副猪嗜血杆菌荚膜多糖合成调节机理及其对致病力的影响

This research will focus on capsular polysaccharide (CPS), a potential virulence factor, of haemophilus parasuis (HPs) which is an important bacterial agent involved in porcine industry. Mechanism of biosynthesis regulation at transcriptional level and effect on pathogenicity of haemophilus parasuis capsular polysaccharide will be explored. CPS export gene Wza deficient mutant and BadM/Rrf2 transcriptional regulator gene deficient mutant of pathogenic HPs will be constructed. The transcription of 14 coding sequences (CDS) in LPS biosynthetic regeion will be detected under capsule-inducing culture conditions, and genes invovled in CPS biosynthesis will be determined based on the relationship between gene transcription and CPS production. Regulation effect of Wza and BadM/Rrf2 transcriptional regulator on CPS biosynthesis will be analyzed through comparison of CPS production in both mutant and wild-type HPs. A promoter detection plasmid with green fluorescence protein coding sequence as reporter gene will be constructed, and the promoter of genes involved in CPS biosynthesis will be determined. Combination capacity of BadM/Rrf2 transcriptional regulator to the regulatory sequence of genes involved in CPS biosynthesis will be detected, which will allow the determination of target gene and the target site of combination. The mechanism of CPS biosynthesis regulation in HPs will be analyzed based on research described above. To elucidate the effect of CPS in the process of infection and on the pathogenicity of HPs, the adherence to the porcine tracheal epithelial cells, and resistance to phagocytosis and serum will be compared for both capsule deficient mutant and wild-type HPs, and the pathogenicity to piglets as well. It would provide new scientific information to the pathogenesis of HPs.

本课题以严重危害养猪业的副猪嗜血杆菌（HPs）的潜在毒力因子荚膜多糖（CPS）为研究对象，探索致病菌株的CPS转录水平的调控机理及其对HPs致病力的影响。拟构建CPS输出基因Wza缺失株和BadM/Rrf2型转录调节因子缺失株，通过比较缺失株及其亲本菌株CPS表达量及其与脂多糖合成基因簇中14个编码序列转录的相关性，鉴定参与CPS合成的相关基因，并分析Wza及BadM/Rrf2在CPS基因转录调控中的作用；构建启动子检测质粒，鉴定CPS合成相关基因的启动子；通过分析BadM/Rrf2型转录调节因子对CPS合成相关基因调控序列的结合能力，鉴定其靶基因及靶位点，阐明其对CPS合成的调控机理；通过比较荚膜缺失株及其亲本菌株对肺泡巨噬细胞的敏感性、气管上皮细胞的黏附能力及血清抗性等差异，结合其对易感仔猪的致病性强弱，分析CPS在HPs致病过程各重要环节中的作用,为揭示HPs致病机理提供新的依据。

副猪嗜血杆菌（HPs）感染是危害养猪业最严重的细菌病之一，但由于缺乏成熟的基因操作系统，因此对其毒力因子及致病机理的研究尚不深入，为防控本病带来很大挑战。荚膜是多种细菌的毒力因子，而HPs的荚膜多糖（CPS）表达调控机制及其在致病过程中发挥何种作用，目前尚不明确。本课题以严重危害养猪业的副猪嗜血杆菌（HPs）的潜在毒力因子荚膜多糖（CPS）为研究对象，探索致病菌株的CPS转录水平的调控机理及其对HPs致病力的影响。本研究首先进行了副猪嗜血杆菌荚膜多糖诱导条件的筛选，发现猪血清是诱导荚膜产生的良好诱导剂。阐明了常规体外培养条件下，副猪嗜血杆菌多糖基因簇相关基因的表达规律。改进了副猪嗜血杆菌遗传操作方法，建立了全新的遗传操作系统，利用所建立的方法，可实现80%临床菌株的成功转化，可在60%的临床分离菌株中进行遗传改造，大大提高了突变菌株的构建效率以及可以进行遗传改造的菌株数，对副猪嗜血杆菌毒力因子及致病机理的研究具有重要作用。构建了IscR缺失株，阐明了转录调节因子IscR对荚膜多糖基因簇相关基因表达的影响，IscR缺失株在模拟体内生存条件下荚膜生成量明显降低，证实了IscR参与副猪嗜血杆菌荚膜多糖的表达调控。构建了荚膜多糖输出蛋白Wza缺失株，证实了Wza缺失对副猪嗜血杆菌的生物学活性及致病力产生诸多影响，比如缺失株的染色形态发生改变、自然沉降速度变快、抗细胞吞噬能力降低、血清抗性降低、对部分单糖（核糖、甘露糖、葡萄糖、蔗糖等）的敏感性增加、对部分抗生素（青霉素、氨苄西林、阿莫西林等）的敏感性增加、对小鼠的致病力明显减弱等。