Secreted enteric antimicrobial activity localises to the mucus surface layer

Objectives: The intestinal mucosa is constantly exposed to a dense and highly dynamic microbial flora and challenged by a variety of enteropathogenic bacteria. Antibacterial protection is provided in part by Paneth cell-derived antibacterial peptides such as the alpha-defensins. The mechanism of peptide-mediated antibacterial control and its functional importance for gut homeostasis has recently been appreciated in patients with Crohn's ileitis. In the present study, the spatial distribution of antimicrobial peptides was analysed within the small intestinal anatomical compartments such as the intestinal crypts, the overlaying mucus and the luminal content. Methods: Preparations from the different intestinal locations as well as whole mouse small intestine were extracted and separated by reversed-phase high-performance liquid chromatography. Antibacterial activity was determined in extracts, and the presence of antimicrobial peptides/proteins was confirmed by N-terminal sequencing, mass spectrometry analysis and immunodetection. Results: The secreted antibacterial activity was largely confined to the layer of mucus, whereas only minute amounts of activity were noted in the luminal content. The extractable activity originating from either crypt/mucus/lumen compartments respectively (given as a percentage) was for Listeria monocytogenes, 48 (4)/44 (4)/8 (8); Enterococcus faecalis, 44 (10)/49 (3)/7 (7); Bacterium megaterium, 56 (4)/42 (3)/2 (1); Streptococcus pyogenes, 48 (4)/46 (3)/6 (6); Escherichia coli, 46 (4)/47 (3)/7 (7); and Salmonella enterica sv. Typhimurium, 38 (3)/43 (7)/19 (10). A spectrum of antimicrobial peptides was identified in isolated mucus, which exhibited strong and contact-dependent antibacterial activity against both commensal and pathogenic bacteria. Conclusion: These findings show that secreted antimicrobial peptides are retained by the surface-overlaying mucus and thereby provide a combined physical and antibacterial barrier to prevent bacterial attachment and invasion. This distribution facilitates high local peptide concentration on vulnerable mucosal surfaces, while still allowing the presence of an enteric microbiota.