Prospecting Peptides Isolated From Black Soldier Fly (Diptera: Stratiomyidae) With Antimicrobial Activity Against Helicobacter pylori (Campylobacterales: Helicobacteraceae)

Abstract

Helicobacter pylori (Marshall & Goodwin) is a widespread human pathogen that is acquiring resistance to the antibiotics used to treat it. This increasing resistance necessitates a continued search for new antibiotics. An antibiotic source that shows promise is animals whose immune systems must adapt to living in bacteria-laden conditions by producing antibacterial peptides or small molecules. Among these animals is the black soldier fly (BSF; Hermetia illucens Linnaeus), a Diptera that colonizes decomposing organic matter. In order to find anti-H. pylori peptides in BSF, larvae were challenged with Escherichia coli (Enterobacteriales: Enterobacteriaceae). Small peptides were extracted from hemolymph and purified using solid-phase extraction, molecular weight cutoff filtration and two rounds of preparative high performance liquid chromatography (HPLC). The anti-H. pylori fraction was followed through the purification process using the inhibition zone assay in brain-heart infusion agar, while peptides from uninoculated larvae had no activity. The inhibition halo of the active sample was comparable to the action of metronidazole in the inhibition zone assay. The purified sample contained four peptides with average masses of approximately 4.2 kDa that eluted together when analyzed by HPLC-mass spectrometry. The peptides likely have similar sequences, activity, and properties. Therefore, BSF produces inducible antibacterial peptides that have in vitro activity against H. pylori, which highlights BSF's position as an important target for further bioprospecting.

Keywords: Helicobacter pylori; Hermetia illucens; antimicrobial peptides; bioprospecting.

Fig. 1.

Schematic of biodirected purification of anti-H. pylori AMPs. Shaded rectangles indicate anti-H. pylori activity is present in the fraction. Distances underneath fraction names report the diameter of the inhibition halo during the inhibition zone assay using 5 µg of sample. (*), 0.63 µg were used for the assay.

Fig. 2.

Results of preparative HPLC, inhibition zone, and mass spectrometry analysis of anti-H. pylori AMPs. (A) HPLC fractionation of the 3–10 kDa fraction. Baseline-adjusted chromatograms of noninoculated sample (blue) and inoculated sample (orange). Vertical lines indicate different fractions collected. Shaded box reports the active fraction (F5). Inoculated and noninoculated chromatograms were also normalized to each other to reflect relative amounts of sample loading. (B) H. pylori inhibition halo of sample F5. The inhibition zone, indicated by absence of colonies and discolored agar, is 2.0 cm wide. (C) Chromatographic separation of F5 (inoculated sample, green). F5.8 with anti-H. pylori activity is highlighted. (D) Inhibition zone of the highlighted fraction in (C). (E) MSqTOF spectrum of the active sample in (C). Peak groupings are due to isotope abundances in the peptides. The most abundant mass over charge ratio for each peptide is indicated. The spacing of the peaks, roughly 0.2 Da, indicates a +5 charge on the peptides. +6 and +4 charges were also observed but not shown.