IX型分泌系统在鸭疫里默氏杆菌致病过程中的作用

Riemerella anatipestifer infection is one of the major infectious diseases in farm ducks worldwide, however, there has been little work on the molecular basis of R. anatipestifer pathogenesis. In our preliminary study, we found that the capacity of adherence to glass surface and the secreted extracellular protease of the deletion mutant of gldN, one of core genes of type IX secretion system (T9SS), were reduced significantly compared with those of the complemented strain and the wild type strain ATCC 11845. Therefore, we deduced that T9SS, the only known secretion system in R. anatipestifer, may play critical roles in the secretion of adhesins and extracellular proteases, and thereby in bacterial pathogenesis. In this study, we will investigate the role of T9SS in the pathogenesis of highly pathogenic R. anatipestifer strain Yb2. Firstly, the proteins secreted by T9SS of strain Yb2 were analyzed through combining differential proteomics between the mutant ΔgldN and the wild type strain Yb2, with bioinformatics prediction; and then the genes coded for the putative adhesins and extracellular proteases were epressed in Escherichia coli and the related polyclonal antibodies were prepared with the purified recombinant proteins respectively; afterwards, the effects of T9SS defect on the secretion of related proteins were analyzed. Finally, the biological characteristics of the putative adhesin or extracellular protease deletion mutants, the related complemented strains and the wild type strain Yb2 were compared to identify the adhesins or extracellular proteases, and to clarify their roles in the virulence of strain Yb2. This study may help us to uncover the molecular pathogenesis of R. anatipestifer infection and to develop novel attenuated vaccine candidates.

鸭疫里默氏杆菌感染是当前危害养鸭业的主要传染病之一，但目前对其分子致病机制还知之甚少。在前期研究中，我们发现ATCC 11845株T9SS核心组分GldN的基因缺失株对玻璃表面的粘附和分泌的胞外蛋白酶均比野生株和回复株显著减低，提示此细菌唯一已知的分泌系统T9SS可能通过分泌粘附素和胞外蛋白酶在细菌致病过程中起着重要作用。本研究将选择强致病性的Yb2株为研究对象，先通过gldN基因缺失株与野生株的差异蛋白质组学和生物信息学预测的方法相结合来分析Yb2株T9SS分泌的蛋白；然后原核表达潜在粘附素和胞外蛋白酶基因，制备相应的多克隆抗体，并分析T9SS缺失对相关蛋白分泌的影响；最后通过比较构建的相关基因缺失株、野生株和互补菌株的生物学特性等，以鉴定粘附素和胞外蛋白酶，并阐明其在Yb2株致病过程中的作用。本项目的研究对于解析鸭疫里默氏杆菌的分子致病机制及研制新型弱毒疫苗等具有重要意义。

鸭疫里默氏杆菌感染是当前危害养鸭业的主要传染病之一，但目前对其分子致病机制还知之甚少。本研究先通过T9SS核心基因gldN缺失株与野生Yb2株的差异蛋白质组和生物信息学预测的方法相结合来分析Yb2株T9SS分泌的蛋白；然后构建了其中预测的3个外膜蛋白编码基因和4个胞外蛋白编码基因的基因缺失突变株和相应的互补菌株，然后通过基因缺失株的生物学特性分析等，以鉴定潜在粘附素和胞外蛋白酶，并阐明其在Yb2株致病过程中的作用。结果表明，3个外膜蛋白编码基因AS87_02965、AS87_00895和AS87_02120的缺失株中，缺失株Yb2Δ2120对雏鸭的致病性没有降低，而缺失株中Yb2Δ0895 和Yb2Δ2965对雏鸭的半数致死量分别上升114和58倍，且感染鸭的血液、肝脏和脑组织中的细菌载量均比野生株感染鸭显著性降低，这两株基因缺失株对Vero细胞的粘附入侵能力和形成生物被膜的能力均较野生株显著性降低，表明AS87_02965和AS87_00895与鸭疫里默氏杆菌的毒力相关，且这两种外膜蛋白可能作为细菌的黏附素。与野生株相比，4个胞外蛋白编码基因AS87_03330、AS87_04355、和AS87_05150和Yb2_07560的缺失株对雏鸭的致病性分别下降了11倍、239倍、127倍和11倍；缺失株Yb2Δ3330和Yb2Δ5150的蛋白水解能力（蛋白酶活性）与野生Yb2株相比无显著性差异，但缺失株Yb2Δ04355和Yb2Δ7560的蛋白水解能力（蛋白酶活性）显著性降低，表明AS87_04355和AS87_07560具有蛋白水解酶活性。另外，研究还表明DhR1(AS87_01955)可调节T9SS的分泌，Yb2ΔdhR1缺失株中T9SS的分泌蛋白编码基因有13个表达上调，2个表达下调。本项目的研究对于解析鸭疫里默氏杆菌的分子致病机制及研制新型弱毒疫苗等具有重要意义。