Disc3 5

After the exposure of vegetative cells to various micromolar concentrations of CXCL10 or CTTC, membrane depolarization was measured by a modification of a previously published protocol (59 (link)). Controls are discussed in Text S2C in the supplemental material. Briefly, B. anthracis cells were grown to mid-log phase as described above. Cells were collected and resuspended in respiration buffer, and 0.1 mM diSC3-5 (AnaSpec, Fremont, CA) was added to cell aliquots. Initial baseline fluorescence (F₀) readings, test antimicrobial fluorescence (F) readings, and maximum depolarization (F_M) readings were collected every 5 s for 150 to 300 s. Using the initial (F₀), test (F), and final, maximum fluorescence readings (F_M), the values of each group were averaged and the following calculation was used to determine percent depolarization by the test molecule compared to complete depolarization by melittin: % Depolarization = [(F − F₀)/(F_M − F₀)] × 100 (59 (link)). In parallel with the above-described depolarization experiments, the numbers of CFU per milliliter were determined to measure bacterial survival (see Text S2C).

Levofloxacin (LVFX), N-methylmorpholine (NMM) and resazurin were purchased from Sigma-Aldrich, Co. LLC (St. Louis, MO, USA). Bicinchoninic acid (BCA) and SYTOX Green were purchased from ThermoFisher Scientific, Inc. (Waltham, MA, USA). Fluoro-N,N,N′,N′-tetramethylformamidinium hexafluorophosphate (TFFH) was purchased from EMD Millipore (a division of Merck KGaA; Darmstadt, Germany), Fmoc-Gly-Wang resin from Peptides International, Inc. (Louisville, KY, USA), and DiSC(3)5 from Anaspec, Inc. (Fremont, CA, USA). Deionized water (dH₂O) was prepared using a Milli-Q Synthesis A10 system (Millipore). The bacterial strains E. coli (#25922) and B. cereus (#11778) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Pep-4 (≥95% purity) was custom synthesized by Genscript USA, Inc. (Piscataway, NJ, USA). Melittin (≥95%) was purchased from Anaspec, Inc., and indolicidin was synthesized (≥95% purity) by AAPPTec, LLC (Louisville, KY, USA). Peptide identities were verified via matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry using a Shimadzu AXIMA Performance. Additionally, Pep-4 and the conjugate were submitted for amino acid analysis (UC Davis Genome Center Proteomics Core Facility) in order to establish molar concentrations.

Membrane potential was monitored using the fluorescent dye DiSC₃(5) (3,3′-dipropylthiadicarbocyanine iodide) (Anaspec, Fremont, CA, USA) according to the following procedures [38 (link)]. S. aureus ATCC25923 was grown to mid-logarithmic phase (0.5 to 0.6 optical density at 600 nm) in MHBc at 37 °C. Cells were collected through centrifugation (5500× g, 10 min) and washed with buffer (5 mM HEPES, 5 mM glucose, pH 7.2). Following resuspension in the same buffer (optical density of 0.05 at 600 nm), DiSC₃ (5) and KCl were added to final concentrations of 0.4 μM and 100 mM, respectively. The mixture was incubated until stabilization of fluorescence and treated with 4× the MICs of lactoquinomycin A (MIC = 0.06 μg/mL) and nisin (MIC = 4 μg/mL), using the latter as a positive control and DMSO as a negative control. Fluorescence was measured using a microplate reader FLx800 (BioTek, Winooski, VT, USA) at wavelengths of 622 nm for excitation and 670 nm for emission.