Short, Synthetic Cationic Peptides Have Antibacterial Activity against Mycobacterium smegmatis by Forming Pores in Membrane and Synergizing with Antibiotics

Abstract

Multicellular organisms are constantly exposed to a multitude of pathogenic microbes. Infection is inhibited in vivo by the innate and adaptive immune system. Mycobacterium species have emerged that are resistant to most antibiotics. We identified several naturally occurring cationic antimicrobial peptides that were active at low micromolar concentrations against Mycobacterium smegmatis. Human-derived cathelicidin LL-37 is well characterized and studied against M. smegmatis; we compared LL-37 with Chinese cobra-derived cathelicidin NA-CATH and mouse cathelicidin (mCRAMP). Two synthetic 11-residue peptides (ATRA-1A and ATRA-2) containing variations of a repeated motif within NA-CATH were tested for their activity against M. smegmatis along with a short synthetic peptide derivative from the human beta-defensin hBD3 (hBD3-Pep4). We hypothesized that these smaller synthetic peptides may demonstrate antimicrobial effectiveness with shorter length (and at less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds or use in combination with antibiotics. These peptides have antimicrobial activity with EC50 ranging from 0.05 to 1.88 μg/mL against Mycobacterium smegmatis. The ATRA-1A short peptide was found to be the most effective antimicrobial peptide (AMP) (EC50 = 0.05 μg/mL). High bactericidal activity correlated with bacterial membrane depolarization and permeabilization activities. The efficacy of the peptides was further analyzed through Minimal Inhibitory Concentration (MIC) assays. The MICs were determined by the microdilution method. The peptide mCRAMP showed the best MIC activity at 15.6 μg/mL. Neither of the effective short synthetic peptides demonstrated synergy with the antibiotic rifampicin, although both demonstrated synergy with the cyclic peptide antibiotic polymyxin B. The peptides LL-37 and mCRAMP displayed synergism with rifampicin in MIC assays, whereas antibiotic polymyxin B displayed synergism with LL-37, ATRA-1A, and hBD3-Pep4. In further studies, polymyxin B synergized with LL-37, ATRA-1A, and hBD3-Pep4 while Rifampicin synergized with LL-37 and mCRAMP for intracellular killing of mycobacteria residing inside macrophages. These studies provide the foundation for the potential development of synthetic cationic antimicrobial peptides with activity against mycobacteria.

Keywords: antibiotics; cationic AMPs; mycobacterium; synergy.

Figure 1

Membrane depolarization activity by LL-37, mCRAMP, hBD3-Pep4, ATRA-1A, ATRA-2, and NA-CATH. Depolarization determined using DiSC₃(5) (50 μg/mL) at multiple concentrations of peptide (50, 25, 5, and 0.5 μg/mL). Experiments were performed three times with n = 2 each time; for each concentration of peptide all the data were combined (n = 6) for the graph and the analysis. Means ± the SD are shown; *: p ≤ 0.001.

Figure 2

Kinetics of permeabilization and binding of ethidium bromide to DNA with 50 μg/mL of peptide concentration. Pore-forming activity by the peptides tested in the studies: hBD3-Pep4, ATRA-1A, mCRAMP, LL-37, and NA-CATH. An increase in fluorescence demonstrates greater binding of DNA by ethidium bromide, which indicates the formation of pores in the bacterial membrane. Permeablization of membrane after 20 min was compared. Experiments were performed in triplicate (n = 3). Means ± the SD are shown; p ≤ 0.01 when comparing each peptide activity at 20 min to untreated control.

Figure 3

Effectiveness of antimicrobial peptides against M. smegmatis. Percent survival was calculated by CFU after 3 h incubation with various peptide concentrations in 10 mM of phosphate buffer (pH 7.4) supplemented with 0.1% of MB7H9 supplemented with OADC. (A) EC₅₀ of ATRA-1A was found to be 0.05 μg/mL; (B) EC₅₀ of mCRAMP was found to be 0.17 μg/mL; (C) EC₅₀ of LL-37 was found to be 0.27 μg/mL; (D) EC₅₀ of NA-CATH was found to be 1.88 μg/mL; (E) EC₅₀ of hBD-3 Pep4 was found to be 0.36 μg/mL; (F) EC₅₀ of Fosmidomycin was found to be 0.14 μg/mL; (G) EC₅₀ of Rifampicin was found to be 0.13 μg/mL; (H) EC₅₀ of polymyxin B was found to be 0.12 μg/mL.

Figure 4

Intracellular killing of mycobacteria in macrophages. Macrophages were infected with M. smegmatis for 12 h and then incubated with antibiotics, antimicrobial peptides, and peptide-drug combinations. Macrophages infected with bacteria alone were used as a control. Cells were lysed, and bacterial intracellular survival was determined by CFU assay. (A) Intracellular survival of M. smegmatis after treatment with antibiotics or antimicrobial peptides; p ≤ 0.05 significantly different compared to untreated; (B) Intracellular survival of M. smegmatis after treatment with peptide-drug combination. Experiments were performed in triplicate (n = 3). Means ± the SD are shown; p ≤ 0.05 significantly different compared to untreated.