Peptide NDBP-5.5 (UniProt P0C8W1) was identified as previously described (Schwartz et al., 2007) and was synthesized by C-terminal amidation using FastBio LTDA (Ribeirão Preto, SP, Brazil). NDBP-5.5 presented >95% of purity. The molecular mass and sequence of the synthetic NDBP5.5 were confirmed by MALDI-TOF/TOF MS (UltraFlex III, BrukerDaltonics, Germany) and LIFTTM (MS/MS) as previously described (Guilhelmelli et al., 2016).

Multiple sequence alignment of the obtained sequences of peptide NDBP-5.5 was performed using ClustalW2 and BioEdit software (Larkin et al., 2007). Structural predictions, as shown in an alpha helix diagram, were generated by the HeliQuest server¹.

Molecular dynamics (MD) simulations were performed with Gromacs 5.1.3 (Berendsen et al., 1995; Yu, 2012) using force field AMBER99SB-ILDN (Yildirim et al., 2010) in order to explore the stability (or instability) of the peptide in water. Initially, the peptide was solvated with a box cubic wall distance of 10 Å using water model TIP3P (Mahoney and Jorgensen, 2000). The system was neutralized by adding the required number of counter ions. The particle mesh Ewald method (Darden et al., 1993) was used to evaluate electrostatic interactions with periodic boundary conditions considered in all directions from box. The system was initially subjected to minimization using the steepest descent energy. The simulations were completed when the tolerance of 1000 kJ/mol was no longer exceeded. The next four stages consisted of a series of equilibration simulations to slowly relax the system without deviating from the initial conformation. The first two stages consisted of 50 ps MD simulations in NVT and NPT ensembled at 300 K with a restraint of 50 kcal/mol/Å on the peptide atoms. In the next stage, the simulations were performed during 0.5 ns without restraint in NPT ensembled at 300 K. Finally, the simulations were performed for 100 ns with a constant temperature of 300 K, 1 atm pressure, time-setup of 2 fs, and without any restriction of peptide conformations. All information concerning the trajectory of these times were collected every 50 ps. The equilibration of the trajectory was evaluated by monitoring the equilibration of quantities, such as the root-mean-square deviation (RMSD) (Coutsias et al., 2004) of the non-hydrogen atoms, kinetic energy, total energy, and potential energy.

The program g_cluster (GROMACS tool) was used to determine the conformations that best represented the peptide structures of the entire trajectory. The algorithm gromos, as described by Daura et al. (1999), was selected for this purpose. A cutoff = 0.2 nm for the clusters was used considering the profile of the RMSD. The clusters were determined using the non-hydrogen atom RMSD values.

The mycobactericidal activity of NDBP-5.5 was determined in vitro using three clinical isolates randomly selected from our laboratory collection: M. abscessus subsp. massiliense GO01, GO06, and GO08 (Cardoso et al., 2008) and the reference strain CRM0020 (Duarte et al., 2009). The bacteria were grown in Mueller–Hinton medium (MH, HIMEDIA) for 3 days at 35°C. Then, the cultures were adjusted to 1.5 × 10⁸ CFU/mL (0.5 McFarland scale) to determine the minimal bactericidal concentration (MBC).

Mycobacterium abscessus subsp. massiliense GO06 was used for the in vivo experiments (mice intravenous infection) (Sousa et al., 2010; das Neves et al., 2016). The bacterial suspension was diluted in PBS 0.05% Tween 80 and adjusted to 1 × 10⁶ CFU/mL.

Macrophages were obtained from the bone marrow of BALB/c mice. IFN-γ KO mice were used to test the effect of peptide NDBP-5.5 on the treatment of M. abscessus subsp. massiliense infection. Female mice, of 7–9 weeks-of-age, were obtained from the animal facilities at the Institute of Tropical Pathology and Public Health at the Federal University of Goiás (IPTSP/UFG). All animal handling procedures and protocols were performed according to the National Counsel for animal experimentation recommendations (Conselho Nacional de Controle de Experimentação Animal -CONCEA). The Animal Ethics Committee at the Federal University of Goias approved the protocols (Comitê de Ética no uso de animais da Universidade Federal de Goiás (No: 016/14).

The MBC determination was adapted from ATS/IDSA guidelines (Griffith et al., 2007). The lyophilized peptide NDBP-5.5 was diluted in 3% DMSO. M. abscessus subsp. massiliense cultures, grown as described previously, were adjusted to 100 CFU/100 μL and added to 96-well plates. The peptide solution was serially diluted in PBS (13–400 μM), and 100 μL of each dilution was added to the wells. Each NDBP-5.5 peptide dilution was tested in triplicate. The plates were incubated for 3 days at 35°C. All test plates contained control wells of medium only, M. abscessus subsp. massiliense isolates as positive controls, or M. abscessus subsp. massiliense isolates treated with CLR (1.34 μM) as a negative control. After 3 days of culture, the wells were homogenized and transferred to an MH agar plate to calculate the number of CFU per treatment. The MBC was determined and the percentage of inhibition in relation to bacterial growth in the medium without treatment was calculated.

The toxicity of NDBP-5.5 against human red blood cells was evaluated as described previously (Ramírez-Carreto et al., 2012). Ten milliliters of peripheral blood from a healthy subject (without any symptoms of disease) were collected. The protocol was approved by the Ethics Committee of the Federal University of Goias (protocol number: 055/2009) and the participant signed a consent form. Briefly, red blood cells were obtained by centrifugation of the blood at 1000 × g. The cells were then washed with PBS, adjusted to 1 × 10⁸ cells/mL, and 100 μL were added to the wells of a 96-well plate. The NDBP-5.5 solution was diluted to 8, 4, 2, and 1× the peptide MBC value (200 μM) and 100 μL was added to the wells. The plates were incubated for 1 h at 37°C, and after this period they were centrifuged (1000 × g) and the absorbance was determined at 540 nm. Red blood cells treated with Triton-100X (0.1%) were used as positive controls and red blood cells only were used as negative controls. The assay was performed in triplicate and repeated twice. The percentage of hemolysis was obtained using the formula: % of red cell lysis = 100× [(test-PBS)/(Triton-PBS)].

The therapeutic index (TI) was determined according to Wang et al. (2009) by dividing the concentration of NDBP-5.5 that caused 10% hemolysis by the MBC value.

BALB/c mice (n = 4) were euthanized and the femur bone marrow was extracted, homogenized and plated with 10 ng/mL of GM-CSF as described previously (Becker et al., 2012). After 3 days of culture, the medium was supplemented with 10 ng/mL of GM-CSF. After 7–10 days of culture, the macrophages were transferred to a 96-well plate at 10⁵ cells/well. After a 1 h incubation at 37°C in a CO₂ incubator, 10⁶ CFU/well of M. abscessus subsp. massiliense were added to the cultures [multiplicity of infection (MOI), 10:1] and further incubated for 3 h. Then, the cell cultures were washed with RPMI-1640 (HIMEDIA), containing 10% fetal bovine serum, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 g/mL streptomycin, 2 mM L-glutamine, and 2 mM non-essential amino acids.

After this procedure, the infected macrophages were treated with 200 μM of NDBP-5.5 (MBC = 200 μM) prepared in RPMI-1640 media. After 72 h, the cells were treated with 200 μL of deionized water, and after homogenization, the suspensions were plated on MH agar plates for culturing to determine the number of CFU. Controls containing only infected macrophages or macrophages treated with CLR were added to all of the 96-well test plates.

IFN-γ KO mice were intravenously infected with 10⁶ CFU of M. abscessus subsp. massiliense. After 18 days of infection, the mice were treated with NDBP-5.5 (2 mg/kg, n = 12 animals; or 1 mg/kg, n = 12 animals) or CLR (200 mg/kg, n = 12 animals) over 8 consecutive days. An infection group treated with PBS (n = 12) was used as a control. The animals were treated by intraperitoneal injection with a volume of 100 μL as previously described (das Neves et al., 2016).

To monitor the infection with M. abscessus subsp. massiliense, the day following infection and 18 days post-infection, three animals per time and group were euthanized, and the lungs, spleens, and livers were collected to determine the bacillary count.

Twenty-seven-days post infection (1 day after the end of treatment), the animals were euthanized (n = 6), and spleens, livers, and lungs were collected to determine the bacillary load.

To establish the bacterial load, the organs were homogenized in PBS 0.05% Tween 80, serially diluted, and plated in MH agar. The percentage reduction in bacterial numbers was calculated using the number of CFU obtained from the organs of animals infected with M. abscessus subsp. massiliense without treatment and the number of CFU from the groups treated with NDBP-5.5 or CLR.

After 28 days of infection, mice were euthanized and the organs were collected. The right lung caudal lobe from each animal was fixed in 10% paraformaldehyde and blocked in paraffin. The tissues were sectioned (at 5-μm), stained with hematoxylin and eosin, and observed under an optical microscope (Axio scope A1; Carl Zeiss, Oberkochen, Germany).

The data were tabulated using Excel software and the average and standard deviation values were calculated. To evaluate statistical differences among groups, the software GraphPad Prism 6.0 was used. The variances among groups were evaluated by one-way ANOVA, followed by the Tukey test. Differences were considered statistically significant when p < 0.05.

A previously described cDNA sequence of a peptide derived from H. gerstchi venom (Schwartz et al., 2007) and comprising 13 amino acids (IFSAIAGLLSNLL) with C-terminal amidation, namely NDBP-5.5, was synthetized and the sequence was confirmed by MALDI-TOF/TOF (Figures 1A,B). The predicted secondary structure¹ revealed an alpha helix with amphipathic characteristics containing a hydrophilic region (A,S,S,G,N,A) and a hydrophobic region (L,F,L,I,L,I,L) (Figure 1C). Sequence alignment of NDBP-5.5 with other scorpion venom peptides from the same family of peptides (from the NDB group) showed highest similarity (69.2%) with Meucin-13 from Mesobuthus eupeus venom (Figure 1D). MD simulations from the PEP-FOLD model were performed in order to verify the stability of the NDBP-5.5 in water under two independent trajectories (different initial conditions). Figures 1E,F shows the RMSD evolution from initial model (PEP-FOLD structure) for MD1 and MD2 simulations. For both simulations, the peptide was not able to remain stable around a particular conformation, even to any conformation other than the helical structure. It can be verified from cluster analysis of the trajectory, that most of the structures were unstable for any time during the simulation (Figure 1F).

NDBP-5.5 inhibited the growth of all M. abscessus subsp. massiliense isolates evaluated (Figure 2). Using a range of clinical isolates and a reference strain (CRM0020), the MBC of NDBP-5.5 was determined to be 200 μM.

As shown in Figure 3, NDBP-5.5 displayed low hemolytic activity. When the peptide was used at a concentration eight-times higher (1600 μM) than that of the MIC, 39% hemolysis occurred (Figure 3). The concentration of NDBP-5.5 that caused 10% hemolysis was calculated (611.8 μM) and, as suggested by Wang et al. (2009), used to estimate the TI, by dividing by the MBC value (200 μM). The TI was determined as 3.05.

Since NDBP-5.5 presented low toxicity, we decided to test its function against phagocytosed bacteria. The treatment of infected macrophages (MOI, 1:10) with NDBP-5.5 or CLR reduced the bacterial load at similar levels (Figure 4). The highest bacterial load reduction was observed with the reference strain (60% reduction; Figure 4E).

Mice susceptible to M. abscessus subsp. massiliense infection were used to test the in vivo activity of NDBP-5.5. Using 2 mg/kg of NDBP-5.5, the bacterial load was significantly reduced in the lungs and liver. The bacterial load reduction observed at this dosage (2 mg/kg) was similar to that observed for CLR (Figures 5A–C). Evaluating the lung inflammatory lesions of the infected animals, it was possible to observe diffuse inflammatory lesions at 28-days-post-infection (Figure 6A), that were reduced when the animals were treated with CLR (Figure 6B). Treatment with NDBP-5.5 at 1 mg/kg resulted in inflammatory lesions that were similar to the lesions observed in the lungs of infected animals that were not treated with NDBP-5.5 (Figure 6C). The lung lesions induced by infection in IFN-γ KO mice were drastically reduced when the animals were treated with NDBP-5.5 at 2 mg/kg (Figure 6D). The score of the lesions were evaluated and showed that mice treated with NDBP-5.5 (2 mg/Kg) had reduced inflammatory lesions when compared to infected and not treated animals (Figure 6E).