人α-防御素-1(HNP-1)抗菌新机制的定量蛋白质组学研究

The widespread use and abuse of the antibiotics led to treatment failure for the infection, which had developed resistance to those commercially available drugs. Antibiotic resistance has become a serious problem for public health worldwide. α-defensin-1, also termed as the human neutrophil protein 1 (HNP -1), has a wide range of antibacterial spectrum and strong antibacterial activity, may become a new generation of antibiotics. However it hs been extremely difficult so far to directly express the mature and active form in recombinant Escherichia coli strains because of its toxicity to the host cells. This is also impeding the in-depth research on antimicrobial mechanisms. Most of the investigators believed that the low expression of α-defensin-1 was resulted from the bactericidal effect of the precursor of HNP-1, i.e. pre-pro-HNP-1. The significant efforts has been put on the optimizing of expression vectors and fusion expression of HNP-1 to diminish the bactericidal activity against the host, but the effect was limited. We found that the mature HNP-1 can be directly and efficiently expressed in recombinant Escherichia coli strain XPX-1 carrying pre-pro-HNP-1 gene. More importantly, the expressed endogenous mature HNP-1 could kill the host more efficiently compared with the same amount of exogenous ones by inducing apoptosis, which is different with the previous hypothesis of cell lysis through hole punch of HNP-1 on the cell membrane. To further understand the mechanism of HNP-1 on the new phenomemom of bactericidal effect, we proposed the SILAC based deep coverage quantitative proteomics established in the lab to compare the XPX-1 proteome samples with or without IPTG inducing for endogenous mature HNP-1. To identify the potential target of mature HNP-1, we planed to investigate the immunoprecipitated samples from IPTG induced XPX-1 and its control as well as the samples collected at different time points after inducing. The novel targets and mechanism of bactericide of mature HNP-1 reveals here may not only support new drugs for resistence bacteria but also shed light on the improvement of production for mature HNP-1 with recombinant Escherichia coli in the near future.

抗生素耐药已成为危害全球公共卫生的严重问题。人α-防御素-1（HNP-1）是人中性粒细胞的主要防御素分子，抗菌谱广，抗菌活性高，但由于其内源表达和大量获得难，阻碍了抗菌机制的基础研究和应用开发。我们发现重组表达pre-pro-HNP-1的大肠杆菌可直接有效可溶表达具抗菌活性的成熟HNP-1，且通过引发宿主细胞凋亡实现抗菌作用，活性显著高于相同剂量外源HNP-1，修正了其主要通过正电荷穿膜导致细胞内容物渗漏杀菌的假说。本申请旨在利用大肠杆菌SILAC完全标记专利技术和正反标策略、深度覆盖精准定量蛋白质组技术、高分辨精准相互作用蛋白质组技术和时间序列相互作用蛋白质组设计，鉴定成熟HNP-1的底物群和受累信号通路，揭示成熟HNP-1的可能新靶点和新抗菌机制，探索其抗耐药菌的可能性和直接高效表达的新途径，为抗耐药菌抗生素的研发和HNP-1物种进化研究提供原始创新性成果和数据支撑。

抗生素耐药已成为危害全球公共卫生的严重问题。人α-防御素-1（HNP-1）是人中性粒细胞的主要防御素分子，具有抗菌谱广、抗菌活性高的优点，但由于其内源表达和大量获得难，阻碍了抗菌机制的基础研究和应用开发。我们发现重组表达pre-pro-HNP-1的大肠杆菌可直接有效可溶表达生成具抗菌有生物学活性的可溶性成熟HNP-1；内源生成的成熟HNP-1通过引发宿主细胞凋亡实现抗菌作用，活性显著高于相同剂量外源HNP-1，修正了其主要通过正电荷穿膜导致细胞内容物渗漏杀菌的假说。本申请旨在利用自主知识产权非营养缺陷型大肠杆菌SILAC蛋白质组完全标记专利技术、SILAC和正反标策略、深度覆盖精准定量蛋白质组技术、高分辨精准相互作用蛋白质组技术和时间序列相互作用蛋白质组设计，鉴定成熟HNP-1的底物蛋白群和受累信号通路，揭示成熟HNP-1的可能新靶点和新抗菌机制，探索其抗耐药菌的可能性及其分子机制，并尝试和直接高效表达成熟HNP-1的新途径，为抗耐药菌抗生素的研发和HNP-1物种进化研究提供原始创新性成果和数据支撑。