Wang Lei, Qin Wanhai, Ruidong Zhai, Liu Shiting, Zhang Hu, Sun Changjiang, Feng Xin, Gu Jingmin, Du Chongtao, Han Wenyu, Langford P R, Lei Liancheng
College of Veterinary Medicine, Jilin University, Changchun, PR China.
Section of Paediatrics, Imperial College London, London, UK.
Microb Pathog. 2015 Jan;78:74-86. doi: 10.1016/j.micpath.2014.11.017. Epub 2014 Nov 28.
Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is the causative agent of porcine pleuropneumonia, a disease that causes serious problems for the swine industry. Successful infection by this bacterium requires breaking the first line of defence in the lungs, the primary alveolar macrophages (PAMs). Therefore, exploring A. pleuropneumoniae-PAM interactions will provide vital groundwork for the scientific control of this infectious disease, which has been little studied up to now. In this work, PAMs were isolated from piglets and co-incubated with A. pleuropneumoniae serovar 5b strain L20 in vitro, and their interaction, PAM cell death, and differential gene expression of A. pleuropneumoniae in response to PAM cell death were observed and analysed using confocal microscopy, electron microscopy, RT-PCR, Western blot, flow cytometry and the use of a gene expression profile chip. A. pleuropneumoniae quickly adhered to and invaded PAMs, inducing apoptosis, which was confirmed using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The highest percentage of apoptosis in cells was confirmed using flow cytometry when the cells were infected at a multiplicity of infection (MOI) of 10 and incubated for 5 h, with higher expression of activated caspase-3 as measured by Western blot. Using microarray gene chips with 2868 probes containing nearly all of the genomic sequence of A. pleuropneumoniae serotype 5b strain L20, a total of 185 bacterial genes were found to be differentially expressed (including 92 up-regulated and 93 down-regulated genes) and involved in the process of apoptosis, as compared with the expression of control bacteria cultured without PAMs in BHI medium (mean expression ratios >1.5-fold, p < 0.05). The up-regulated genes are involved in energy metabolism, gene transcription and translation, virulence related gene such as LPS, Trimeric Autotransporter Adhesin, RTX and similar genes. The down-regulated genes are involved in amino acid, cofactor, and vitamin metabolism, and also include ABC transporters. These data demonstrate that A. pleuropneumoniae induces apoptosis of PAMs and undergoes complex changes in gene transcription, including expression changes in known and potential virulence factors. Some potentially novel virulence targets have been identified, suggesting new strategies for the development of vaccines and medicines for both preventive and clinical use.
胸膜肺炎放线杆菌（A. pleuropneumoniae）是猪胸膜肺炎的病原体，这种疾病给养猪业带来了严重问题。该细菌成功感染需要突破肺部的第一道防线，即肺泡巨噬细胞（PAMs）。因此，探索胸膜肺炎放线杆菌与肺泡巨噬细胞的相互作用将为科学控制这种传染病提供至关重要的基础工作，而目前对此研究甚少。在这项研究中，从仔猪中分离出肺泡巨噬细胞，并在体外与胸膜肺炎放线杆菌血清型5b菌株L20共同孵育，使用共聚焦显微镜、电子显微镜、逆转录-聚合酶链反应（RT-PCR）、蛋白质免疫印迹法（Western blot）、流式细胞术以及基因表达谱芯片，观察并分析它们之间的相互作用、肺泡巨噬细胞的细胞死亡以及胸膜肺炎放线杆菌对肺泡巨噬细胞死亡的差异基因表达。胸膜肺炎放线杆菌迅速黏附并侵入肺泡巨噬细胞，诱导细胞凋亡，这一结果通过透射电子显微镜（TEM）和扫描电子显微镜（SEM）得以证实。当细胞以感染复数（MOI）为10进行感染并孵育5小时时，通过流式细胞术证实细胞凋亡的比例最高，蛋白质免疫印迹法检测显示活化的半胱天冬酶-3表达更高。使用含有胸膜肺炎放线杆菌血清型5b菌株L20几乎所有基因组序列的2868个探针的基因芯片微阵列，与在脑心浸液（BHI）培养基中不与肺泡巨噬细胞共同培养的对照细菌的表达相比（平均表达比率>1.5倍，p<0.05），共发现185个细菌基因差异表达（包括92个上调基因和93个下调基因），并参与细胞凋亡过程。上调的基因涉及能量代谢、基因转录和翻译、与毒力相关的基因如脂多糖（LPS）、三聚体自转运黏附素、重复毒素（RTX）及类似基因。下调的基因涉及氨基酸、辅因子和维生素代谢，还包括ABC转运蛋白。这些数据表明，胸膜肺炎放线杆菌诱导肺泡巨噬细胞凋亡，并在基因转录方面发生复杂变化，包括已知和潜在毒力因子的表达变化。已鉴定出一些潜在的新型毒力靶点，为开发预防和临床用的疫苗及药物提供了新策略。
