCKKERCMILKRRV, net charge = + 7.9) were synthesized by FMOC solid-phase peptide synthesis chemistry, aliquoted (1 mg/tube), and supplied as trifluoroacetate salt with over 95% purity (Peptide 2.0 Inc., Chantilly, VA). Lyophilized peptides were dissolved in Dulbecco’s phosphate-buffered saline (PBS, pH 7.2), aliquoted, and stored at − 20 °C until used.
Peptide concentration determination
Concentrations of all NK-lysin peptides and Sc-NK2A were determined by quantitative amino acid analysis at the Protein Chemistry Laboratory, Texas A&M University and the Center for Biotechnology, University of Nebraska-Lincoln.
Animals and ethics
The authors complied with the ARRIVE guidelines. All animal procedures were conducted in strict accordance with the Animal Welfare Act, and all procedures were approved by the Institutional Animal Care and Use Committee at the National Animal Disease Center, Ames, IA. Blood samples collected from 4 years old healthy two Holstein cows from National Animal Disease Center were used to isolate red blood cells.
Mouse study
To determine whether NK-lysin peptides show in vivo cytotoxic effects, a mouse study was conducted. Briefly, thirty 5–6 weeks old female Swiss-Webster mice (Envigo, Indianapolis, IN) were housed in groups in six individual cages (5 mice per cage) in ABSL2 facilities with ad libitum access to food and water in a 12-h light:dark cycle. After 1 week of acclimation, mice were intraperitoneally injected with a single dose of 100 µg of NK-lysin peptides (NK1, NK2A, NK2B, NK2C, or Sc-NK2A, ~ 4 mg/kg of body weight) in PBS (1 mL). Mice injected with PBS (1 mL) served as a negative control for the study. Mice were daily observed for any signs of toxicity and were euthanized 2 weeks post peptide administration.
Circular dichroism (CD) analysis
CD analysis of all four bovine NK-lysin peptides were previously described24. Therefore, CD analyses were performed here using the same methods for NK2A and Sc-NK2A in potassium phosphate buffer and liposomes using Jasco J-815 CD spectrophotometer (Jasco, Easton, MA).
Hemolytic activity assay
The hemolytic activity of all four bovine NK-lysin peptides, as the amount of hemoglobin released by the lysis of cattle red blood cells (RBCs), were previously determined20. Therefore, only hemolytic activity of Sc-NK2A was compared with NK2A (final concentrations for both peptides at 2, 5, 10, and 20 µM) using cattle RBCs in this study as described previously20. Triton X-100 and PBS were used as positive and negative control for the assay, respectively.
Nanoparticle synthesis
SA monomer was purchased from Sigma Aldrich (St. Louis, MO). CPH and CPTEG monomers were synthesized as previously described52,53. The 20:80 CPTEG:CPH, 20:80 CPH:SA, and 10:90 CPTEG:SA copolymers were synthesized by melt condensation to number average molecular weights of approximately 7.5, 17.5, and 17.5 kDa, respectively49,52,53,54, and composition and molecular weight were confirmed by 1H NMR analysis using a Varian MR-400 (Varian, Inc., Palo Alto, CA). Nanoparticles were synthesized by flash nanoprecipitation29. Nanoparticles were collected by vacuum filtration, scanning electron microscopy (SEM, FEI Quanta 250, Hillsboro, OR) was used to image all formulations, and size distributions were calculated using Fiji image analysis software55. Nanoparticle Zeta potential was measured (see below) using a Zetasizer Nano (Malvern Instruments Ltd., Worcester, UK).
Nanoparticle release kinetics
Nanoparticle release kinetics and encapsulation efficiency were assayed in PBS, pH 7.4 as previously described29,30. Released NK2A peptide concentrations were quantified by micro bicinchoninic acid (microBCA) assay (Pierce Biotechnology, Rockford, IL). Kanamycin was quantified by o-pthalaldehyde derivatization56 (Fluoraldhehyde crystals, ThermoFisher Scientific, Carlsbad, CA) followed by absorbance spectroscopy at 340 nm (SpectraMax M3, Molecular Devices, San Jose, CA). Drug release kinetics are presented as fraction released, where the cumulative payload mass released is normalized by the total encapsulated payload mass.
MDR Salmonella isolates and culture conditions
MDR Salmonella outbreak isolates (SX238, SX231, and SB395) as well as a non-MDR Salmonella isolate (SB377) used in this study are listed in Table 1. Bacterial freezer stocks were streaked individually onto Luria–Bertani (LB Lennox) agar (Sigma Aldrich) and incubated in a 37 °C incubator overnight. Bacterial cultures were prepared by growing each isolate from a single colony in 3 mL LB Lennox broth (ThermoFisher Scientific, Wilmington, DE) overnight at 37 °C in a shaker-incubator at 200 rpm.
MDR Salmonella-NK-lysin peptide susceptibility assays
Overnight cultures of a MDR Salmonella outbreak isolate (SX238) and one non-MDR Salmonella isolate (SB377) were diluted 1:1000 in LB broth to obtain approximately 1 × 106 colony forming units per milliliter (CFU/mL). One hundred microliters of each diluted culture were transferred into honeycomb® 100-well plates (~ 1 × 105 CFU/well) (Growth Curves USA, Piscataway, NJ). Bovine NK-lysin peptides (NK1, NK2A, NK2B, and NK2C) were added at the final peptide concentrations of 20, 10, 5, 3.5, 2 µM in PBS (total volume = 20 µL) with a final bacterial-peptide volume of 120 µL/well. The plates were covered with lids and placed in an automated growth curve reader (Bioscreen C; Growth Curves USA, Piscataway, NJ) which was programmed for continuous shaking at 37 °C, and optical density readings at 600 nm (OD600) were recorded every hour for 24 h. Bacteria incubated with kanamycin and PBS were used as positive and negative control for the assay, respectively. The optical density data were analyzed, and growth curve graphs for each isolate were generated using MS Excel. Because NK2A peptide showed the strongest Salmonella growth inhibition, this peptide was selected for further studies. Twenty-four-hour growth curve assays for Salmonella isolate SX238 were repeated at different bacterial CFU (~ 1 × 105, 1 × 106, 1 × 107, and 1 × 108 CFU/mL) with NK2A (5 µM and 10 µM). Sc-NK2A was included as a control for the assay.
The bactericidal effect of NK2A peptide against MDR Salmonella (SX238) was also determined. Overnight SX238 culture was centrifuged (10,000 rpm for 2 min) and diluted 1:1000 in LB broth and/or PBS to obtain ~ 1 × 106 CFU/mL; 100 µL of diluted bacterial culture was placed in a Honeycomb plate (~ 1 × 105 CFU/well), NK2A (20, 5, 2 and 0 µM) was added and incubated at 37 °C in the reader with continuous shaking for 3 h. Bacteria in each well was 10 × serially diluted in LB broth, 10 µL was plated on Xylose-Lysine-Tergitol 4 (XLT4) (ThermoFisher) agar plates and incubated at 37 °C overnight. Bacterial colonies were enumerated for each NK2A dilution.
Antimicrobial activity of NK2A (final concentration ~ 5 µM) encapsulated nanoparticles (CPTEG:CPH, CPH:SA and CPTEG:SA) were tested against Salmonella isolate SX238 as previously described32. To prepare 10 µM NK2A stock solutions, required amounts of nanoparticles was measured, added into 1 mL PBS and briefly sonicated as previously described32. One hundred microliters of NK2A nanoparticles and 100 µL of diluted bacterial culture in PBS was added into a Honeycomb plate (~ 1 × 105 CFU/well) and incubated at 37 °C in the reader with continuous shaking for 3 h. Bacteria in each well was 10 × serially diluted in LB broth, 10 µL was plated on XLT4 agar plates and incubated at 37 °C overnight. Kanamycin (final concentration = 16 µg/mL) encapsulated and empty nanoparticles were used as positive and negative control for the assay, respectively. Bacterial colonies were enumerated for each nanoparticle treatment.
The percentage of bacterial killing was calculated using the following Eq. (1 − [average number of live bacteria in NK2A-treated sample/average number of live bacteria in the negative control sample] × 100). The antimicrobial assays for Salmonella isolates were repeated at least three times in duplicate wells.
Zeta potential measurements
Salmonella isolates were prepared for Zeta potential measurements via the diffusion barrier technique as previously described but with some minor modifications35. Briefly, overnight cultures of Salmonella in LB broth were centrifuged (10,000 rpm for 2 min), washed twice in Milli-Q water and resuspended in Milli-Q water (~ 1 × 109 CFU/mL). Bacteria were then diluted 1:1000 in Milli-Q water supplemented with 10% PBS, then transferred to clear disposable folded capillary Zeta cells filled with 10% PBS via gel loading pipette tip (Malvern Instruments Ltd). The Zeta potential measurements (mV) of bacterial isolates were immediately measured using a Zeta potential analyzer equipped with He–Ne 633 nm laser (Zetasizer Nano, Malvern) at a constant voltage of 150 V at room temperature. The Zeta potential changes of Salmonella isolates following incubation with NK2A were also recorded. Bacterial isolates (~ 106 CFU in 1 mL Milli-Q water) were incubated with 2 µM NK2A (in PBS) at 37 °C for 20 min. Capillary Zeta cells were washed with Milli-Q water between samples and refilled with Milli-Q water containing 10% PBS before measuring the Zeta potentials. Zeta potential changes of one Salmonella isolate (SB395) was also similarly tested with Sc-NK2A (2 µM) peptide. This method was also used for assessing NK-lysin peptide Zeta potentials. A total of five Zeta potential measurements of 40 runs each with 30 s cooling cycles for each sample was performed.
Confocal laser scanning microscopy (CLSM)
The viabilities of bacteria following incubation with NK2A and Sc-NK2A were determined using culture-independent (function of the membrane integrity of the bacteria) staining technique by using LIVE/DEAD™ BacLight™ bacterial viability kit (for microscopy) as described previously20. Bacteria in each treatment was visualized using a Nikon A1R + laser scanning confocal microscope (Nikon Instruments, Melville, NY) as described previously20. Images were obtained using plan Apo 60 × objective lens (oil) at numerical aperture 1.4. Final figures were prepared using Adobe Photoshop Elements 11.
Transmission electron microscopy (TEM)
Morphological changes of bacteria following incubation with NK2A and Sc-NK2A peptides were studied by TEM. An overnight culture of Salmonella (isolate SX238) was centrifuged and resuspended in PBS. One hundred microliters of bacterial suspension were placed in a Honeycomb plate (~ 1 × 107 CFU/well) and NK2A, Sc-NK2A (20 µM), and PBS were added and incubated at 37 °C with continuous shaking for 1 h. The bacterial suspensions were mixed with an equal volume of 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) and processed for TEM as described before24. Sections were examined with a FEI Tecnai G2 Biotwin (FEI Co., Hillboro, OR) TEM and images were taken with Advanced Microscopy Technologies (AMT Inc., Danvers, MN) imaging camera.
Propidium iodide (PI) uptake assay
The kinetics of NK2A-induced damage to Salmonella cell membranes were evaluated using PI uptake assay as described previously24. Briefly, an overnight culture of MDR Salmonella outbreak isolate SX238 was centrifuged, washed with PBS, and resuspended in PBS. Ninety microliters of bacterial suspension was transferred to a 96-well opaque plate (~ 1 × 107 CFU/well), mixed with 10 µL of PI (final concentration = 3 µM) and incubated at 37 °C for 5 min. PBS, polymyxin B (2, 4, 25, 50 and 100 µg/mL), NK2A or Sc-NK2A (both at 2 and 5 µM) were added and PI fluorescent intensities in each well was continuously measured (15 s intervals, flex mode) using a FlexStation 3 multi-mode (fluorescent) microplate reader (Molecular Devices LLC., San Jose, CA) for up to 6 h. The excitation and emission wavelengths for PI was set up at 530 nm and 620 nm, respectively.
NK2A-Lipopolysccharides (LPS) binding studies
The binding of NK2A to Salmonella LPS was studied by growth curve assay and TEM. Five micromolar NK2A (~ 2.2 µg/well) was preincubated with different amounts of Salmonella enterica serotype Typhimurium LPS (Sigma; LPS = 0, 0.5, 1.1, 2.2, 4.4, 8.8 µg/well, final volume of LPS and NK2A = 20 µL) at 37 °C in a honeycomb plate with continuous shaking for 30 min. Overnight Salmonella (isolate SX238) culture was diluted to ~ 1 × 106 CFU/mL and 100 µL was transferred into wells containing NK2A-LPS and further incubated at 37 °C with continuous shaking for 24 h. The optical density (OD600) data were analyzed, and growth curve graphs were generated.
The direct binding of NK2A to Salmonella LPS/membranes was also examined by TEM using gold nanoparticle-conjugated NK2A (NK2A-AuNPs). Ten nanometer (diameter) NHS-activated gold nanoparticles (AuNPs) were conjugated to NK2A as described by the manufacturer (Cytodiagnostics Inc., Burlington, ON, Canada). One hundred microliters of Salmonella isolate SX238 was placed in a Honeycomb plate (~ 1 × 107 CFU/well) and incubated with NK2A-AuNPs (20 µM) or AuNPs (without NK2A) at 37 °C with continuous shaking for 15–45 min. Bacterial samples were fixed with 3% glutaraldehyde and processed for TEM as described above.
Statistical analyses
The data analysis for this paper was generated using SAS® software, Version 9.4 of the SAS System for Windows. Copyright © 2002–2012. SAS Institute Inc. SAS and all other SAS Institute Inc. product or service names are registered trademarks or trademarks of SAS Institute Inc., Cary, NC, USA. When significant, mean comparisons were performed using the predicted differences (PDIFF) option. Significant differences were established at P ≤ 0.05.
