溶血素在鸭疫里默氏杆菌HXb2株获铁和致病过程中的作用

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 R. anatipestifer strains showed haemolytic activity in blood agar, and strain HXb2, which was highly pathogenic to ducklings, showed stronger haemolytic activity than that of other R. anatipestifer strains.On the other hand, we also found that strain HXb2 did not carry sip gene or secret siderophore (or the amount of secreted siderophore was very low). Therefore, we deduced that hemolysin of strain HXb2 may play critical roles in iron acquisition and its pathogenesis. In this study, we would analyze iron acquisition of strain HXb2 by iron utilization test, detection of siderophore production and transcriptomics. Then the hemolysin gene(s) was (were) screened and identified using random transposon mutagenesis and real-time PCR methods, and moreover, the effects of iron and different culture conditions on hemolytic activity and expression of hemolysin of strain HXb2 were also evaluated. Finally, the biological characteristics of hemolysin deletion mutant, the complemented strain and the wild type strain HXb2 were compared to analyze the effects of hemolysin deletion on iron acquisition and the virulence. This study may help us to uncover the molecular pathogenesis of Riemerella 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，但目前人们对其分子致病机制等还知之甚少。在前期的研究中，我们发现鸭疫里默氏杆菌具有溶血活性，且对雏鸭致病性很强的HXb2株溶血活性也比其他菌株强；此外还发现HXb2株不携带铁载体相互作用蛋白基因sip，也不分泌铁载体（或分泌的量很低）。因此，我们推测溶血素可能在HXb2株获铁和致病过程中起着重要的作用。本研究将先通过铁利用实验、铁载体的检测和转录组学研究等来分析HXb2株的获铁情况；再通过转座子随机突变和实时定量PCR等方法筛选和鉴定HXb2株的溶血素基因；然后分析铁和不同的培养条件对HXb2株溶血活性和溶血素表达的影响；最后通过比较构建的溶血素基因缺失株、互补菌株和野生株HXb2的生物学特性等，来分析溶血素缺失对HXb2株获铁和致病性的影响。本项目的研究对于解析鸭疫里默氏杆菌的分子致病机制以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。但目前人们对其毒力相关基因和分子致病机制还知之甚少。本研究先分析了鸭疫里默氏杆菌的溶血特性，然后通过构建转座子随机突变库来筛选和鉴定与溶血相关的基因，最后通过分析重组蛋白和基因缺失突变株等的溶血性来鉴定溶血素，并分析了溶血素在致病过程中的作用。研究结果表明：（1）sip-的鸭疫里默氏杆菌HXb2和NJ-1株比sip+的CH3、SX株等对限铁环境敏感；HXb2和CH3株等鸭疫里默氏杆菌以Fe2+比Fe3+作为唯一铁源时生长快；转录组学分析表明在限铁条件下CH3株有两个预测的操纵子（M949_0525～M949_0535和M949_0609～M949_0612）中的基因全部上调，一个预测操纵子（M949_2159～M949_2161）中的基因全部下调；而HXb2株在限铁条件下只有一个预测的操纵子（AB406_0175～AB406_0185，同M949_0525～M949_0535）中的基因上调；（2）优化后的鸭血平板检测结果表明，33/51株（64.7%）鸭疫里默氏杆菌在血平板上具有溶血活性；鸭疫里默氏杆菌的培养上清和外膜蛋白均具有溶血活性；只有活的鸭疫里默氏杆菌才具有溶血活性；相差显微镜观察的结果表明，在血平板上不溶血的鸭疫里默氏杆菌也具有对鸭红细胞膜的穿孔能力。提示所有检测的dba+和dba-鸭疫里默氏杆菌都具有溶血活性；（3）通过构建SX株的转座子随机突变库，筛选并鉴定到了23个与鸭疫里默氏杆菌溶血相关的基因；（4）溶血试验的结果表明，重组蛋白rOmpA1467具有溶血活性；且缺失株CH3ΔompA失去溶血活性，回复株的溶血活性野生株相近。并进一步鉴定到OmpA1- 405是溶血素OmpA1467中与溶血相关的结构域；（5）重组蛋白rRiean_0620和rRiean_0415具有溶血活性；缺失株HXb2ΔRiean_0620和HXb2ΔRiean_0415的溶血活性较野生株HXb2株减弱，且回复菌株可以在一定程度上回复缺失株的溶血活性。野生株HXb2株、缺失株HXb2ΔRiean_0620和HXb2ΔRiean_0415，及相应回复株对雏鸭的致病性结果表明，溶血素Riean_0620和Riean_0415均与鸭疫里默氏杆菌的毒力相关。本研究为进一步研究该细菌的分子致病机制及研制新型弱毒疫苗等奠定了基础。