【作者】 徐欢；

【导师】 刘金林； 祁超；

【作者基本信息】 华中师范大学 ， 生物化学与分子生物学， 2022， 硕士

【摘要】 胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)是一种革兰氏阴性菌,为巴氏杆菌科放线杆菌属菌种。其引发的猪胸膜肺炎是猪的主要呼吸道疾病,该病的急性爆发以出血坏死性肺炎和纤维蛋白性胸膜炎为特征,具有高传染率和致死率,给全球猪养殖业造成了巨大的经济损失。该病发病快,传染力强,且在感染一些猪呼吸系统疾病后,容易继续感染猪胸膜肺炎,多种因素使猪胸膜肺炎防御和管控难度大。硫是致病菌繁殖和感染宿主所不能缺乏的重要化学物质,在细胞中发挥着多种功能,如参与构成细胞组分、信号传导、氧化还原等过程。APP作为一种猪的致病菌,其感染宿主致病能力与营养物质转运相关。目前对APP硫转运特别是谷胱甘肽的转运机制及其与致病力的关系还不清楚,为阐明APP营养物质的转运及代谢在感染中的作用,本研究对可能的二肽转运蛋白(Dipeptide Transporter)DppA1、DppA2在硫转运过程中的功能进行了分析,主要内容与结果如下:1.突变株的构建及鉴定以APP血清7型菌株WF83为亲本菌,利用同源重组和负向筛选法,构建了dppA1和dppA2的单基因缺失突变株ΔdppA1、ΔdppA2,双基因缺失突变株ΔdppA1ΔdppA2-1、ΔdppA1ΔdppA2-2;通过电转化和抗性筛选法,分别将dppA1、dppA2、dppA1dppA2基因导入到ΔdppA1ΔdppA2-1菌株中,构建回复突变株ΔdppA1ΔdppA2-1-CΔA1、ΔdppA1ΔdppA2-1-CΔA2 和ΔdppA1ΔdppA2-1-CΔA1A2。2.突变株体外生长特性分析将WF83、ΔdppA1、AdppA2、ΔdppA1ΔdppA2-1接种于TSB培养基和含不同硫源的化学限制性培养基(chemically defined medium,CDM)中,测定生长曲线,分析dppA1、dppA2基因缺失对APP生长水平的影响。结果显示,三种突变株与野生型相比,在TSB中生长能力无明显区别;在仅含谷胱甘肽的CDM中,ΔdppA1、ΔdppA2的生长水平与野生型WF83菌株一致,双基因缺失突变株ΔdppA1ΔdppA2-1有少量生长,低于亲本菌的生长水平,回复突变株ΔdppA1ΔdppA2-1-CΔA1、ΔdppA1ΔdppA2-1-CΔA2和AdppA1ΔdppA2-1-CAA1A2在谷胱甘肽CDM中的生长水平不能恢复至野生型水平,与ΔdppA1ΔdppA2-1相当。另外在仅含谷胱甘肽的CDM中,ΔdppA1ΔdppA2-2与WF83生长能力无明显区别。3.AdppA1ΔdppA2-1致病性分析本研究初步分析了谷胱甘肽利用受阻对APP致病性的影响。测定了 WF83野生型和双基因缺失突变株ΔdppA1ΔdppA2-1在小鼠肺竞争感染能力,结果显示,ΔdppA1ΔdppA2-1与野生型WF83在小鼠肺部竞争感染指数为1.35,高于致弱临界值(0.2)。为进一步验证突变株的致病力,本研究还通过鼻腔接种法感染仔猪,测定了ΔdppA1ΔdppA2-1和WF83野生型在猪肺部中的竞争感染能力。结果表明,两种菌在肺部组织中的竞争感染指数为0.91,高于致弱临界值(0.2),表明dppA1和dppA2基因的缺失不影响APP的致病性。综上,本研究初步表明,dppA1和dppA2基因连同之间的间隔序列同时缺失后,影响了 APP在谷胱甘肽CDM中的生长能力,间接表明这段序列与APP谷胱甘肽利用相关,但可能并不是因为DppA1和DppA2的贡献。不过,虽然这段序列的缺失影响了 APP对谷胱甘肽的利用,但缺失株对小鼠和仔猪的致病力并没有显著变化。本研究虽未能鉴定DppA1、DppA2与APP谷胱甘肽转运的直接关系,但为后续研究这两个基因在其他短肽转运中的作用,以及阐明APP谷胱甘肽转运与细菌感染致病的关系奠定了基础。更多还原

【Abstract】 Actinobacillus pleuropneumoniae(APP)is a Gram-negative bacterium belonging to the genus Pasteurella.Porcine pleuropneumonia caused by A.pleuropneumoniae that is one of the most important respiratory diseases of pigs.The disease is mainly characterized by necrotic pneumonia and fibrinous pleuritis.Due to high mortality and mortality rate,the outbreak of porcine pleuropneumonia causes huge economic losses to the pig industry all over the world.Porcine pleuropneumonia is a highly contagious disease which is easily co-infected some other porcine respiratory pathogens.The control of porcine pleuropneumonia is not easy since the complex and un-clarified pathogenesis of A.pleuropneumoniae infection.Sulfur is an important nutrient that is essential for pathogenic bacteria survival in vitro and in vivo.At present,the mechanisms of sulfur nutrients transport in A.pleuropneumoniae,especially glutathione transport and its relationship with bacterial pathogenicity is still unclear.In this study,roles of possible dipeptide transporters DppAl and DppA2 in the process of sulfur transport were investigated.The main contents and results are as follows:1.Construction and identification of mutant strainsIn this study,single-gene deletion mutants AdppAl and ΔdppA2,and double-g ene deletion mutants ΔdppA1ΔdppA2-1 and ΔdppA1ΔdppA2-2 were constructed by homologous recombination and negative screening,separately,by using A.pleurop neumoniae standard strain WF83(serovar 7)as the wild type(WT)strain.Then c omplementation strains were obtained,by introduction of dppAl,dppA2,dppA1dpp A2 genes into the ΔdppA1ΔdppA2-1 strain,respectively,and named as ΔdppA1Δdp pA2-1-CΔA1,ΔdppA1ΔdppA2-1-CΔA2 and ΔdppA1ΔdppA2-1-CΔA1A22.Analysis of the growth ability of mutant strains in vitroThe growth ability of A.pleuropneumoniae strains WF83,ΔdppAl,ΔdppA2,ΔdppA1ΔdppA2-1 were evaluated in TSB medium and chemically defined medium(CDM)with various sulfur sources.The results showed that there was no significant difference between these three mutant strains and WT when cultured in TSB.When use glutathione as the sole sulfur source,the growth levels of ΔdppA1 and ΔdppA2 were nearly the same as WT,however,the growth ability of the double gene deletion mutationΔdppA1ΔdppA2-1 was significantly lower than that of the WT after lag phase.Trans-complementation of DppAl,DppA2 or DppA1DppA2 was unable to recover the growth disability of ΔdppA1ΔdppA2-1 in CDM supplemented with glutathione.In addition,the growth curves indicated that the growth ability of ΔdppA1ΔdppA2-2 was not significantly different from WT when use glutathione as the only sulfur source.3.Pathogenicity of ΔdppA1ΔdppA2-1As we known that the ability of WF83 and double gene deletion mutantΔdppA1ΔdppA2-1 is disabled to use glutathione.Therefore,the relationship between the ability of glutathione utilization and virulence were determined,by using mouse and piglet infection models.The results showed that the competitive infection index ofΔdppA1ΔdppA2-1 and WT in mouse lung tissues was 1.35,which was higher than the attenuation cutoff value(0.20).And the competitive indices of AdppA1ΔdppA2-1 and WT in pig lung tissues were determined.The results showed that average CI value was 0.91,which was higher than the weakening threshold(0.20),indicating that the deletion of dppAl and dppA2 genes did not decrease the pathogenicity of A.pleuropneumoniae,and the utilization of glutathione is probably not regarded to the virulence of A.pleuropneumoniae.In conclusion,this study preliminarily shows that deletion of dppAldppA2 as well as the sequence between these two genes affects the growth ability of A.pleuropneumoniae in glutathione CDM medium,indicating that this deleted sequence is related to A.pleuropneumoniae glutathione utilization.However,DppAl and DppA2 probably not contribute to this process.Although the deletion of this sequence affected the utilization of glutathione by A.pleuropneumoniae,the pathogenicity in mice and piglets was not significantly decreased.Although our present study was unable to provide direct evidence between DppAl DppA2 and glutathione utilization in A.pleuropneumoniae,it offers some materials and clues for further investigation of the roles of DppAlDppA2,and the relationship between sulfur nutrients transport and bacterial pathogenesis of A.pleuropneumoniae.更多还原