Final Report Summary - DEFENSINACTIVITY (The influence of environmental factors on antimicrobial activity of human intestinal defensins)
The human body is constantly exposed to a multitude of microorganisms. Antimicrobial peptides (AMPs), such as defensins, protect human surfaces from commensal and pathogenic microorganism and shape the microbiota at different sites. As such, antimicrobial defensins are an integral part of natural innate barrier protection. Compromised expression and function of this defense is linked to different chronic inflammatory disorders such as inflammatory bowel diseases. In this project, we contributed to establish the understanding of how strong local environmental conditions can influence host defense and microbiome shaping via complex posttranslational changes of antimicrobial host defense peptides. We hypothesized that local conditions like pH and reducing environment which can be found in the absence of oxygen can affect antimicrobial activity of different defensins against different bacteria. For this purpose, we studied human redox-systems in human intestinal samples for their ability to reduce native hBD1 (hBD1ox). We found two redox-systems catalyzing this conversion: thioredoxin-1 (TRX) was most efficient, while glutarredoxin-1 (GRX) also showed efficiency but to a lower extend. This reaction takes place at the surface of epithelia cells in extracellular colonic mucus and is mediated mainly by the TRX-system. Furthermore, in this project we found a compromised expression of TRX in human colonic biopsies from inflammatory bowel disease patients, providing a new mechanism of attenuated host defense in disease. We extended these investigations including samples from Crohn’s disease (CD) and ulcerative colitis (UC) patients, differentiating between acute inflammation and non-inflamed tissue from colon and ileum. Our results revealed a reduced mRNA expression of TRX in the colon of CD and UC patients and an increased expression of GRX. In healthy controls we confirmed the co-localization of reduced hBD1 and TRX localize to extracellular colonic mucus. By mimicking the intestinal milieu, we tested the effect of a mild acidic pH and used anaerobic bacterial stains, which are commensal in the human intestine. For the first time, we described potent antibacterial activity of Paneth cell derived peptide HD6. Of note, the activity of HD6 is strongly depends on reduction, similar to hBD1. While incubation of human with HD5 resulted in a multitude of shorter HD5 fragments, HD6 appeared resistant to proteolytic degradation. In contrast, the human beta defensin 1 (hBD1) is continuously produced and we could show that after reduction of its disulfide bridges, it becomes a potent antimicrobial agent against bacteria, while the oxidized form (hBD1ox) shows a specific activity against gram-negative bacteria, depending on aerobic growth. We identified that hBD1ox could use an outer membrane protein FepA to enter the periplasmic space, where its activity depends on presence of periplasmic redox proteins DsbA and DsbB. Taken together, the results of our research in the reporting time support our hypothesis that AMP activity depends on environmental conditions found at the given body site. We believe that better understanding of the mode of action will help to find new antimicrobial strategies against upcoming multi resistant bacteria.