Identification, isolation and characterisation of novel antimicrobial peptides from human tissue as well as the development of therapeutically applicable lead structures

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Human surfaces are permanetly challenged by microbial colonization. To protect themselves against infections, these surfaces produce endogenous antibiotics, the so called antimicrobial peptides (AMPs). Because of their broad range of activity these AMPs are considered to be suitable candidates for the development of new antibiotic drugs. Therefore, the identification of new AMPs in the stomach, the gall and the bile ducts, as well as a better understanding of already known AMPs, is one goal of our project. For this purpose we will extract proteins from the respective tissues, separate and purify them by liquid chromatography and analyze them for antimicrobial activity against commensal and pathogenic bacteria. As we could show recently, the antimicrobial activity of human beta-defensin 1(hBD-1) and human defensing 6 (HD-6) depends significantly on environmental conditions. Therefore, we will include to asses redox-status, pH and salt concentrations in our experiments. Furthermore we could show that reduced (active) hBD-1 can be degraded by endogenous proteases and duodenal secretion, e. g. in the stomach. This is not possible using the oxidized, almost inactive, form of hBD-1. Degradation of reduced hBD-1 generated a fragment (octapeptide), which correlates to the last eight amino acids of hBD-1 carboxy terminus and shows a similar spectrum of antimicrobial activity as the full-length peptide. By chemical modification we could increase peptide stability and change the antimicrobial range. As increasing resistance of pathogens against antibiotic drugs is a major reason for the importance of our work, we aim to investigate the risk of resistance development against AMPs. Another goal of our proposal is the further characterization of the octapeptide, and other AMPs, in terms of cytotoxicity and immunogenicity, to evaluate the possibility for their application in humans. In this context, we want to apply the peptides to cell cultures (in vitro) as well as to an ex vivo human whole blood model. Cytotoxicity will be determined by quantification of lactate dehydrogenase and WST-assay. To analyze unspecific immunogenicity we will investigate a specific marker molecule on the surface of lymphocytes (CD69) by flow cytometry and quantify cytokine release by ELISA. Finally, we want to prove the efficiency of our peptides in various models of infection, in vitro (cell culture) and in vivo (larvae of the greater wax moth Galleria mellonella). Taken together, with this proposal we aim to improve the understanding of endogenous antimicrobial substances with the goal of developing new antimicrobial components.