Fragmentation of Human Neutrophil α-Defensin 4 to Combat Multidrug Resistant Bacteria

Abstract

The occurrence and spread of multidrug-resistant bacteria is a prominent health concern. To curb this urgent threat, new innovative strategies pursuing novel antimicrobial agents are of the utmost importance. Here, we unleashed the antimicrobial activity of human neutrophil peptide-4 (HNP-4) by tryptic digestion. We identified a single 11 amino acid long fragment (HNP-4_{1 - 11}) with remarkable antimicrobial potential, exceeding that of the full length peptide on both mass and molar levels. Importantly, HNP-4_{1 - 11} was equally bactericidal against multidrug-resistant and non-resistant strains; a potency that was further enhanced by N- and C-terminus modifications (acetylation and amidation, respectively). These observations, combined with negligible cytotoxicity not exceeding that of the full length peptide, presents proteolytic digestion of innate host-defense-peptides as a novel strategy to overcome the current health crisis related to antibiotic-resistant bacteria.

Keywords: HNP-4; host defense peptides; multidrug resistance; proteolytic digestion; α-defensins.

FIGURE 1

Proteolytic digestion of reduced HNP-4 by trypsin produced different fragments. (A) Displays an overview of the chromatogram from an incubation of reduced HNP-4 with trypsin after reduction with 2 mM TCEP. All detectable fragments were marked in red or gray (a–j) and listed due to their retention time. Panel (B) show the mass-to-charge (m/z) graphs of all detected fragments. In all mass-to-charge graphs we pointed out the neutral mass based on the detected ions. All peptides marked in red were chose for synthesis and further investigations.

FIGURE 2

HNP4-derivates display a high antimicrobial activity against commensal and pathogenic bacteria. We analyzed the antimicrobial potential of the identified fragment and its modified version against commensal and pathogenic bacteria. In this heat map, we listed all bacteria and the activity of the fragments in RDA against them. We used 2 μg of the full-length peptide and 4 μg of each fragment. An inhibition zone greater than 8 mm was determined as highly active, between 2.5 and 8 mm as low active, while a diameter of 2.5 mm (diameter of the punched well) was marked as no activity. The heat map is based on three independent experiments.

FIGURE 3

Comparison of the potency (MIC) and efficacy (killing rate) of HNP-4_fl, HNP-4_1–11 and HNP-4_1–11mod. (A) The minimal inhibitory concentration (MIC) in μM and μg/ml as a concentration without any bacterial growth. Peptides were incubated with tested bacteria and changes in optical density (OD₆₀₀) were measured after 12 h at 37°C. If we were able to observe an antimicrobial effect but did not detect a total inhibition of bacterial growth we marked it with “>>>.”Each experiment was carried out three independent times. (B) Killing of E. coli ATCC25922 after 0–120 min exposure to 6.25 μM (1× MIC) HNP-4_fl, HNP-4_1–11 and HNP-4_1–11mod. Results are expressed as the number of viable bacteria (in log10 CFU) per milliliter. Values are means of three independent experiments.

FIGURE 4

Reduction as well as proteolysis of HNP-4_1–11 and HNP-4_1–11mod have no influence on the antimicrobial activity. (A) Changes in the antimicrobial activity against E. coli ATCC25923 were analyzed in the presence of a protease inhibitor cocktail. (B) The minimal inhibitory concentration of HNP-4_1–11 and HNP-4_1–11mod was determined against E. coli ATCC25922 under reducing conditions due to the optical density after 12 h. Results from three independent experiments with ±SEM are represented. (C) ESI-MS analysis of HNP-4_1–11 to detect potential dimer’s after peptide dilution. (D) Analysis of HNP-4_1–11mod using ESI-MS to detect potential dimer’s after peptide dilution.

FIGURE 5

HNP-4_1–11 and HNP-4_1–11mod show only minor cytotoxic and hemolytic activity at high concentrations. We investigated the cytotoxic activity of HNP-4_1–11 and HNP-4_1–11mod against (A) CaCo2/TC7 or (B) HT29 MTX E 29 cells. We seeded 1500 cells per well and treated them after 24 h with different peptide concentrations. Living cells were determined after 96 h treatment using a CellTiter Glo2.0 assay. (C) Hemolytic activity on human erythrocytes of the peptides compared to 0.1% Triton-X treatment. (A,B) Results from three independent experiments with ±SEM are shown, and (C) Results from two independent experiments with ±SEM are represented.