【作者】 周洋；

【导师】 周锐； 陈焕春； 金梅林； 郭爱珍； 徐卓菲；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2013， 博士

【摘要】 猪传染性胸膜肺炎是由胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae, APP)引起的一种高度传染性呼吸道疾病,给世界各地的养猪业造成了巨大的经济损失。APP通过飞沫传播或感染动物接触传播,定植于猪下呼吸道表皮细胞。APP血清型众多,根据表面多糖抗原性差异可将该病原分为15个血清型。胸膜肺炎放线杆菌在不同国家和地区的流行情况具有一定差异,我国已分离到APP血清1、2、3、4、5、7和8型菌株,其中以7型最多,其次为1型、2型和3型。APP致病过程涉及众多毒力因子,目前围绕其致病机制开展了大量研究工作,然而APP早期定植感染的致病机理尚不明确。Ⅳ型菌毛在多种病原菌致病过程中发挥重要作用,其中包括介导粘附定植、突破免疫屏障、影响生物被膜形成、颤搐运动、DNA摄取等。为了更好地控制和预防APP导致的疾病,研究APP致病机理,尤其是APP早期粘附定植的作用机制具有重要意义。本课题对APPⅣ型菌毛的功能和在致病过程发挥的作用进行探讨,并对Ⅳ型菌毛结构蛋白白ApfA的免疫原性与免疫保护力进行了研究。1.胸膜肺炎放线杆菌Ⅳ型菌毛介导粘附和定植粘附是细菌建立感染的第一步,在致病过程中起着决定性作用。因此本课题对APPⅣ型菌毛是否参与APP宿主粘附定植过程进行研究,研究结果如下：首先,通过荧光定量PCR的方法对APP与猪肺上皮细胞(SJPLC)互作后Ⅳ型菌毛操纵子基因表达水平进行检测,发现与细胞互作后apf操纵子整体表达水平显著上升。其次,本研究构建了Ⅳ型菌毛结构蛋白基因缺失突变株4074△apfA,以及互补菌株C40740△apfA。以猪髋动脉内皮细胞(PIEC)和SJPLC为粘附模型,通过粘附和粘附抑制实验共同证明Ⅳ型菌毛在APP粘附宿主细胞的过程中发挥作用。同时,通过共聚焦显微镜对粘附结果进行观察,发现4074ΔapfA基因缺失突变株粘附能力显著减弱,而互补菌株与野生株粘附能力相当,再次证明Ⅳ型菌毛参与粘附过程。最后,在小鼠活体感染模型中证明Ⅳ型菌毛在APP肺部定植过程中发挥重要作用,并影响APP致病力。以上结果表明APPⅣ型菌毛是细胞接触诱导表达型,参与了APP定植感染过程,在致病过程中发挥重要作用。2.胸膜肺炎放线杆菌Ⅳ型菌毛负调控生物被膜的形成Ⅳ型菌毛参与粘附外的多种致病机制,本研究对APPⅣ型菌毛可能参与的其他致病机制进行探讨。为了研究Ⅳ型菌毛是否与APP生物被膜形成调控相关,我们通过结晶紫染色方法对野生菌株和4074ΔapfA基因缺失突变株的生物被膜形成能力进行定量比较,结果发现4074ΔapfA基因缺失突变株生物被膜形成能力显著增强。该结果表明Ⅳ型菌毛负调控APP生物被膜的形成,我们推测Ⅳ型菌毛可能参与了群体感应调节因子LuxS对于生物被膜形成的负调控过程。此外,我们还对比了野生菌株和4074ΔapfA基因缺失突变株溶血活性和细胞毒性的差异,结果发现Ⅳ型菌毛的缺失不影响APP溶血毒素和细胞毒素的分泌。3.胸膜肺炎放线杆菌Ⅳ型菌毛潜在受体的寻找以上研究证明了Ⅳ型菌毛主要是通过介导细胞粘附在APP早期感染过程中发挥重要作用。在奈瑟球菌属中,Ⅳ型菌毛的已知细胞粘附受体是CD46分子,为了验证CD46分子是否为APP Ⅳ型菌毛的特异性受体,我们进行了以下研究：首先,构建pcDNA-CD46真核表达质粒并转染幼仓鼠肾传代细胞(BHK)(CD46分子阴性细胞),通过流式细胞术检测转染效率,证明CD46分子在BHK细胞中表达。通过粘附实验发现,CD46分子的表达并没有显著提高APP对细胞的粘附效率。其次,用CD46抗体处理SJPLC和PIEC细胞(CD46分子阳性细胞),进行粘附抑制实验,结果显示CD46分子抗体封闭不影响APP对细胞的粘附作用。因此,上述结果证明CD46并非APP特异性粘附受体。为了进一步寻找Ⅳ型菌毛的可能性受体,我们通过酵母双杂交的方法来进行研究：构建pGBKT7-apfA作为诱饵载体,转化酵母Y187菌株后与猪肺部cDNA酵母文库进行杂交,经过三轮筛选获得4个阳性克隆。对阳性克隆进行测序并分析,结果发现其中一个可能是Ⅳ型菌毛互作受体蛋白。该蛋白与猪Talinl蛋白同源,是一种细胞骨架蛋白,其与APPIV型菌毛的互作关系还有待进一步的验证。4.胸膜肺炎放线杆菌Ⅳ型菌毛结构蛋白ApfA是良好的保护性抗原本研究对Ⅳ型菌毛基因簇在APP13个血清型中的分布情况和同源性进行比较分析,结果发现编码ap/A基因的结构蛋白在12个血清型中高度保守；Ⅳ型菌毛基因簇在11个血清型中高度保守。为了检测Ⅳ型菌毛结构蛋白ApfA的免疫原性,我们首先表达纯化了重组ApfA (rApfA)蛋白,然后用纯化的rApfA免疫小鼠,发现能够诱发小鼠产生免疫应答,产生较高抗体水平。其次,我们用rApfA-ELISA对猪APP感染血清和阴性血清中anti-ApfA抗体水平进行检测,发现感染血清中的抗体水平显著高于阴性血清。以上结果说明Ⅳ型菌毛结构蛋白ApfA不仅存在于所有血清型中,序列高度保守,并且具有很强的免疫原性。以上研究发现APP IV型菌毛结构蛋白ApfA具有较好的免疫原性并且高度保守,有可能发展成为是良好的保护性抗原。因此,我们接下来对菌毛结构蛋白ApfA能否提供有效的免疫保护力进行评估。首先,通过rApfA主动免疫小鼠后感染致死剂量APP,发现该抗原能够对APP血清1型4074菌株感染提供90%的保护,对APP血清7型WF83菌株感染提供80%的保护。其次,对免疫血清中anti-rApfA抗体进行分型,发现rApfA能够诱导Th2型主导的免疫反应。用rApfA免疫血清通过静脉注射被动免疫小鼠,发现该免疫血清能够对APP血清1型4074菌株感染提供40%的保护,对APP血清7型WF83菌株感染提供60%的保护。以上结果表明Ⅳ型菌毛结构蛋白ApfA不仅是一种高效的保护性抗原,能够激发小鼠产生Th2型为主导的免疫应答,对于中国流行血清型1型和7型APP感染提供有效保护,而且ApfA诱导的抗体也具有被动免疫保护力,对于APP不同血清型的感染能够提供一定保护。更多还原

【Abstract】 Actinobacillus pleuropneumoniae (APP) is the etiological agent of porcine contagious pleuropneumonia, a highly contagious respiratory disease of pig which causes serious economic losses in the pig farming industry worldwide. The A. pleuropneumoniae organism is transmitted via respiratory droplets or through direct contact with infected animals. It colonizes the epithelial cells of the lower respiratory tract of pigs. A total of15serotypes of A. pleuropneumoniae have been identified on the base of surface polysaccharide antigens. Serotype prevalence varies in different countries and regions. Strains of serovar1,2,3,4,5,7, and8have been isolated in China, among which serovar7is the most prevalent serotype and followed by serovar1,2and3.APP enrolls multiple virulence factors during the infection process. Currently, a lot of study focuses on its pathogenesis. However, the mechanism of colonization in early infection phase is still unclear. Type Ⅳ pili of different pathogenic bacteria play important roles during infection process, including mediating adherence, breaking immunity barrier, affecting biofilm formation, twitching motility and DNA uptaking. Consequently, to better prevent and control the diseases caused by APP, study of the APP pathogenesis, especially the colonization mechanism in early infection phase is meaningful. In this study we investigate the role of APP type Ⅳ pili play in the infection process and also evaluate the pilus structural protein ApfA as a protective antigen.1. Type Ⅳ pili of APP involve in adherence and colonizationAdherence is the first step to establish infection, which plays a critical role in the infection process. Therefore, in this study, we examined if type Ⅳ pili are involved in APP colonization to host. The results are as follows:First, we analyzed the expression level of type Ⅳ pili operon after contacting with St. Jude porcine lung cells (SJPLC) via RT-PCR analysis. The results showed that after cell contacting, the expression level ofapf operon genes significantly increase. Second, type Ⅳ fimbrial protein mutant strain4074△apfA and complementary strain C4074△apfA are constructed in this study. With adherence cell models of porcine iliac artery endothelial cell (PIEC) and SJPLC, we certified type Ⅳ pili involve in APP adherence to host cells by adherence and adherence inhibition assay. Meanwhile, we observed significantly reduced adherence capacity of4074△apfA mutant strain by confocal microscopy, while the apfA complementary strain had this capability restored, which proved the involvement of type Ⅳ pili in adherence process. Third, in mice infection model, type Ⅳ pili were proved to be involved in colonization and virulence in early infection stage. So far, these results showed that type IV pili of APP are cell-contact induced and play an important role in colonization during infection process, which contribute critically to the pathogenicity.2. APP type Ⅳ pili involve in negative regulation of biofilm formationType IV pili are involved in multiple pathogenic mechanisms. In this study, we further explored other roles type Ⅳ pili may play in the pathogenesis during APP infection. To determine whether type Ⅳ pili involve in the regulation of biofilm formation, the capacity of parent and mutant strains to form biofilm is quantificationally compared by crystal violet staining assay. The mutation of apfA significantly enhanced the biofilm formation ability of APP, implying that type Ⅳ pili take part in the negative regulation of biofilm formation. We speculated that type Ⅳ pili of APP could involve in the negative regulation of biofilm formation by LuxS, a quorum sensing regulator. Furthermore, we compared4074△apfA with parent strain in hemolytic and cytotoxicity assays. The results showed no differences between the two strains in hemolysis and cytotoxicity, which indicates that the absence of type Ⅳ pili didn’t affect the secretion of hemolysins and cytotoxins.3. Searching for the potential receptors for APP type Ⅳ piliType IV pili are proved to mediate colonization in early stage of APP infection. In Neisseria, CD46is proved to be the specific receptor of type IV pili. To verify whether CD46is the specific receptor of APP type IV pili, we constructed pcDNA-CD46eukaryotic expression vector and transfected it into baby hamster kidney (BHK) cells (CD46negative). CD46is verified to be expressed in transfected BHK cells by flow cytometry analysis. However, APP didn’t show enhanced adherence to BHK cells with CD46expression. In adherence inhibition assay, blocking SJPLC and PIEC cells (CD46positive) with CD46specific monoclone antibody didn’t reduce the adherence by APP. These results indicated that CD46is not a specific receptor for APP adherence.We further applied yeast two-hybrid system to look for potential receptors for APP type IV pili. Bait vector pGBKT7-apfA was constructed and transformed into yeast strain Y187. Four positive clones were captured in porcine lung cDNA yeast library by yeast mating. Sequencing analysis found that only one of the four positive clones could be the potential receptor of type IV pili. The protein is homologous with Talinl, a cytoskeletal protein. The interaction between type Ⅳ pili and Talinl remains to be further verified. 5. Type Ⅳ pilius structural protein ApfA is a good protective antigenWe analyzed the homology of type Ⅳ cluster genes in13APP strains of different serotypes and found that ApfA is conserved in12serotypes and type Ⅳ pili operon is conserved in11serotypes. To investigate the immunogenicity of ApfA, recombinant ApfA (rApfA) is expressed in E. coli and purified. Then mice immunized with purified rApfA were induced an elevated humoral immune response and produced high levels of specific antibody. Then pig convalescent serum samples and control serum samples were examined with a rApfA-based ELISA. The results showed that the titers of anti-rApfA antibody in convalescent serum are significantly higher than that in control serum. These results suggest that structural protein ApfA of type Ⅳ pili, which exists in all A. pleuropneumoniae serotypes, is not only highly conserved in sequences, but also highly immunogenic.APP type Ⅳ pilus structural protein ApfA, with good immunogenicity and conserved identity is a promising protective antigen. In next study, we evaluated the protection provided by ApfA in mice. The rApfA showed a90%protective efficacy against lethal infection of serovar1strain4074. For serovar7strain WF83, rApfA conferred80%protection. Antibody typing found that rApfA mainly induces Th2type immune response. Then, naive mice were passively immunized with anti-rApfA serum via i.v. injection and the results showed that the anti-rApfA serum provided protection against both strain WF83(serovar7,60%) and strain4074(serovar1,40%). So far, the results suggested that type Ⅳ pilus structural protein ApfA is a highly effective protective antigen, mainly induce Th2type humoral immune response, which confer significant protection against APP infection of Chinese prevalent serotypes. In addition, the antibody raised against ApfA is effective in passive immunization, which could also provide protection against APP infection of different serotypes.更多还原