Peptides

Clavanin A, clavanin-MO and cathelicidin LL-37 (Table S1) were synthesized by Shanghai Hanhong Chemical (R.P. of China) using the solid-phase with the N-9-fluorenylmethyloxycarbonyl (Fmoc) strategy and purified by high-performance liquid chromatography (HPLC) (GE, USA)10,34. The identity of each peptide was confirmed by MALDI ToF MS (Bruker, Germany). Peptide purity used in biologic assays was higher than 95%.

Strains and growth conditions

Strains used included clinical isolates Escherichia coli KPC-positive ID N°.181244635, Escherichia coli multiresistant ID N°.210112335 and carbapenemase-producing Klebsiella pneumoniae 1825971 (KPC971), as well as reference strains Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 12953, Staphylococcus aureus ATCC 29213, Methicillin-Resistant Staphylococcus aureus ATCC 33591, Streptococcus pyogenes ATCC 19615, Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 13885, Proteus mirabilis ATCC 25933, Pseudomonas aeruginosa ATCC 15442 and Salmonella enterica ATCC 14028. Bacteria were plated on brain heart infusion (BHI) agar (Himedia, India) from a frozen stock. Following 24 h incubation, three isolated colonies were transferred to 1 mL of BHI broth. The broth culture was incubated overnight (12–16 h) at 37 °C with shaking36.

Minimum Inhibitory Concentration (MIC) Determination

Minimum inhibitory concentration (MIC) of peptides and antibiotics was evaluated using the broth microdilution technique in BHI with an initial inoculum of 5 × 105 cells in non-treated polystyrene microtiter plates (Corning, USA) as described by Wiegand et al.37. The MICs were interpreted as the lowest concentration of peptide or antibiotic that completely inhibited the visible growth of bacteria after 12 h of incubation at 37 °C. Each agent was tested in triplicate in at least three independent experiments12.

Antibiofilm assays

Experiments were performed as described previously6,15. Briefly, flow cell chambers (dimensions of 1 × 4 × 40 mm) were inoculated with bacteria by injecting 400 μl of an overnight culture. After inoculation, the chambers were left without flow for 2 hours to enable initial bacterial adherence. Biofilms were grown in BM2 glucose minimal medium flowing through the cells at a constant rate of 2.4 ml/h for 48 hours at 37 °C. The matured biofilms were then exposed to clavanin-MO or media alone for an additional 24 hours. Biofilm cells were stained using the LIVE/DEAD BacLight Bacterial Viability kit (Molecular Probes, USA) prior to microscopy experiments. Microscopy was performed using a confocal laser-scanning microscope (Olympus, Fluoview FV1000, USA), and 3D reconstructions were generated using the Imaris software package (Bitplane AG).

Thin Layer Chromatography

Thin layer chromatography (TLC) assays were used to measure ppGpp levels in biofilms as described previously6,15. Briefly, untreated or bacteria treated with the peptides were labelled with 10 μCi/ml 32P for 3 hours. Samples were then extracted with frozen 13 M formic acid by three cycles of freeze-thaw. Aliquots (7.5 μl) of the supernatants were applied to 20 × 20 cm PEI cellulose TLC plates and resolved with 1.5 M KH 2 PO 4 (pH 3.4) for 4 hours. After chromatography, nucleotides were visualized by autoradiography and quantified with MolecularImager FX PhosphorImager and Quantity One software (Bio-Rad, USA).

Cell cultures

Murine macrophage-like cells RAW 264.7 and human embryonic kidney cells 293 (HEK-293) were purchased from the Rio de Janeiro Cell Bank and were maintained in supplemented Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, USA) (4 mM L-glutamine, 10% fetal bovine serum (FBS), 2 mM nonessential amino acids, 50 mg.mL−1, gentamicin, and 100 units.mL−1, penicillin/streptomycin) in the presence of 5% CO 2 at 37 °C. Cells were stimulated by adding 0.1 ng/mL LPS (E. coli 0111:B4) (Sigma, USA) into the medium specified above. Peptides were added at a concentration of 2 μM were 30 min after addition of LPS. After 6, 12, 24 and 48 h of incubation, plates were centrifuged for 6 min at 400× g and the supernatants were collected and kept frozen at −20 °C until used for analysis of IL-10, IL-12p70 and TNF-α production by ELISA (see below for detailed experimental procedure)12,38.

Hemolysis assays

The hemolytic activity of clavanin A and clavanin-MO was evaluated against fresh mouse red blood cells (mRBCs) by measuring the peptide-induced change in the optical density (OD) at 540 nm (Victor X, Perkin-Elmer, Germany)39 of a mRBC cell suspension [a 20% (v/v) erythrocytes/phosphate buffered saline (PBS) suspension]. Maximum lysis (100%) was determined by analyzing the supernatant of erythrocytes that had been incubated with 1% Triton X-100 while PBS was used as a negative control12,39,40,41.

Cytotoxicity assays

Cellular cytotoxicity was measured by a colorimetric assay that makes use of thiazolyl blue tetrazolium bromide (MTT: Sigma, USA). A sub-confluent monolayer culture of L929, RAW264.7 and Human Embryonic Kidney HEK-293 cells were collected by scraper in supplemented DMEM (Invitrogen, USA). Cells were seeded in 96-well microtiter plates at a density of 1.0 × 105 cells per well, with different concentrations of peptide or antibiotic controls (1–600 μM). Cells were incubated at 37 °C in the presence of 5% CO 2 for 48 h. Following incubation, MTT was added to the cells (10 μL at 5 mg.mL−1). The plate was incubated for 4 hours in the presence of 5% CO2 at 37 °C. Formazan crystals was dissolved by the addition of 100 μL of 100% DMSO (Mallinckrodt Chemical, USA) per well. Plates were then gently swirled for 5 min at room temperature to dissolve the precipitate. Absorbance was monitored at 575 nm using a microplate spectrophotometer (Victor X, PerkinElmer, Germany)12,37,38. Maximum cytotoxicity (100%) was determined by cells incubated with 1% Triton X-100, PBS was used as a negative control.

Animal experiments

Six-week-old female C57BL/6 mice (Central Bioterium of the USP/Ribeirão Preto) were used for all studies. Mice used in this study were housed in an animal facility at the Catholic University of Brasilia (UCB). Mice were provided standard food and water ad libitum.

Ethics Statement

The use of mice was conducted in accordance with the regulations set forward by the respective national animal protection committees and in accordance with European Community Directive 86/609 and the U.S. Association for Laboratory Animal Care recommendations for the care and use of laboratory animals. All the techniques/procedures have been refined to provide for maximum comfort/minimal stress to the animals. Experiments performed have been approved by Animal Ethics Committees of the Catholic University of Brasilia (AECs/UCB), number 005/13.

In vivo toxicity assays

The in vivo toxicity of clavanin A and clavanin-MO were evaluated by intraperitoneal (i.p.) injection of each peptide to groups of 10 C57BL/6 mice as described42. Each mouse was injected with a 0.5-ml solution of freshly prepared peptide in PBS. Doses of peptide administered per mouse were 0, 10, 30, 50, 70 and 90 mg.kg−1 of body weight. Animals were directly inspected for adverse effects after 6 hours, and mortality was monitored for 7 days thereafter12,42.

Murine systemic infection model

Based on preliminary experiments to determine an effective challenge bacterial inoculum [E. coli ATCC 8739 and E. coli KPC-positive ID N°.1812446 or S. aureus ATCC 29213 and S. aureus (MRSA) ATCC 33591] that resulted in consistent systemic infection without rapidly killing the mice (data not shown), mid-log-phase bacteria were diluted to ~2 × 107 CFU/mouse in PBS for Gram-negative bacteria and ~2 × 109 CFU/mouse in PBS for Gram-positive bacteria12,13,35. Mice were challenged by i.p. injection with 200 μL of bacteria suspended in PBS. The day of challenge was designated as day 1 of the experiment. Three hours after bacterial injection, mice (n = 10) were treated by i.p. injection with 10 mg.kg−1 of peptides, gentamicin, imipenem or PBS for 8 days and treated every 24 hours12,43. For bacterial load evaluation, 5 mice/group were humanely killed 24 h after bacterial administration. To measure bacterial colonization, peritoneal lavage was obtained by washing the cavity with 5 ml of sterile PBS. Samples of these washes were serially diluted and 100 μl of each dilution was spread in duplicate on appropriate agar plates to count colonies44.

Isolation of leukocytes from the peritoneal cavity of mice

C57BL/6 mice were injected i.p. with 10 mg.kg−1 of peptide suspended in sterile saline. Mice were euthanized, and peritoneal lavage was carried out at different times post-injection. Additionally, leukocyte counts present in the in peritoneal cavity of mice infected with E. coli ATCC 8739 or S. aureus ATCC 29213 were determined. Animals infected and treated with peptides were euthanized 3 and 24 hours after treatment, and peritoneal lavage was performed to count leukocytes cells in the peritoneal cavity45. Animals were sacrificed, and cells present in the peritoneal cavity were harvested by introducing 3.0 ml of PBS containing 1 mM EDTA. Total cell counts were performed with a cell counter (Coulter AC T series analyzer), and differential cell counts were conducted on cytocentrifuge slides (Cytospin 3; Thermo Shandon) stained by the May-Grünwald-Giemsa (Rosenfeld) method. The results are expressed as the number of cells per cavity46.

ELISA

Cultured cells or peritoneal lavage samples were centrifuged at 1000 × g for 10 min to obtain cell-free samples and stored at −20 °C. Cytokine levels were measured by ELISA using anti-mouse IL-10, IL-12p70 and TNF-α (Peprotech, USA). Cytokine levels were analysed according to the manufacturer’s instructions35.

Molecular modelling studies

Initially, the clavanin-MO sequence was submitted to QUARK ab initio modelling server47 in order to obtain a full folded clavanin-MO structure. Then, the ab initio structure was used together with the NMR structure of clavanin A14 as templates for construction of the final model of clavanin-MO. One hundred molecular models were constructed by comparative molecular modelling through MODELLER 9.1448. The models were constructed using the default methods of auto-model and environ classes from MODELLER. The final models were selected according to the discrete optimized protein energy score (DOPE score). This score assesses the energy of the models and indicates the best probable structures. The best models were evaluated through PROSA II49 and PROCHECK50. PROCHECK checks the stereo-chemical quality of a protein structure through the Ramachandran plot, where good quality models are expected to have more than 90% of amino acid residues in most favored and additional allowed regions, while PROSA II indicates the fold quality. Structure visualization was done in PyMOL (http://www.pymol.org).

Molecular Dynamics

The three-dimensional structures of clavanin A and MO were placed into a DOPC (1,2-dioleolyl-sn-glycero-3-phosphocholine) membrane using the CHARMM-GUI server51. The principal axis of the peptide was aligned to Z-axis and then inserted in a rectangular bilayer of 50 Å1 (X and Y axis) using the insertion method for system building. The system peptide-membrane was solvated with Single Point Charge water model52. The analyses were performed using the GROMOS96 53a6 force field extended for Berger lipids52 and the computational package GROMACS 453. Chlorine ions were added in order to neutralize the system charge. The geometry of water molecules was constrained using the SETTLE algorithm54. All atom bond lengths were linked by using the LINCS algorithm55. Electrostatic corrections were made according to the Particle Mesh Ewald algorithm56, with a cut-off radius of 1.4 nm in order to minimize the computational time. The same cut-off radius was also used for van der Waals interactions. The list of neighbours of each atom was updated every 10 simulation steps of 2 fs. The system underwent an energy minimization using 50,000 steps of the steepest descent algorithm. After that, the system temperature was normalized to 310 K for 100 ps, using the velocity-rescaling thermostat (NVT ensemble). Then, the system pressure was normalized to 1 bar for 100 ps, using the Parrinello-Rahman barostat using semiisotropic pressure and the thermostat was changed to Nosé-Hoover one (NPT ensemble). The systems with minimized energy, balanced temperature and pressure were simulated for 100 ns by using the leap-frog algorithm57. The structures were saved every 2 ps of simulation.

Statistical analyses

Data are presented as mean ± SD of all samples. Statistical significance of fatality rates between different groups was analysed by Kaplan–Meier test. In vivo assays were analysed using the Fisher´s exact test. The other data were submitted to one-way analysis of variance (ANOVA) followed by Bonferroni correction. Values of p < 0.05 were considered statistically significant. GraphPad Prism software v5.0 (GraphPad Software, USA) was used for all statistical analyses12.