多杀性巴氏杆菌Lon蛋白酶的致病机理研究

Pasteurella multocida, a ubiquitous animal pathogen that is associated with a range of diseases including hemorrhagic septicemia in cattle and buffalo, atrophic rhinitis in swine, and fowl cholera in poultry, and snuffles in rabbits. The lon gene of P. multocida in infected rabbit livers was identified and compared with that from in vitro culture by selective capture of transcribed sequences. The ATP-dependent Lon protease plays the major roles in proteins quality control and the specific control of several regulatory proteins and associates with the virulence of bacteria. This research was to construct the lon delete mutant strain of P. multocida strain, C51-17, and evaluate its pathogencity in rabbits. The growth rates and biofilm formations of lon delete mutant strain were tested on stress conditions, for example, high temperature, H2O2 concentrate and acid tolerance. The roles of adherence and invade to cell of mutant strain was checked in rabbit kidney cell line (RK-13). The relations of Lon protease and other proteins were analysis by RNA-sequences and differentially expressed genes were verified by real-time reverse transcriptase-polymerase chain reaction. These results were contributed to reveal the pathogenesis and regulations of Lon protease in P. multocida.

多杀性巴氏杆菌能够引起多种动物感染的一种急性、败血性细菌传染病的病原菌，利用选择性捕获转录序列技术对多杀性巴氏杆菌感染兔肝脏组织筛选到的差异表达基因中有lon基因。Lon是一种ATP依赖的蛋白酶，可降解异常蛋白和特异性调控许多调控蛋白，与病原菌的致病机制有关。本研究拟以A型兔多杀性巴氏杆菌C51-17为研究对象，构建lon缺失突变株和互补突变株，研究其在多杀性巴氏杆菌中的致病作用以及突变株在温度、H2O2、酸性环境等应激条件下的生长状态和生物被膜形成状态，验证突变株的细菌粘附和侵袭细胞作用。利用转录组学测序检测Lon对其他蛋白的影响和荧光定量PCR方法验证相关蛋白的基因表达水平的变化。本研究将阐明Lon蛋白酶在多杀性巴氏杆菌中的致病作用和初步验证其对相关蛋白的调控作用，也为进一步验证Lon蛋白酶对相关蛋白的调节机制奠定基础。

多杀性巴氏杆菌属于巴氏杆菌科巴氏杆菌属，是革兰氏阴性菌，能够引起多种动物感染的一种急性、败血性细菌传染病的病原菌，利用选择性捕获转录序列技术对多杀性巴氏杆菌感染兔肝脏组织筛选到的差异表达基因中有 lon 基因。 Lon 是一种 ATP 依赖的蛋白酶，可降解异常蛋白和特异性调控许多调控蛋白，与病原菌的致病机制有关。本研究主要以 A 型兔多杀性巴氏杆菌 C51-17 为研究对象，扩增表达了Lon蛋白，分析其ATPase活性；构建了lon缺失突变株，研究突变株遗传稳定性、致病性等生物学特性；利用转录组学测序检测了 Lon 对其他蛋白的影响和荧光定量 PCR 方法验证了相关蛋白的基因表达水平的变化。利用PCR克隆表达了多杀性巴氏杆菌C51-17株lon基因，SDS-PAGE结果表明，重组Lon蛋白分子量大小97 ku，主要以可溶性蛋白形式表达；western blot检测结果表明，重组蛋白能够被P.multocida阳性血清识别。纯化的重组蛋白Lon具有ATPase活性，其ATPase的酶活性可达到27 708.47µmol/mg•min。扩增C51-17株lon基因的上、下游同源臂，将卡那霉素抗性基因表达盒克隆至lon的上、下游同源臂之间，构建重组转移质粒pBC-Δlon。电转化至C51-17感受态细胞中，成功构建了多杀性巴氏杆菌Δlon突变菌。Δlon突变株具有良好的遗传稳定性和生长特性；生化实验结果表明，Δlon突变菌与亲本菌的生化特性一致。Δlon突变菌和亲本菌对RK13细胞的粘附分别为9.10±0.75×104 CFU/孔和9.7±0.69×103CFU/孔。小鼠的致病性试验结果表明，Δlon突变株的LD50为63.1 CFU，而亲本菌C51-17的的LD50为26.24 CFU。提取多杀性巴氏杆菌亲本菌C51-17和Δlon突变株的RNA，进行转录组测序，结果表明：发现上调基因180个，下调基因144个。利用荧光定量PCR验证部分基因表达量与转录组测序的结果相一致。本研究将阐明 Lon 蛋白酶在多杀性巴氏杆菌中的致病作用和初步验证其对相关蛋白的调控作用， 也为进一步验证 Lon 蛋白酶对相关蛋白的调节机制奠定基础。