PhoP/PhoQ介导禽致病性大肠杆菌抑制巨噬细胞自噬-溶酶体降解途径的分子机制

Avian Pathogenic Escherichia coli (APEC) is an important pathogen of poultry. Intracellular macrophage survival is a key part of APEC pathogenicity during the early stages of avian colibacillosis. Research currently available is not sufficient to reveal the molecular mechanisms for APEC blocking macrophage autophagosome fusion with degradative lysosome. HlyF-mediated APEC outer membrane vesicles (OMVs) secretion as the research entry point, the applicant proposes to explore the mechanism for PhoP/PhoQ-HlyF signaling pathway to regulate APEC OMVs secretion by Northern Blot, EMSA and qPCR. To identify that whether phoP and hlyF deletion could have effect on blocking macrophage autophagosome fusion with the lysosome during APEC intracellular survival by immunofluorescence labeling. Isolation and identification of APEC OMVs containing secretory proteins by mass spectrometry, and confirming that the PhoP/PhoQ-HlyF pathway could regulate the transcriptional expression of OMVs-related secretory proteins. The research is trying to confirm that APEC PhoP/PhoQ system might be involved in blocking fusion of the autophagosome with the lysosome. A better understanding of the mechanisms by which APEC manipulate autophagy will contribute to anti-bacterial drugs/vaccines design to defense APEC infections.

禽致病性大肠杆菌(APEC)是禽的重要细菌性病原,在禽巨噬细胞内存活是APEC致病的关键环节，目前研究成果不足以阐明APEC抑制巨噬细胞自噬-溶酶体降解途径的分子机制。在已有研究的基础上,申请者拟以HlyF介导APEC外膜囊泡(OMVs)分泌为研究切入点，通过EMSA、Northern Blot和qPCR等分子生物技术鉴定PhoP/PhoQ二元系统调控HlyF表达的分子机制，揭示APEC调控外膜囊泡(OMVs)分泌的PhoP/PhoQ-HlyF信号途径；通过免疫荧光标记等技术探究phoP和hlyF等基因缺失对APEC抑制巨噬细胞自噬-溶酶体降解的影响，质谱测序分析APEC OMVs含有的分泌性蛋白质，鉴定PhoP/PhoQ二元系统是否调控OMVs分泌性蛋白质的转录表达；阐明PhoP/PhoQ介导APEC抑制巨噬细胞自噬-溶酶体降解途径的分子机制，从而为研发抗细菌药物和疫苗防疫提供潜在靶标。

通过EMSA和qPCR等分子生物技术鉴定PhoP/PhoQ二元系统调控HlyF表达的分子机制，识别PhoP转录结合hlyF基因的启动子序列。提取APEC野生株、缺失株和各个基因互补株的OMVs，通过透射电镜鉴定发现缺失PhoP和HlyF能显著降低APEC的外膜囊泡分泌水平，而过表达HlyF能显著提高外膜囊泡的分泌水平，研究揭示PhoP-PhoQ能通过调控HlyF的表达来控制APEC外膜囊泡的表达，本研究首次发现了细菌可以通过二元调控系统操纵OMVs的分泌。吞噬溶酶体是细菌主要的杀菌途径，能够在巨噬细胞内存活的致病菌往往能进行吞噬溶酶体逃匿，研究发现缺失PhoP和HlyF同样能显著提高ExPEC与吞噬溶酶体标记物的共定位水平。APEC在巨噬细胞内增殖时，PhoP-PhoQ能通过调控HlyF的表达进行吞噬溶酶体逃匿，即能阻碍吞噬-溶酶体降解途径。通过免疫荧光试验鉴定发现缺失PhoP和HlyF能显著降低ExPEC与LC3的共定位率，由于ExPEC具有在巨噬细胞内存活的能力，推测APEC可能利用自噬过程进行胞内存活。本研究发现了新的APEC感染巨噬细胞的调节通路，APEC通过二元调控系统PhoP/PhoQ操纵OMVs的分泌，OMVs能介导APEC在巨噬细胞内的存活和增殖，APEC可以通过产生OMVs而引起巨噬细胞损伤，OMVs可能促进APEC从吞噬溶酶体逃匿至细胞质。上述结果揭示APEC感染巨噬细胞的重要分子机制，为APEC新药物靶标的筛选和弱毒疫苗的开发提供理论依据。