Defensins protect human barriers from commensal and pathogenic microorganisms. Human α-defensin 6 (HD-6) is produced exclusively by small intestinal Paneth cells but, in contrast to other antimicrobial peptides (AMPs) for HD-6, no direct antibacterial killing activity has been detected so far. Herein, we systematically tested how environmental factors, like pH and reducing conditions, affect antimicrobial activity of different defensins against anaerobic bacteria of the human intestinal microbiota. Remarkably, by mimicking the intestinal milieu we detected for the first time antibacterial activity of HD-6. Activity was observed against anaerobic gut commensals but not against some pathogenic strains. Antibiotic activity was attributable to the reduced peptide and independent of free cysteines or a conserved histidine residue. Furthermore, the oxidoreductase thioredoxin, which is also expressed in Paneth cells, is able to reduce a truncated physiological variant of HD-6. Ultrastructural analyses revealed that reduced HD-6 causes disintegration of cytoplasmic structures and alterations in the bacterial cell envelope, while maintaining extracellular net-like structures. We conclude that HD-6 is an antimicrobial peptide. Our data suggest two distinct antimicrobial mechanisms by one peptide: HD-6 kills specific microbes depending on the local environmental conditions, whereas known microbial trapping by extracellular net structures is independent of the reducing milieu.

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.

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Microbial resistance against clinical used antibiotics is on the rise. Accordingly, there is a high demand for new innovative antimicrobial strategies. The host-defense peptide human beta-defensin 1 (hBD-1) is produced continuously by epithelial cells and exhibits compelling antimicrobial activity after reduction of its disulphide bridges. Here we report that proteolysis of reduced hBD-1 by gastrointestinal proteases as well as human duodenal secretions produces an eight-amino acid carboxy-terminal fragment. The generated octapeptide retains antibiotic activity, yet with distinct characteristics differing from the full-length peptide. We modified the octapeptide by stabilizing its termini and by using non-natural D-amino acids. The native and modified peptide variants showed antibiotic activity against pathogenic as well as antibiotic-resistant microorganisms, including E . coli , P . aeruginosa and C . albicans . Moreover, in an in vitro C . albicans infection model the tested peptides demonstrated effective amelioration of C . albicans infection without showing cytotoxity on human cells. In summary, protease degradation of hBD-1 provides a yet unknown mechanism to broaden antimicrobial host defense, which could be used to develop defensin-derived therapeutic applications.