Paneth cell α-defensins HD-5 and HD-6 display differential degradation into active antimicrobial fragments

Abstract

Antimicrobial peptides, in particular α-defensins expressed by Paneth cells, control microbiota composition and play a key role in intestinal barrier function and homeostasis. Dynamic conditions in the local microenvironment, such as pH and redox potential, significantly affect the antimicrobial spectrum. In contrast to oxidized peptides, some reduced defensins exhibit increased vulnerability to proteolytic degradation. In this report, we investigated the susceptibility of Paneth-cell-specific human α-defensin 5 (HD-5) and -6 (HD-6) to intestinal proteases using natural human duodenal fluid. We systematically assessed proteolytic degradation using liquid chromatography-mass spectrometry and identified several active defensin fragments capable of impacting bacterial growth of both commensal and pathogenic origins. Of note, incubation of mucus with HD-5 resulted in 255-8,000 new antimicrobial combinations. In contrast, HD-6 remained stable with consistent preserved nanonet formation. In vivo studies demonstrated proof of concept that a HD-5 fragment shifted microbiota composition (e.g., increases of Akkermansia sp.) without decreasing diversity. Our data support the concept that secretion of host peptides results in an environmentally dependent increase of antimicrobial defense by clustering in active peptide fragments. This complex clustering mechanism dramatically increases the host's ability to control pathogens and commensals. These findings broaden our understanding of host modulation of the microbiome as well as the complexity of human mucosal defense mechanisms, thus providing promising avenues to explore for drug development.

Keywords: antimicrobial peptides; host–microbiota interaction; intestinal barrier; proteolytic digestion; α-defensins.

Fig. 1.

HD-6 nanonet formation is not affected by duodenal fluid. (A) Here we show the chromatograph of only exogenously added HD-6 incubated with duodenal fluid after reduction with 2 mM TCEP. We detected only the oxidized and reduced full-length peptides due to their retention time with their m/z graphs and their two-, three-, four-, five- and sixfold protonated ions and neutral masses. (B) We incubated beads with 200 µg/mL reduced HD-6 or 0.01% HAc (control) and duodenal fluid or 0.9% NaCl (control). The nanonet formation of HD-6 seemed to be unaffected by the duodenal fluid, because it still forms nanonets comparable to those in the 0.9% NaCl control. (Scale bar: 0.2 µm.)

Fig. 2.

Incubation of HD-5 and duodenal fluid produced many different fragments. We incubated only HD-5 with duodenal fluid after reduction with 2 mM TCEP. (A) Here we show the overview of the chromatogram from an incubation of reduced HD-5 with duodenal fluid. All detectable fragments were marked in red or gray (a–m). All peptides marked in red were chosen for synthesis and deeper investigation of their abilities. (B) The chosen fragments with their name and amino acid sequence and their distribution over the HD-5 sequence.

Fig. 3.

HD-5 fragments are antimicrobially active peptides against commensal bacteria. (A) We tested different commensal bacteria due to their susceptibility to HD-5 fragments. In this heat map, we listed all bacteria and the activity of the fragments in RDA against them. In the RDA, we used 2 µg of the full-length peptide and 4 µg of each fragment. An inhibition zone greater than 5 mm was determined as high activity, between 2.5 and 5 mm as low activity, and 2.5 mm (the diameter of the punched well) as no activity. (B) To investigate the mode of action of the different peptides, we incubated E. coli MC1000 with all the different fragments, performed transmission electron microscopy, and analyzed the resulting phenotypes. [Scale bars of all pictures are 0.5 µm except the full-length peptide (1 µm).]

Fig. 4.

HD-5_1–9 treatment did not affect the microbiota diversity but has an influence on certain bacteria strains. We treated chow-fed wild-type mice for 7 d orally with 7.19 µg/mouse HD-5_1–9 or PBS (six mice per group). After this period, we stopped the treatment. Fecal samples were collected for days 0, 7, and 14. Additionally, small intestine samples were taken after 14 d. (A) Here we show a principal coordinate analysis (PCoA including group mean) of fecal or small intestine microbiota composition using Weighted UniFrac Distances at days 0, 7, and 14, respectively. There was no difference in baseline microbiota between the groups at day 0 (Adonis PERMANOVA P = 0.65). After 7 d of treatment, the fecal microbial abundances were significantly different between the groups (Adonis PERMANOVA P = 0.02), but not when comparing each group to its baseline (Adonis PERMANOVA P > 0.05). At day 14, 7 d after washout, neither the fecal nor the small intestine microbial abundance was significant between the groups (Adonis PERMANOVA P = 0.09 and P = 0.36, respectively). (B) PCoA including group mean of fecal or small intestine microbiota composition using Unweighted UniFrac Distances at days 0, 7, and 14, respectively. The baseline microbiota did not differ between the groups regarding absence and presence of bacteria (Adonis PERMANOVA P = 0.11). After 7 d of treatment, the groups differed significantly in fecal microbiota calculated by Unweighted UniFrac Distances (Adonis PERMANOVA P = 0.009), which persisted after the 1-wk washout in fecal samples (Adonis PERMANOVA P = 0.003) and small intestine samples (Adonis PERMANOVA P = 0.004). Despite this, the groups did not at any time point differ significantly from their baseline composition by Unweighted UniFrac Distance (Adonis PERMANOVA P > 0.05). (C) HD-5_1–9 treatment affects the abundance of bacterial genera. Using a linear mixed-effect model, we identified a significant increase of Akkermansia sp. in the feces samples in mice treated with HD-5_1–9 (P = 0.024), while this increase was not observed in PBS-treated mice. For our small intestine samples, we observed a significantly higher abundance of Akkermansia sp. in the HD-5_1–9–treated than in the PBS-treated group (P = 0.008).