LAUSR

Significance Paneth cells provide intestinal host defense against pathogens and control the healthy microbiota by secreting antimicrobial peptides. We show that the most abundant secreted Paneth cell products, human defensin HD-5 and HD-6, show a distinct susceptibility to proteolytic digestion by human duodenal fluid. While HD-5 is digested in many fragments, HD-6 is stable and still able to form nanonets. The occurring fragments of HD-5 were antimicrobially active against microorganisms. We provide proof of concept about microbiome modulating capacities in vivo, which includes an increase of Akkermansia sp. Our results indicate that fragmentation of defensins increases antimicrobial diversity and further adds to the complexity of host microbial interaction at interfaces. Fragmentation could lead to new antimicrobial peptides with possible therapeutic usage. 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.