Date Published: March 21, 2017
Publisher: Public Library of Science
Author(s): Judith Raschig, Daniela Mailänder-Sánchez, Anne Berscheid, Jürgen Berger, Adam A. Strömstedt, Lioba F. Courth, Nisar P. Malek, Heike Brötz-Oesterhelt, Jan Wehkamp, Denise M. Monack.
Ever since the discovery of endogenous host defense antimicrobial peptides it has been discussed how these evolutionary conserved molecules avoid to induce resistance and to remain effective. Human ß-defensin 1 (hBD1) is an ubiquitously expressed endogenous antimicrobial peptide that exhibits qualitatively distinct activities between its oxidized and reduced forms. Here, we explore these antimicrobial mechanisms. Surprisingly, using electron microscopy we detected a so far unknown net-like structure surrounding bacteria, which were treated with the reduced but not the oxidized form of hBD1. A transmigration assay demonstrated that hBD1-derived nets capture bacteria and inhibit bacterial transmigration independent of bacterial killing. The presence of nets could completely prevent migration of hBD1 resistant pathogens and are stable in the presence of human duodenal secretion with a high amount of proteases. In contrast to HD6, cysteins are necessary for net formation. This redox-dependent function serves as an additional mechanism of action for hBD1 and differs from net formation by other defensins such as Paneth cell-derived human α-defensin 6 (HD6). While hBD1red and hBD1ox have distinct antimicrobial profiles and functions, only the reduced form provides additional host protection by entrapping bacteria in extracellular net structures preventing bacterial invasion. Better understanding of the modes of action of endogenous host peptides will help to find new antimicrobial strategies.
The innate immune system is the primary barrier against commensal invasion and microbial infections. Antimicrobial peptides (AMPs) are key effector molecules of the innate immune system protecting the human body from bacterial overgrowth, thereby retaining a balanced microbiota and fending off commensals and pathogens [1,2]. Some of the most important molecules within the innate immune system are the defensins. These are small cationic peptides of about 3 kDa characterized by three intramolecular disulphide-bridges [3–5]. According to the arrangement of these disulphide-bonds, defensins are classified as alpha- and beta-defensins, respectively . Defensins show antimicrobial activity against various microbes, including bacteria, fungi and some viruses, whereby they shape the composition of the human microbiota . While native human alpha-defensins 5 and 6 (HD5/ HD6) are expressed by Paneth cells in the small intestine and human beta-defensin 2 (hBD2) is mainly induced in case of inflammatory situations, human beta-defensin 1 (hBD1) is constitutively expressed by all human epithelia [8–10], indicating its important role in innate immunity. In a previous study, we could show that hBD1red is localized in human extracellular colonic mucus, suggesting that hBD1 can prevent infections in vivo . However, the antimicrobial activity of hBD1 was previously undervalued. By using reducing conditions, as they are found in the human intestine, we could show antimicrobial activity of hBD1 against numerous members of the human intestinal microbiota  as well as against several pathogenic bacteria. In contrast, oxidized hBD1 (hBD1ox), which contains three closed disulfide bonds, showed no antimicrobial activity against most microbes, with the exception of a few Gram-negative bacteria including E. coli . It is known that different AMPs have different bacterial target sites or mechanisms of activity, as recently reviewed by Brogden . For example, Sass et al. showed that human ß-defensin 3 kills S. aureus by interfering with membrane-bound multienzyme-machineries such as the electron transport chain and the cell wall biosynthesis complex . Chileveru et al. described a different mechanism for human Paneth cell alpha-defensin 5 (HD5) . They reported that HD5 enters the cytoplasm of E. coli and other Gram-negative bacteria, leading to morphological changes like blebbing, clumping and cell elongation , followed by the loss of bacterial viability. Paneth cell HD6 was reported to form net-like structures, entrapping S. typhimurium, thereby preventing the translocation of these bacteria into the intestinal epithelium .
If a conventional antibiotic is used repeatedly, microbial resistance against this antibiotic is the rule rather than the exception. Multiresistant bacteria are increasing and more and more become a serious threat for our world in the modern antibiotic era [25,26]. Ever since the discovery of endogenous host defense antimicrobial peptides it has been discussed why these evolutionary conserved molecules do not evoke resistance, especially in a constant use situation. For a long time after the discovery of hBD1, scientists wondered why an antimicrobial peptide exhibits so little antimicrobial activity. HBD1 is likely one of the most abundant known antimicrobial peptides because it is constantly expressed not only by all epithelial surfaces but also by circulatory cells and cells of the reproductive tract. Polymorphisms of hBD1 are associated with an inefficiency to clear potential harmfully microbes like MRSA , and various diseases like periodontitis , psoriasis , colonic inflammatory bowel diseases [30,31] and infertility . Recently, we have shown that hBD1 becomes a potent AMP after reduction of its disulphide bonds . Furthermore, we could show that this reduction can be performed by the thioredoxin system, which is present in the human intestine, where it co-localizes with hBD1red . The data provided here demonstrate additional fundamental principles of the permanent war at mucosal surfaces where the host has to be protected against pathogens as well commensal overpopulation . These data highlight the contrast between most conventional antibiotics, primarily of bacterial origin, and human endogenous host defense molecules such as hBD1, the latter providing multifunctional activities, including distinct antimicrobial mechanisms from a single peptide. Herein, we show that, post-translationally, hBD1 forms functionally distinct structures, which are controlled by the local microenvironment. Our data show that hBD1ox is only active against some Gram-negative species (S. enteritidis, E. coli) in (solid) radial diffusion assays (Fig 1A). Interestingly, E. coli showed decreased sensitivity to hBD1ox in a turbidity (liquid) broth assay. While hBD1red was bactericidal and sterilized the culture, viable cells remained in the presence of hBD1ox and the treated culture eventually reached the same turbidity as the untreated control (Fig 1B + 1C).