The animal experimental procedures were approved by the Ethics Committee of Animal Care and Welfare of the Institute of Medical Biology, Chinese Academy of Medical Sciences (CAMS) & Peking Union Medial College (PUMC) (Permit Number: SYXK (dian) 2010-0007), in accordance with the animal ethics guidelines of the Chinese National Health and Medical Research Council (NHMRC) and the Office of Laboratory Animal Management of Yunnan Province, China. All efforts were made to minimize animal suffering.

All participants submitted a signed informed consent form to participate in the study. The protocol complied with the Helsinki Declaration and was approved by the Institutional Review Board of the Institute of Medical Biology, CAMS & PUMC.

Female BALB/c mice (6–8 weeks old, 16–18 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd., and raised and maintained in the Central Animal Care Services of our institute under specific pathogen-free (SPF) conditions. Mice were anesthetized, and blood samples were collected by cardiac puncture. After being kept at 37°C for 1 h and then at 4°C overnight, blood samples were centrifuged at 3,000 × g for 15 min, and sera were collected. A portion of the serum was inactivated by boiling in a water bath for 20 min. Escherichia coli strain DH5α was grown in Luria-Bertani (LB) medium at 37°C with constant shaking at 220 rpm overnight to reach the middle of the logarithmic growth phase and diluted with LB to 3 × 10⁵ CFU/mL before use. For tests, cathelicidin-BF (purity ≥95%, synthesized by GL Biochem Ltd, Shanghai, China) was dissolved in sterile deionized water and mixed with serum, inactivated serum or sterile deionized water at a volume ratio of 1:4 to achieve a final concentration of 2 mg/mL. After incubation at 37°C, aliquots were taken at each time point, and the MIC for DH5α was taken as the lowest peptide concentration at which no microbial growth was observed visually after 18 h of incubation at 37°C.

Cell viability was measured using cell proliferation kit II (XTT) (Roche). Cells, including A549 (adenocarcinomic human alveolar basal epithelial cells), 293FT (human embryonic kidney cells) and L929 (murine fibroblast cells), were cultured in Dulbecco's minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, 100 U penicillin/mL, and 100 μg streptomycin/mL in a humidified 5% CO₂ atmosphere at 37°C. After digestion with trypsin, the cells were diluted in serum-free DMEM without phenol red to a final concentration of 2 × 10⁵ cell/mL, seeded in 96 well plates (100 μl/well) and cultured overnight until adhesion. Cathelicidin-BF dissolved in serum-free DMEM without phenol red was added to wells, and the plates were incubated for 24 h as previously described. Subsequent procedures were performed according to the kit. Briefly, XTT labeling reagent (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis-4-methoxy-6-nitro) benzene sulfonic acid hydrate) in Roswell Park Memorial Institute (RPMI) 1640 medium without phenol red was mixed with electron-coupling reagent PMS (N-methyl dibenzopyrazine methyl sulfate) at a volume ratio of 50:1 to make the working solution. Fifty microliters of the XTT labeling mixture was added to each well and incubated at the same conditions for 6 h. Absorbance [A492 nm-A690 nm] stands for the quantification of viable cells.

For the hemolysis assay, blood samples from mice were mixed with Alsever's solution (8 g/L sodium citrate, 0.55 g/L citric acid, 20.5 g/L glucose, 4.2 g/L NaCl, pH 6.1) at a volume ratio of 1:5, centrifuged at 1,000 × g for 10 min, and washed three times with 0.9% saline. Erythrocytes were suspended in 0.9% saline at a volume ratio of 1:50. Cathelicidin-BF dissolved in 0.9% saline was added and incubation continued at 37°C for 30 min. Then, the samples were centrifuged at 1,000 × g for 15 min, the supernatants were diluted four times with 0.9% saline, and the absorbance at 540 nm was measured. Using 1% Triton X-100 (v/v) to determine 100% hemolysis and 0.9% saline as the negative control, the hemolysis rate of cathelicidin-BF is expressed as [(Absorbance _sample-Absorbance _control)/(Absorbance_100%-Absorbance _control)] × 100.

One MDR clinical isolate, P. aeruginosa 1409, was identified with a Vitek 32 system (bioMerieux, France) and further verified by sequencing of 16s rDNA with universal primers 27f and 1492R. For tests, the bacteria were incubated in LB at a concentration corresponding to an OD₆₀₀ value of 0.5, and then cathelicidin-BF was added to 100 μL of culture to obtain final concentrations of 4 × MIC (32 μg/mL) or ¼ × MIC (2 μg/mL). Levofloxacin (Tokyo Chemicals Industry Co. Ltd.,) with final concentrations of 4 × MIC (64 μg/mL) or ¼ × MIC (4 μg/mL) was used as a control. After 1 h of incubation at 25°C, the culture was centrifuged at 3,000 × g for 5 min and resuspended in PBS (phosphate-buffered saline, 8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na₂HPO₄, 0.24 g/L KH₂PO₄, pH 7.4). Then, PI was added to a final concentration of 10 μg/mL. After 30 min of incubation at 25°C, the cells were washed 3 times with PBS and immediately imaged with a fluorescence microscope (Olympus, Japan) (Yu et al., 2015).

One MDR clinical isolate, A. baumannii 1408, was identified with a Vitek 32 system (bioMerieux, France) and further verified by sequencing 16s rDNA with universal primers 27f and 1492R. For tests, bacterial strains A. baumannii 1408 and P. aeruginosa 1409 were incubated in LB at a concentration corresponding to an OD₆₀₀ value of 0.5, and then cathelicidin-BF conjugated with FITC at its N terminus (purity ≥95%, synthesized by GL Biochem Ltd, Shanghai, China) was added to a final concentration of ¼×MIC (i.e., 4 μg/mL for A. baumannii 1408 and 2 μg/mL for P. aeruginosa 1409) and incubated at 25°C for 1 h. The culture was centrifuged at 3,000 × g for 5 min, resuspended in PBS, and incubated at room temperature for 20 min with Hoechst (Sigma) diluted with PBS to a final concentration of 20 μg/mL. Next, the culture was centrifuged at 3,000 × g for 5 min, incubated with SynaptoRed C2 (Tocris Bioscience), diluted with Hank's solution (8 g/L NaCl, 0.4 g/L KCl, 1 g/L glucose, 60 mg/L KH₂PO₄, 47.5 mg/L Na₂HPO₄, pH 7.2) to a final concentration of 20 μg/mL, and maintained on ice for 1 min. Microscopy was performed with excitation and emission wavelengths, respectively, of 488 nm and 530 nm for FITC, 352 nm and 461 nm for Hoechst, and 515 nm and 640 nm for SynaptoRed C2 (Olympus, Japan) (Wang et al., 2015).

Bacterial strains A. baumannii 1408 and P. aeruginosa 1409 were grown overnight in LB medium at 37°C with constant shaking at 220 rpm to reach the middle of their logarithmic growth phase. Cathelicidin-BF was added to a final concentration of 1/2 MIC (i.e., 8 μg/mL for A. baumannii 1408 and 4 μg/mL for P. aeruginosa 1409), and the cultures were incubated at 37°C for 2 h. Samples were collected by centrifugation at 4,000 × g for 5 min at 4°C and washed 3 times with PBS. All samples were homogenized in lysis buffer (4% SDS, 1 mM DTT, 150 mM Tris-HCl, pH 8.0, protease inhibitor). After 5 min incubation in boiling water, the homogenate was sonicated on ice. The crude extract was then incubated in boiling water again and clarified by centrifugation at 16,000 × g at 25°C for 10 min before the supernatants were collected. The protein concentration in the supernatants was determined using the BCA protein assay (Beyotime, China).

Protein digestion was performed based on a filter-aided sample preparation procedure (Wisniewski et al., 2009). The resulting peptide mixtures were labeled with the 4-plex iTRAQ reagent according to the manufacturer's instructions (Applied Biosystems). Briefly, 200 μg of proteins for each sample was incorporated into 30 μl STD buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl, pH 8.0). The detergent, DTT and other low-molecular-weight components were removed using UA buffer (8 M urea, 150 mM Tris-HCl, pH 8.0) by repeated ultrafiltration (Microcon units, 30 kD). Then, 100 μl of 0.05 M iodoacetamide in UA buffer was added to block reduced cysteine residues, and the samples were incubated in darkness for 20 min. The filters were washed with 100 μl UA buffer three times and then with 100 μl DS buffer (50 mM triethylammonium bicarbonate at pH 8.5) twice. Finally, the protein suspensions were digested with 2 μg trypsin (Promega) in 40 μl DS buffer overnight at 37°C, and the resulting peptides were collected as a filtrate. The peptide content was estimated by UV light spectral density at 280 nm. A standard pool comprising a mixture of an equal amount of protein derived from all samples served as an internal control (IS). For labeling, each iTRAQ reagent was dissolved in 70 μl of ethanol, added to the respective peptide mixture, and then multiplexed and vacuum dried.

iTRAQ-labeled peptides were fractionated by SCX chromatography using the AKTA Purifier system (GE Healthcare). The dried peptide mixture was dissolved in 2 mL buffer A (10 mM KH₂PO₄ in 25% acetonitrile, pH 3.0) and loaded onto a Polysulfoethyl column (4.6 × 100 mm, 5 μm, 200 Å, PolyLC Inc.). The peptides were eluted at a flow rate of 1 mL/min with a gradient of 0–10% buffer B (500 mM KCl, 10 mM KH₂PO₄ in 25% acetonitrile, pH 2.7) for 7 min, 10–20% buffer B for 10 min, 20–45% buffer B for 5 min, and 45–100% buffer B for 5 min. The eluates were monitored by absorbance at 214 nm and collected every 1 min. The collections were pooled in groups of 4 fractions and desalted separately on C18 Cartridges (Empore™ SPE Cartridges C18, standard density, bed I.D. 7 mm, volume 3 mL, Sigma). Each final fraction was dried in a vacuum concentrator and reconstituted in 40 μl of 0.1% (v/v) trifluoroacetic acid. All samples were stored at −80°C before the next analysis.

MS experiments were performed on a Q Exactive mass spectrometer that was coupled to a nanoflow HPLC instrument (Easy nLC, Thermo Fisher Scientific). The peptide mixture (5 μg) was loaded onto a C18-reversed phase column (Thermo Scientific Easy Column, 10 cm long, 75 μm diameter, 3 μm resin) in buffer A (0.1% formic acid) and separated in a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min over 140 min, controlled by IntelliFlow technology. MS data were acquired using a data-dependent “top10” method, dynamically choosing the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation. Determination of the target value was based on predictive Automatic Gain Control (pAGC). The dynamic exclusion duration was 60 s. Survey scans were acquired at a resolution of 70,000 at m/z 200, and the resolution for the HCD spectra was set to 17,500 at m/z 200. The normalized collision energy was 30 eV, and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. The instrument was run with peptide recognition mode enabled.

MS/MS spectra were searched using the MASCOT engine (Matrix Science, London, UK, version 2.2.) embedded into Proteome Discover 1.4 (Thermo Electron, San Jose, CA) against Uniprot_A. baumannii, Uniprot_P. aeruginosa and the corresponding decoy databases. Proteins were identified with the following parameters: Peptide mass tolerance = 20 ppm; MS/MS tolerance = 0.1 Da; Enzyme = trypsin; Missed cleavage = 2; Fixed modification: Carbamidomethyl (C), iTRAQ 4plex (K), iTRAQ 4plex (N-term); Variable modification: Oxidation (M). All reported data were based on 99% confidence intervals for protein identification as determined by a false discovery rate (FDR) ≤0.01 (Zheng et al., 2014).

The final ratios of proteins were normalized to the median average protein ratio of a mixture of equal volumes of differently labeled samples. Differentially expressed proteins were specified by a ratio of > ±1.2 and p < 0.05 (Cox and Mann, 2008). Screened proteins were loaded into Blast2GO (Version 2.7.0) for GO mapping and annotation (Ashburner et al., 2000; Gotz et al., 2008). These proteins were also mapped to KEGG pathways for further analysis (Kanehisa et al., 2012). Fisher's exact test was used to calculate p-values, and p < 0.05 indicated GO or KEGG pathways that were significantly enriched in differentially expressed proteins compared to the untreated group (Blüthgen et al., 2005).

Although cathelicidin-BF was stable after incubation in sterile deionized water at 37°C for nearly 24 h, reflected by the fact that the MIC against DH5α increased slightly from 8 mg/L to 16 mg/L, it almost completely lost its antibacterial activity after 1 h incubation in mouse serum, reflected by the fact that the MIC against DH5α increased to more than 128 mg/L (Figure 1). This process was so rapid that cathelicidin-BF lost half of its antibacterial activity immediately after being mixed with mouse serum, reflected by the fact that the MIC against DH5α was 8 mg/L after being mixed with water, while the MIC against DH5α was 16 mg/L after being mixed with serum at incubation time 0. When the serum was heat inactivated, the antimicrobial activity of cathelicidin-BF was relatively stable, as indicated by the slight increase in the MIC against DH5α from 8 mg/L at time 0 to approximately 13.3 mg/L after 6 h of incubation. These results are consistent with previous reports that linear AMPs are unstable in vivo because of endogenous mammalian proteases and proteases secreted by pathogens (Pasupuleti et al., 2009; Li et al., 2012).

Less than 1% hemolysis was observed when cathelicidin-BF was at a concentration of 800 mg/L (Figure 2A). Regarding other cells, cathelicidin-BF inhibited the proliferation of human embryonic kidney 293FT cells at concentrations >50 mg/L (Figure 2B) while leaving the murine fibroblast cell line L929 and adenocarcinomic human alveolar basal epithelial cell line A549 intact at 400 mg/L (Figures 2C, D). Our results are consistent with previous reports that a cathelicidin-BF mutant is toxic to Madin-Daby canine kidney (MDCK) cells at 20 μM (~72 mg/L) and may cause renal injury when applied systemically (Tian et al., 2013).

PI is a DNA-binding fluorescent dye that can penetrate broken membranes but not intact membranes. As shown in Figure 3, neither levofloxacin nor cathelicidin-BF at low concentrations (¼ × MIC) affects the integrity of bacterial membranes, as no red dyes were detected under these conditions. When the concentrations increased to 4 × MIC (a lethal concentration), both levofloxacin- and cathelicidin-BF-treated bacteria showed red fluorescence after incubation with PI, which indicated that the membranes of these bacteria were broken and that PI entered these cells and formed complexes with the DNA inside. Cathelicidin-treated membranes were noticeably more thoroughly disrupted than levofloxacin-treated ones. We are not sure whether these differences are due to the greater efficiency of cathelicidin-BF on bacteria or its potential actions on membranes or DNA compared with that of levofloxacin.

Indeed, cathelicidin-BF passed through bacterial membranes without disruption at low concentrations (Figure 4). Rather than localizing at membranes (stained with SynaptoRed C2 and shown in red in Figure 4, S) as we had previously expected according to the rupture thesis, FITC-tagged cathelicidin-BF (green in Figure 4, F) localized at nuclear regions, which is indicated by its co-localization with DNA (stained with Hoechst and shown in blue in Figure 4, H). Notably, FITC-tagged cathelicidin-BF seems to be more concentrated compared with the distribution of DNA at the nuclear region, suggesting that cathelicidin-BF may act on specific regions of the nucleoid.

The functional interpretation of differentially expressed proteins (Tables S1–S3) was enriched via GO analysis. Interestingly, cathelicidin-BF- and levofloxacin-treated P. aeruginosa shared many GO categories concerning the transcription process (Table 1). These similarities also existed in cathelicidin-BF-treated A. baumannii, with shared processes, including core RNA polymerase binding (Table 2). Notably, both cathelicidin-BF- and levofloxacin-treated P. aeruginosa have their own specific GO categories. For example, GO categories concerning nucleic acids from cathelicidin-BF-treated P. aeruginosa mainly involve RNA (i.e., tRNA 3′-terminal CCA addition, RNA repair, etc.,), while GO categories concerning nucleic acids from levofloxacin-treated P. aeruginosa primarily involved DNA (i.e., primosome complex, replisome, double-strand break repair, etc.,) (Table 3). These results are consistent with the mechanisms of levofloxacin-mediated killing of bacteria (inhibition of two type II topoisomerases, namely, DNA gyrase and topoisomerase IV, which are involved in DNA separation and supercoiling, respectively) and implied that although both molecules target the nucleoid, cathelicidin-BF may have different mechanisms from levofloxacin (Drlica and Zhao, 1997; Ferrandiz and de la Campa, 2014).

KEGG pathway maps represent experimental knowledge on metabolism and various other functions of the cell and organism. Localization of differentially expressed proteins in these pathways may offer hints to how drugs work by interfering with key metabolic activities. As seen in Table 4, differentially expressed proteins from P. aeruginosa after cathelicidin-BF or levofloxacin treatment shared many pathways, including amino acid synthesis and pyrimidine and purine metabolism. Some of these pathways were further confirmed by their existence in cathelicidin-BF treated A. baumannii (KEGG categories with MapIDs are underlined in Table 4). Notably, while some of these pathways were not “enriched” (p > 0.05), they share some differently expressed proteins with other enriched pathways. For example, proteins Q9HVA1 and Q9I3S7 are shared in six pathways, although they are considered enriched in only 4 of them. Q9HVA1 is the acetolactate synthase isozyme III small subunit with functions in branched-chain amino acid biosynthesis and metabolism of cofactors and vitamins. It is overexpressed in both cathelicidin-BF- and levofloxacin-treated P. aeruginosa. Q9I3S7 is a likely decarboxylase that may interact selectively and non-covalently with thiamine pyrophosphate (the diphosphoric ester of thiamine, TPP). It is overexpressed in levofloxacin-treated P. aeruginosa but downregulated in cathelicidin-BF-treated P. aeruginosa. A similar phenomenon occurred with Q9HUU8, the urease subunit gamma that is involved in arginine biosynthesis and purine metabolism.

Unique pathways were identified for each treatment. Q9I5V3, the multifunctional CCA protein, is downregulated in cathelicidin-BF-treated P. aeruginosa and enriched in the RNA transport pathway. Ubiquinone and other terpenoid-quinone biosynthesis pathways were enriched in levofloxacin-treated P. aeruginosa. Q9I298, a putative 3-methylglutaconyl-ConA hydratase, and Q9HTV3, a 3-octaprenyl-4-hydroxybenzoate carboxy-lyase, were downregulated in this pathway. Notably, Q9I5V3 was downregulated in levofloxacin-treated P. aeruginosa, and both Q9I298 and Q9HTV3 were downregulated in cathelicidin-BF-treated P. aeruginosa but they are not categorized as “differentially expressed proteins” according to the criteria set in the method section.

Interestingly, we found one protein, Q9I523, a nucleoside-triphosphate diphosphatase that is involved in both purine and pyrimidine metabolism, that was downregulated in cathelicidin-BF-treated P. aeruginosa. This protein was reported to interact with Era, an essential GTPase identified in various bacteria and some eukaryotes (Zhang and Inouye, 2002). Recently, a study reported that AMPs can kill bacteria by inhibiting E. coli ATP synthase (Azim et al., 2016). Considering that Q9I523 could hydrolyze all eight of the canonical ribo- and deoxynucleoside triphosphates to their respective monophosphates and PP (i), downregulation of this “energy switch” may play an important role in regulating the energy supply of a bacterial or tumor cell.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.