In Vitro Evaluation of Antimicrobial Peptides from the Black Soldier Fly (Hermetia Illucens) against a Selection of Human Pathogens

Abstract

Antimicrobial peptides (AMPs) are being explored as alternatives to traditional antibiotics to combat the rising antimicrobial resistance. Insects have proven to be a valuable source of new, potent AMPs with large structural diversity. For example, the black soldier fly has one of the largest AMP repertoires ever recorded in insects. Currently, however, this AMP collection has not yet undergone antimicrobial evaluation or in-depth in vitro characterization. This study evaluated the activity of a library of 36 black soldier fly AMPs against a panel of human pathogens (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, and Aspergillus fumigatus) and a human cell line (MRC5-SV2). The activity profile of two cecropins (Hill-Cec1 and Hill-Cec10) with potent Gram-negative activity, was further explored by characterizing their hemolysis, time-to-kill kinetics, membrane-permeabilization properties, and anti-biofilm activity. Hill-Cec1 and Hill-Cec10 also showed high activity against other bacterial species, including Klebsiella pneumoniae and multi-drug resistant P. aeruginosa. Both AMPs are bactericidal and have a rapid onset of action with membrane-permeabilizing effects. Hill-Cec1 and Hill-Cec10 were also able to prevent P. aeruginosa biofilm formation, but no relevant effect was seen on biofilm eradication. Overall, Hill-Cec1 and Hill-Cec10 are promising leads for new antimicrobial development to treat critical infections caused by Gram-negative pathogens such as P. aeruginosa. IMPORTANCE With the ever growing antimicrobial resistance, finding new candidates for antimicrobial drug development is indispensable. Antimicrobial peptides have steadily gained attention as alternatives for conventional antibiotics, due to some highly desirable characteristics, such as their low propensity for resistance development. With this article, we aim to upgrade the knowledge on the activity of black soldier fly antimicrobial peptides and their potential as future therapeutics. To achieve this, we have evaluated for the first time a library of 36 synthetically produced peptides from the black soldier fly against a range of human pathogens and a human cell line. Two selected peptides have undergone additional testing to characterize their antimicrobial profile against P. aeruginosa, a clinically important Gram-negative pathogen with a high established resistance. Overall, this research has contributed to the search for new peptide drug leads to combat the rising antimicrobial resistance.

Keywords: Gram-negative bacteria; Hermetia illucens; Pseudomonas aeruginosa; antimicrobial agents; antimicrobial peptides.

FIG 1

Primary sequence alignment of the BSF AMPs from the cecropin family. The color of the amino acids indicates the extent of conservation among the different peptides. The figure was constructed using the PRALINE software available at https://www.ibi.vu.nl/programs/pralinewww.

FIG 2

(a) Helical wheel projection of Hill-Cec1. (b) Helical wheel projection of Hill-Cec10. Projections were constructed using the online Galaxy CPT software available at https://cpt.tamu.edu/galaxy-pub. Polar residues with a positive charge are indicated in blue, negatively charged polar residues are red. Uncharged polar amino acids are indicated in gray, and hydrophobic residues have a yellow color.

FIG 3

Time-to-kill curves showing the log₁₀ reductions in P. aeruginosa ATCC 9027 caused by the selected cecropins. (a) Killing kinetics of Hill-Cec1 at 1 μM, 2 μM, 4 μM, and 8 μM. (b) Killing kinetics of Hill-Cec10 at 2 μM, 4 μM, 8 μM, and 16 μM. Graphs represent the mean of five independent experiments.

FIG 4

Anti-biofilm activity of cecropins Hill-Cec1 and Hill-Cec10 against P. aeruginosa ATCC 15442. (a) Effect of Hill-Cec1 and Hill-Cec10 on P. aeruginosa biofilm formation as measured by biofilm mass. (b) Effect of Hill-Cec1 and Hill-Cec10 on P. aeruginosa biofilm formation as measured by biofilm viability. (c) Effect of Hill-Cec1 and Hill-Cec10 on preformed P. aeruginosa biofilms as measured by biofilm mass. (d) Effect of Hill-Cec1 and Hill-Cec10 on preformed P. aeruginosa biofilms as measured by biofilm viability. Bars represent the mean ± SD of three independent experiments. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 5

Membrane activity of Hill-Cec1 and Hill-Cec10. (a) Outer membrane (OM) permeabilization of P. aeruginosa caused by Hill-Cec1 measured with N-phenyl-naphthylamine (NPN). (b) OM permeabilization of P. aeruginosa caused by Hill-Cec10 measured with NPN. NPN uptake is expressed as a percentage of the maximal uptake recorded with a high dose (16 μM) of polymyxin B. (c) Fluorescence caused by propidium iodide (PI) uptake in P. aeruginosa after addition of Hill-Cec1. (d) Fluorescence caused by PI uptake in P. aeruginosa after addition of Hill-Cec10. Values were normalized with the negative control. (e) Fluorescent signal of 3,3′-dipropylthiadicarbocyanine iodide (diSC₃(5)) as an indicator of cytoplasmic membrane depolarization of P. aeruginosa caused by Hill-Cec1. (f) Fluorescent signal of diSC₃(5) as an indicator of cytoplasmic membrane depolarization of P. aeruginosa caused by Hill-Cec10. Fluorescent signals were normalized with the negative control. AU, arbitrary units; S, start of measurement immediately after dye exposure.