Enhancing the antibacterial activity of antimicrobial peptide PMAP-37(F34-R) by cholesterol modification

Abstract

Background: The problem of increasing resistance against conventional antibiotics has drawn people's attention. Therefore, the development of novel antibacterial agents with effective and safe therapeutic effects is imminent. Antimicrobial peptides (AMPs) are considered a promising class of antibacterial agents due to their broad antibacterial spectrum.

Results: In this study, on the basis of our previously studied peptide PMAP-37(F34-R), a novel antimicrobial peptide Chol-37(F34-R) was developed by N-terminal cholesterol modification to increase hydrophobicity. We observed that the N-terminal cholesterol-modified Chol-37(F34-R) showed higher antimicrobial activity than PMAP-37(F34-R) in vitro. Chol-37(F34-R) also exhibited effective anti-biofilm activity and may kill bacteria by improving the permeability of their membranes. Chol-37(F34-R) exerted high stability in different pH, salt, serum, and boiling water environments. Chol-37(F34-R) also showed no hemolytic activity and substantially low toxicity. Furthermore, Chol-37(F34-R) exhibited good potency of bacteria eradication and promoted wound healing and abscess reduction in infected mice. Meanwhile, in S. aureus ATCC25923-infected peritonitis model, Chol-37(F34-R) exhibited an impressive therapeutic effect by reducing the decrease in systemic bacterial burden and alleviating organ damage.

Conclusions: Our findings suggested that the N-terminal cholesterol modification of PMAP-37(F34-R) could improve antibacterial activity. Chol-37(F34-R) displayed excellent bactericidal efficacy and impressive therapeutic effect in vivo. Thus, Chol-37(F34-R) may be a candidate for antimicrobial agents against microbial infection in the clinic.

Keywords: Antibacterial activity; Antimicrobial peptide PMAP-37(F34-R); Cholesterol; Hydrophobicity; Therapeutic efficacy.

Fig. 1

Peptides timeline and Chol-37(F34-R) molecular structure. a shows the timeline of peptide design from PMAP-37 to PMAP-37(F34-R) to Chol-37(F34-R). b shows the structure of Chol-37(F34-R). Chol-37(F34-R) was synthesized using Fmoc solid-phase peptide synthesis protocols. After removal of the Fmoc and coupling of the subsequent amino acids, the monocholesteryl ester of carbonic acid was linked to the only “-NH₂” group of the first amino acid (Gly) of the antimicrobial peptide PMAP-37(F34-R). The blue dashed frame indicates a linker. The yellow dashed frame indicates Arg(R) substitution at position 34 of PMAP-37

Fig. 2

HPLC and MS analysis of Chol-37(F34-R). a, b, and c show the HPLC of PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R), respectively. d, e, and f show the MS of PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R), respectively

Fig. 3

Biofilm inhibition of Chol-37(F34-R). a, b, and c respectively show the effect of Chol-37(F34-R) on the biofilm formation of S. aureus ATCC25923, S.typhimurium SL1344, and P. aeruginosa GIM1.551. The data are presented as means ± standard deviation (SD) of results (n = 5). *, P < 0.05 and **, P < 0.01, compared with PMAP-37. #, P < 0.05 and ##, P < 0.01, compared with PMAP-37(F34-R)

Fig. 4

Biofilm eradication of Chol-37(F34-R). a, b, and c respectively show the effects of Chol-37(F34-R) on biofilm eradication of S. aureus ATCC25923, S. typhimurium SL1344, and P. aeruginosa GIM1.551. d, e, and f respectively show the killing effect of Chol-37(F34-R) against S. aureus ATCC25923, S. typhimurium SL1344, and P. aeruginosa GIM1.551 under the biofilm. The number of bacteria (CFU) is displayed in the form of log. The data are presented as means ± SD of results (n = 5). *, P < 0.05 and **, P < 0.01, compared with control. ns, nonsignificant difference

Fig. 5

Membrane permeability against bacteria by Chol-37(F34-R). Fluorescence inverted microscopy imaging of S. aureus ATCC25923, L. monocytogenes CICC21634, S. typhimurium SL1344, and P. aeruginosa GIM1.551 after treatment with PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) for 20 min. Original magnification, × 40. The measured resolution of each image was 300 × 300 DPI and the resolution of the combined image was 300 × 300 DPI

Fig. 6

Antibacterial activity of Chol-37(F34-R) treated at different pH. pH stability test was performed using Chol-37(F34-R) treated at different pH (2–13) by disk diffusion. a, b, c show the pH stability curves of PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) treated with different pH solutions (2–13) with S. aureus ATCC25923 as the test strain, respectively. d, e, and f show the pH stability curves of PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) treated with different pH solutions (2–13) with P. aeruginosa GIM1.551 as the test strain, respectively. The data are presented as means ± SD of results (n = 5)

Fig. 7

Heat-stable determination of Chol-37(F34-R) by boiling. Thermal stability test was performed using Chol-37(F34-R) boiled at different times (0, 10, 20, 30, 40, 50, 60, 90, 120 min) by disk diffusion. a, b, and c show the thermal stability curves of PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) with S. aureus ATCC25923 as the test strain, respectively. d, e, and f show the thermal stability curves of PMAP-37, PMAP-37(F34-R), and Chol-37(F34-R) with P. aeruginosa GIM1.551 as the test strain. The data are presented as means ± SD of results (n = 5)

Fig. 8

Hemolytic activity and cytotoxicity of Chol-37(F34-R). a shows the hemolytic activity of peptides measured by the release of hemoglobin from mouse erythrocytes. b shows the cytotoxicity of peptides determined by MTT assays. Mouse embryo fibroblast cell line NIH 3 T3 cells were used as target cells. The data are presented as means ± SD of results (n = 5)

Fig. 9

In vivo analysis of Chol-37(F34-R) against P. aeruginosa GIM1.551-infected mouse knife injury model. a shows typical photographs of wound at day 7 after different treatments. b shows normalized number of viable P. aeruginosa GIM1.551 isolated from wound sites at day 7 after different treatments. AS, ampicillin sodium. The data are presented as means ± SD of results (n = 5). The number of bacteria (CFU) is displayed in the form of log. *, P < 0.05 and **, P < 0.01 compared with PBS. ##, P < 0.01 compared with PMAP-37(F34-R)

Fig. 10

In vivo therapeutic effect of Chol-37(F34-R) against S. aureus ATCC25923 or P. aeruginosa GIM1.551-infected mouse abscess model. a shows typical photographs of S. aureus ATCC25923 or P. aeruginosa GIM1.551-infected abscess sites on day 3 after different treatments. b and c show the normalized number of viable S. aureus ATCC25923 or P. aeruginosa GIM1.551 isolated from abscess sites on day 3 after different treatments. BP, benzylpenicillin potassium. AS, ampicillin sodium. The data are presented as means ± SD of results (n = 5). The number of bacteria (CFU) in the PBS group was defined as 100%, and the other groups are displayed proportionally compared with the PBS group.*, P < 0.05 and **, P < 0.01, compared with PBS. ##, P < 0.01, compared with PMAP-37(F34-R). ns, nonsignificant difference

Fig. 11

Therapeutic efficacy of Chol-37(F34-R) in S. aureus ATCC25923-infected mouse peritonitis model. a shows the lesions in lung and spleen of challenged mice treated with Chol-37(F34-R). b shows the pathological changes in the liver and spleen sections of mice challenged with S. aureus ATCC25923. c and d show histological scores of liver and spleen sections, respectively. Original magnification, Liver sections, × 40, spleen sections, × 40. The areas indicated by the arrows represent the type of lesion (a, necrosis; b, congestion; c, lymphocytic infiltration; d, hemorrhage; e, intumescentia). The measured resolution of each image was 300 × 300 DPI and the resolution of the combined image was 300 × 300 DPI. e and f show bacterial load of liver and spleen in mice, respectively. BP, benzylpenicillin potassium. The data are presented as means ± SD of results (n = 5). The number of bacteria (CFU) in the PBS group was defined as 100%, and the other groups are displayed proportionally compared with the PBS group. *, P < 0.05 and **, P < 0.01, compared with PBS. #, P < 0.05, compared with PMAP-37. §§, P < 0.01, compared with PMAP-37(F34-R). ns, nonsignificant difference