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

人体表面长期受到微生物定植的挑战。为了保护自己免受感染，这些表面产生内源性抗生素，即所谓的抗菌肽（AMP）。由于其广泛的活性范围，这些AMP被认为是用于开发新的抗生素药物的合适候选物。因此，在胃、胆囊和胆管中鉴定新的AMP，以及更好地理解已知的AMP，是我们项目的一个目标。为此，我们将从相应的组织中提取蛋白质，通过液相色谱法对其进行分离和纯化，并分析其对真菌和病原菌的抗菌活性。正如我们最近所展示的，人β-防御素1（hBD-1）和人防御素6（HD-6）的抗微生物活性显著依赖于环境条件。因此，我们将在我们的实验中评估氧化还原状态，pH值和盐浓度。此外，我们可以表明，还原（活性）hBD-1可以降解内源性蛋白酶和十二指肠分泌，如。G.在胃里。这是不可能使用氧化的，几乎无活性的hBD-1形式。还原的hBD-1的降解产生了一个片段（八肽），它与hBD-1羧基末端的最后8个氨基酸相关，并显示出与全长肽相似的抗菌活性谱。通过化学修饰可以提高多肽的稳定性，改变抗菌范围。由于病原体对抗生素药物的耐药性增加是我们工作重要性的主要原因，我们的目标是调查对AMP的耐药性发展的风险。我们建议的另一个目标是进一步表征八肽和其他AMP，在细胞毒性和免疫原性方面，以评估其在人类中应用的可能性。在这种情况下，我们希望将肽应用于细胞培养物（体外）以及离体人全血模型。将通过乳酸脱氢酶定量和WST试验测定细胞毒性。为了分析非特异性免疫原性，我们将通过流式细胞术研究淋巴细胞表面的特异性标记分子（CD 69），并通过ELISA定量细胞因子释放。 最后，我们希望证明我们的肽在各种感染模型中的效率，在体外（细胞培养）和体内（大蜡螟的幼虫）。总之，通过这项提案，我们的目标是提高对内源性抗菌物质的理解，以开发新的抗菌成分。

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.