双组份系统CpxA/CpxR介导胸膜肺炎放线杆菌抗热应激的分子机制研究

胸膜肺炎放线杆菌（APP）是一种能够引起猪传染性胸膜肺炎的重要呼吸道传染病原菌，在感染过程中会遭遇来自宿主体内外环境中的各种热应激。我们前期研究发现，双组份CpxA/CpxR通过调控IVa型菌毛蛋白ApfA介导APP抗热应激，但具体机制未知。因此，本研究将在前期工作基础上，通过生物质谱、显微成像、免疫生物学手段解析CpxA/CpxR与ApfA在热应激条件下维持细胞内蛋白质稳态的协同机制；利用EMSA、细菌单杂交、DNaseI足迹法、定点突变、Capping-RACE等分子生物学技术，阐释CpxA/CpxR双组份调控ApfA表达的分子细节；阐明APP通过CpxA/CpxR-ApfA信号途径抗热应激的分子机制以及感染宿主的生物学意义，从而为研发抗细菌药物和疫苗防疫提供新的策略和思路。

Actinobacillus pleuropneumoniae (APP), an important endemic pathogen, is the causative agent of porcine pleuropneumonia (PCP) responsible for huge economic losses to the pig industry worldwide. This pathogen is well-accommodated to diverse physiological environments, particularly with the ability to resist heat stress conditions present in the environment and in the host. Our previous study demonstrated that CpxA/CpxR is involved in the heat stress resistance of APP by influencing the expression of the type IVa pilus protein ApfA. However, the underlying mechanism remains unknown. In this study, antibody preparation, MALDI-TOF, Western blot and transmission electron microscopy will be employed to reveal the synergistic mechanism between CpxA/CpxR and ApfA under heat stress conditions to maintain proper cellular protein homeostasis. Furthermore, EMSA, bacterial one-hybrid, DNase I, site-directed mutagenesis and Capping-RACE assays will be performed to identify apfA regulation triggered by CpxR. This study will shed light on the mechanism of roles of CpxA/CpxR-ApfA signal transduction pathway under heat stress conditions, which will be helpful for APP to establish infection within the host, paving a new way for the development of vaccine to combat PCP.