禽致病性大肠杆菌ompT质粒编码基因的特殊致病作用及其成因研究

Outer membrane protein T (OmpT) encoded by the ompT gene located on plasmid ColⅤ of avian pathogenic Escherichia coli (APEC) strain E058 was found unable to inactivate the protamine which is the main substrate of the outer membrane protease encoded by chromosomal ompT gene of E. coli. When half of the OmpT encoding gene of ColⅤ plasmid origin (pompT) was knocked out from the APEC strain E058, the virulence of the mutant E058Δ50% pompT declined at 10-fold level evaluated by 50% lethal dose determination assay compared to the wild-type strain E058. Meanwhile, whole pompT gene was knocked out from the strain E058, the virulence of the mutant E058 Δ100% pompT was as equal as that of the wild-type strain. To explore the mechanism of OmpT encoded by pompT (pOmpT) lost its activity of protamine inactivation, the crystal structure analysis of pOmpT will be performed using the expressing pOmpT, and the promoter of pompT was replaced by that of the chromosomal ompT gene, to see whether the activity of protamine inactivation was obtained by the reconstructed pOmpT or not. RNA sequencing was employed to discover the both up-regulated expression genes and down-regulated genes and the potential regulation network of virulence genes governed by pompT under both in vivo and in vitro conditions. For in vivo, E058 Δ50% pompT and E058 Δ100% pompT were inoculated into 1-day-old specific pathogen free (SPF) chickens, respectively. Bacteria were collected from the blood of infected birds at 3 hours post-challenge, and total RNA from these bacteria was extracted and purified. For in vitro, total RNA was extracted and purified from bacteria grown in Luria-Bertani medium, then the total RNAs mentioned above were subjected to RNA sequencing. E058 Δ50% pompT was significantly attenuated based on the 50% lethal dose determination in 1-day-old SPF chickens, and the results of the colonization and persistence assay carried out in 35-day-old SPF birds. 25%, 15% and 5% fragments among 50% pompT mentioned above were selected and knocked out from the ColⅤ plasmid of strain E058, and E058Δ25%pompT、E058Δ15%pompT and E058Δ5%pompT mutants were generated, and 50% lethal dose determination and the colonization and persistence assay were also performed for these mutants. The mutant with both the attenuated virulence and the shortest knockout gene fragment, together with its revertant strain will be evaluated both in 50% lethal dose determination and the colonization and persistence assay to confirm the virulence complementation for the revertant strain. RNA sequencing will also be employed for the mutant with the shortest knockout gene mentioned above which grown in vivo and in vitro, to confirm the RNA sequencing results derived from E058Δ50%pompT. This study is some of help to develop the antimicrobial drug related to the inactivation of OmpT, to explore the pathogenesis of pompT and to provide a new target for both the pathogenesis study and the immunology study of avian colibacillosis.

禽致病性大肠杆菌（APEC）E058株质粒编码的外膜蛋白OmpT（pOmpT）不能灭活抗菌肽，但其染色体编码的OmpT能灭活抗菌肽；敲除50% OmpT质粒编码基因（pompT）的突变株（E058 Δ50% pompT）毒力下降10倍，而敲除100%该基因的突变株毒力不变。通过蛋白晶体结构分析和启动子替代试验，剖析pOmpT不能灭活抗菌肽的结构与分子机制；通过E058 Δ50% pompT突变株体内、体外转录组测序分析，揭示pompT主导的APEC毒力调控机制；对50% pompT基因片段进行截短突变，获得显著影响E058株毒力的更短pompT基因片段突变株，通过进一步的转录组学分析，精炼pompT主导的APEC毒力调控机制。本研究有助于厘清APEC pompT基因与其特殊致病作用的关系，为APEC致病、免疫机理的研究提供新靶标。

禽致病性大肠杆菌E058株质粒编码的外膜蛋白OmpT（pOmpT）不能灭活鱼精蛋白，但其染色体编码的OmpT（cOmpT）能灭活鱼精蛋白。本研究表达出无自水解活性的点突变蛋白pOmpT K217G (赖氨酸(K)→甘氨酸(G))，通过优化结晶条件，最终获得可以正常衍射的蛋白晶体，发现pOmpT与已发表的cOmpT结构存在明显差异。通过融合PCR，实现cOmpT、pOmpT 5个环的互换和第5环中差异氨基酸位点的单互换和双互换，并在重组菌中表达嵌合蛋白进行鱼精蛋白裂解试验。发现cOmpT的第5环是决定鱼精蛋白裂解能力的关键环，经过多重试验证实，cOmpT与pOmpT灭活鱼精蛋白能力差异的原因在于cOmpT的267位天冬氨酸（D）和276位丝氨酸（S）为丝氨酸蛋白酶活性位点，而pOmpT的267位丝氨酸（S）和276位苏氨酸（T）非丝氨酸蛋白酶活性位点，导致其不能裂解鱼精蛋白。对50% pompT、100% pompT突变株进行转录组测序，发现50% pompT突变株中受影响的差异基因较多，涉及摄铁系统/铁离子载体、生物被膜形成等可能影响细菌毒力的相关基因。构建pompT系列截短突变株，LD50测定和体内定居试验结果显示，25%pompT为影响上述致病性的最短pompT基因片段。25%pompT突变株体内、外转录组测序结果表明，与亲本株E058相比，25% pompT突变株中受影响的差异基因数量比50% pompT突变株更多，但仍主要涉及摄铁系统/铁离子载体、生物被膜形成等可能影响毒力的相关基因。本研究主要发现了pOmpT与cOmpT结构存在显著差异；同时，发现cOmpT的267位天冬氨酸（D）和276位丝氨酸（S）为丝氨酸蛋白酶活性位点；明确了25%pompT为影响APEC E058株致病性的最短pompT基因片段，从而为深化禽大肠杆菌病致病机理、防控提供了新视角。