氨基酸饥饿状态下(p)ppGpp调控胸膜肺炎放线杆菌致病性研究

Actinobacillus pleuropneumoniae （APP）is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease leading to great economic losses to the global swine industry. The strigent response regulated by (p)ppGpp is one of the strategies adopted by bacteria when encounted enviromental and vegetative stress. (p)ppGpp is a well known intracellular signal that mediates bacterial stringent response to environment stresses through the change of its concentration level thus controls many important cellular processes for bacterial survival. S-8 strain was isolated from swine lung in China by our lab, RelA and SpoT, which encodes both (p)ppGpp synthetase and hydrolase, were identified from the draft genome sequence sequenced by our lab, ppGpp is thought to bind to and alter the affinity of RNA polymerase for various promoters, such as those associated with adaptation to adverse conditions. However, whether the ppGpp also could regulate the duration ability to hash environments and pathogenesis of Actinobacillus pleuropneumoniae under nutrient deprivation in lung was unknown. In this project, we constructed the mutants deficient in stringent response by homologous recombination, and then analyze the physiological function of ppGpp by comparing the growth character,the ability of forming biofilm, the carbohydrate metabolic ability,the morphology change of wild-type and mutant under different conditions, and analyze the regulatory mechanism by identifying the difference of proteome and transcriptome, and determined the influence to pathogenesis by infected piglets with wild-type and mutant. We hope that our work could shed new light to the pathogenesis of APP by investigating the physiological function and regulatory mechanism of ppGpp, and then providing new theoretical foundation and ideas for the control, drug discover, vaccine development of APP.

(p)ppGpp 介导的严谨反应是细菌应对环境或营养胁迫的多种策略之一，对逆境生存起到了重要作用。本实验室通过对胸膜肺炎放线杆菌(APP)S-8株全基因组测序，鉴定到与启动细菌严谨反应的信号素分子[(p)ppGpp]合成与分解相关的relA/spoT基因，其是否能够调控APP在肺部氨基酸饥饿状态下的宿主适应性及致病性，目前尚不清楚。本项目拟通过接合转移同源重组的方法，构建S-8株的relA/spoT双基因缺失株，通过比较野生株、缺失株及功能互补株在饥饿状态下生长曲线、生物被膜形成能力、糖代谢能力、菌体形态等生理适应性差异，分析ppGpp的生理功能；通过比较仔猪感染试验，确定ppGpp对APP致病性的影响；通过比较转录组及蛋白质组学差异，探讨其调控机制。通过对ppGpp的生理功能和调控机制的研究，增加对APP适应性及致病机制的认识，为猪传染性胸膜肺炎的防治及疫苗研究提供新的理论基础和思路。

(p)ppGpp 介导的严谨反应是细菌应对环境或营养胁迫的多种策略之一，对逆境生存起到了重要作用。本实验室通过对胸膜肺炎放线杆菌(APP)S-8株全基因组测序，鉴定到与启动细菌严谨反应的信号素分子[(p)ppGpp]合成与分解相关的relA/spoT 基因，其是否能够调控APP 在肺部氨基酸饥饿状态下的宿主适应性及致病性，目前尚不清楚。本项目通过接合转移同源重组的方法，构建S-8 株的relA基因（负责合成ppGpp）缺失株，然后比较了野生株、缺失株及功能互补株在饥饿状态下生长曲线、生物被膜形成能力、糖代谢能力、菌体形态等生理适应性差异，发现ΔrelA突变株与野生型相比较生长速率降低，24小时后活菌数大大降低，表明ppGpp能够影响APP的生长。电镜观察结果表明野生型随着培养时间的延长菌体逐渐拉长，ΔrelA突变株无此现象；ΔrelA突变株与野生型相比较具有更强的形成生物膜能力并丧失了利用多种糖的能力；氨基酸缺失实验结果表明，常见的20种氨基酸中有10种氨基酸是可以作为营养饥饿的条件的信号分子被胸膜肺炎放线杆菌感知，启动严谨效应。通过测定relA基因缺失后ΔrelA突变株与野生株的半数致死量发现，ΔrelA突变株的毒力降低3倍；同时对肺泡巨噬细胞（PAM）侵袭能力降低。同时组学分析表明，与野生型相比，突变株体内共有405个基因差异表达，其中380个上调，25个下调。上调表达差异表达基因主要涉及核糖体结构和形成；氨基酸的转运和代谢；细胞壁（膜）合成。蛋白质组学分析结果基本与转录组结果相吻合。以上数据表明当APP在肺部环境下10种氨基酸可以作为营养饥饿的信号分子启动严谨效应合成(p)ppGpp，该小分子调控下游参与氨基酸代谢相关的基因，调节其生长速率，改变生物膜形成状态，同时下调毒力基因的表达影响其致病能力，初步阐明胸膜肺炎放线杆菌在氨基酸饥饿状态下的致病机制。