【作者】 党晓群；

【导师】 周泽扬；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2012， 博士

【副题名】家蚕微孢子虫类枯草杆菌蛋白酶NbSLP1的功能分析

【摘要】 病原微生物的分泌蛋白酶主要参与自身发育和代谢调节。近年来,分泌性蛋白酶作为致病性真菌的重要毒力因子已经进行了深入的研究,发现这些酶不仅参与粘附和入侵过程,还可以与宿主的免疫系统相互作用。作为丝氨酸蛋白酶家族的枯草杆菌蛋白酶,广泛存在于昆虫病原性真菌中,是通过降解昆虫体壁帮助病原真菌入侵宿主的重要蛋白酶之一。家蚕微孢子虫是家蚕微粒子病的病原,其致病机理仍不清楚。人们普遍认为,家蚕微孢子虫在增殖时通过释放某些蛋白酶,破坏组织或器官失去正常的生理功能。近年来,本研究室对家蚕微孢子虫进行了基因组测序,从获得的数据中,我们鉴定到了一组类枯草杆菌蛋白酶(subtilisin-like protease, SLP)家族基因,进一步调查发现,在已报道的兔脑炎微孢子虫、蝗虫微孢子虫、比氏肠道微孢子虫、蜜蜂微孢子虫和肠脑炎微孢子虫基因组中均存在类枯草杆菌蛋白酶家族基因。微孢子虫基因组中存在的类枯草杆菌蛋白酶家族究竟扮演什么样的功能?迄今尚不清楚。有鉴于此,本研究以家蚕微孢子虫类枯草杆菌蛋白酶1基因Nbslp1为对象,系统分析了其序列及结构特征,并进行了原核重组蛋白的表达和抗体制备,检测了重组蛋白的酶活特性,进一步应用间接免疫荧光、免疫组化、免疫胶体金定位等技术对家蚕微孢子虫类枯草杆菌蛋白酶1NbSLP1的亚细胞定位进行了分析；同时运用生物信息学分析方法、抗体封闭、免疫共沉淀、细胞转染和酵母双杂交对家蚕微孢子虫NbSLP1的功能进行了探索研究。主要研究内容包括以下三方面：1.家蚕微孢子虫类枯草杆菌蛋白酶基因Nbslps的鉴定本研究利用生物信息学方法和软件对公共数据库和各种微孢子虫基因组中的蛋白酶进行序列比对和结构域预测。在已报道基因组的微孢子虫中均存在两类SLPs,一类具有前肽Inhibitor_I9结构域和丝氨酸蛋白酶Peptidase_S8结构域,命名为SLP1,另一类仅含有丝氨酸蛋白酶Peptidase_S8结构域和C端跨膜结构域,命名为SLP2,两类SLPs的活性位点为Asp192,HiS224和Ser413。在家蚕微孢子虫基因组中共鉴定出了3个slp基因(?)Vbslpl和Nbslp2-1, Nbslp2-2。我们重点对(?)Vbslpl的序列特征进行了分析。NbSLP1具有N端抑制结构域Inhibitor_I9和Peptidase_S8结构域。NbSLP1蛋白N端30个氨基酸残基为信号肽,含有一个Ca2+结合位点,暗示该酶是金属离子依赖型蛋白酶。糖基化预测显示NbSLP1可能为糖蛋白。微孢子虫的SLP1序列的多重比对结果显示,不同微孢子虫种间SLP1的结构域非常保守。共线性分析表明,SLPs在微孢子虫基因组中的位置相对保守。系统发育分析表明,微孢子虫的slp1基因和slp2基因各聚为一支,暗示二者的功能可能发生分化。NbSLP1的活性结构催化三联体的空间排布与枯草芽孢杆菌的枯草杆菌蛋白酶相一致,推测NbSLP1发挥功能的方式与枯草芽孢杆菌的枯草杆菌蛋白酶相似；Inhibitor_I9结构域延伸至酶活性中心内部,暗示NbSLP1的活性发挥需要去除Inhibitor_I9结构域。RT-PCR检测发现,Nbslp1基因在感染家蚕后的1-5天均有转录,推测NbSLP1在孢子发育和增殖中发挥作用。2.家蚕微孢子虫类枯草杆菌蛋白酶NbSLP1的表达及定位分析基于以上对Nbslp1基因序列特征的研究结果,针对各结构域,我们设计了相应的原核表达引物,包括全长(NbslpI)、活性结构域(Nbslp1c)和抑制结构域(Nbslp1p),将这些片段插入到表达载体pET30a中,转入表达宿主菌E. coli BL21,于30℃经IPTG诱导表达,分别获得了预期的重组蛋白质(rNbSLP159kDa, rNbSLPIC41kDa, rNbSLPIP17kDa),但主要以包涵体形式存在。通过镍柱亲和层析纯化后,我们获得了较纯的融合His标签的融合蛋白。复性后的蛋白在最适温度为30℃、最适pH为8.0时酶活力单位为58U/mL。将纯化的融合蛋白作为抗原免疫小鼠和家兔,制备相应的抗体。双向Western blotting结果显示,成熟孢子中NbSLP1主要以酶原形式存在。采用间接免疫荧光试验(IFA)和胶体金免疫电镜(IEM)等方法对NbSLP1进行了定位分析。共聚焦显微镜观察显示,在成熟孢子中NbSLP1位于孢壁；孢子发芽后,NbSLP1的成熟酶位于孢子顶端,推测当孢子发芽时,该酶可能被激活成为成熟酶,水解固定盘相关蛋白,以利于孢子极丝的弹出。重组蛋白能够与几丁质标准品和家蚕微孢子虫的几丁质层发生粘附,进一步支持了NbSLPl定位于孢壁的结果。以感染家蚕微孢子虫的Sf9细胞进行IFA时,可以检测到具有现较强荧光的孢子；以感染家蚕微孢子虫的家蚕丝腺组织为材料的免疫组化实验显示,在丝腺细胞的细胞质中存在荧光信号,暗示家蚕微孢子虫在宿主细胞增殖过程中可能会分泌NbSLP1。3.家蚕微孢子虫NbSLP1的功能分析主要通过NbSLP1在家蚕微孢子虫侵染中的作用以及分析其互作蛋白来研究NbSLP1的功能。第一,家蚕微孢子虫NbSLP1侵染中的作用分析。由于类枯草杆菌蛋白酶属于丝氨酸蛋白酶家族,本研究采用丝氨酸蛋白酶抑制剂PMSF对家蚕微孢子虫孢子进行处理,统计孢子发芽率。分析表明,PMSF可使孢子的发芽率降低20%,差异极显著(p<0.01)；经PMSF处理的孢子添食家蚕后,可降低家蚕死亡率,差异显著(p>0.01)。这些结果反映了家蚕微孢子虫类枯草杆菌蛋白酶参与了孢子的发芽过程并发挥重要作用。第二,家蚕微孢子虫NbSLPl互作蛋白的分析。一方面,以家蚕微孢子虫为对象。采用免疫共沉淀差异带的二维线性离子阱质谱鉴定结果显示,三磷酸甘油脱氢酶、翻译起始因子2C、ATP酶、内质网型ATP酶、孢壁蛋白1、假定孢壁蛋白7、热激蛋白HSP70和极管蛋白2可能为NbSLP1的互作蛋白。通过对这些蛋白的结构域预测和潜在枯草杆菌蛋白酶剪切位点的分析,发现剪切位点恰好位于ATP酶的两个结构域之间,推测NbSLP1可能参与了ATP酶的加工成熟过程,使ATP酶水解,产生能量ATP。另一方面,以宿主细胞为研究对象。首先,细胞转染结合双向电泳差异分析表明,转染了NbSLP1的草地贪夜蛾细胞总蛋白缺失了7个蛋白点,经MALDI-TOF/MS质谱鉴定,暗示NbSLP1可能与N-乙酰转移酶、表皮蛋白和血淋巴蛋白互作。其次,选取生物信息学预测底物中的跨膜蛋白(BmTIR)为候选靶标。该蛋白结构特征与家蚕Toll蛋白样受体(TLR)相似,含有胞外亮氨酸重复区域和胞内参与信号传导的TIR结构域。TLR通过参与对不同病原体的病原分子相关模式进行识别,在抗感染天然免疫中发挥重要作用。实验表明,NbSLP1的抗体与感染家蚕微孢子虫的家蚕组织总蛋白共孵育,可将BmTIR沉淀出来。同时,重组表达含TIR结构域的蛋白rBmTIR,复性的rNbSLP1C可水解rBmTIR。这些结果表明,NbSLP1与BmTIR之间存在互作。如果NbSLP1能水解Toll受体,破坏其TIR结构域,Toll受体便不能把免疫识别信号传递至下游效应因子,从而减弱宿主家蚕的免疫防御,更有利于家蚕微孢子虫的寄生。因此,我们推测家蚕微孢子虫NbSLP1可抑制家蚕的免疫通路。综上所述,本论文对家蚕微孢子虫NbSLP1的定位进行了细致的研究,探讨了NbSLP1的功能,丰富了病原微生物中类枯草杆菌蛋白酶SLPs的研究。本研究为深入解析该蛋白在微孢子虫侵染寄生中的功能和作用奠定了基础。更多还原

【Abstract】 Extracellular proteases of pathogens including serine protease are mainly participated in regulation of both parasites’ development and metabolism. In the last decades, extracellular enzymes as virulence factors have been intensively studied. Numerous enzymes have confirmed their involvement in the infection process and interaction with immune system of host. Nosema bombycis is the pathogen of pebrine which bring heavy losses to sericulture every year. To date, the pathogenic mechanism of N. bombycis is thought of plundering host’s nutrition by virtue of secreted protease during schizonts stages, which ultimately result in physiological dysfunction and host cell disruption. As research moved forward on microsporidia, the genome databases including Encephalitozoon cuniculi, Encephalitozoon intestinalis, Enterocytozoon bieneusi, Antonospora locustae, Nosema ceranae have been released and provided us a powerful platform to investigate microsporidia more intensively. In our study, we characterized the subtilisin-like protease (slp) which existed in all microsporidia genomes mentioned above. Subtilisin-like proteases are widespread in prokaryotes and eukaryotes and have been well researched. In the pathogenic fungi, subtilisin proteases were involved in degrading the cuticle proteins and helped fungi penetrate host cuticle. It was reported that high concentration of subtilisin in haemolymph caused the host phenoloxidase over-expression, which was one of main reasons for insect death. Our work represented the characterization of a putative subtilisin-like serine protease NbSLPl and its predicted three dimensional structure. Subsequently, heterologous expression was conducted and the NbSLPl enzymatic activity was tested with the refolded recombinant protein. Furthermore, multiple techniques including indirect immuno-fluorescence assay, immunoelectron microscopy and immunohistochemistry were employed to probe the localization of NbSLPl. Meanwhile, antibody blocking, co-immunoprecipitation, host cell transfection and yeast two hybrid were used to screen interacted proteins of NbSLPl. The main results are summarized from three aspects as following:1. Identification of Subtilisin-like proteases from microsporidia genomes and comparative genomics analysisThe typical subtilase possesses propeptide and Peptidase_S8domain. The MEORPS database and genome databases of six microsporidia were downloaded for searching subtilisin-like proteases (slp) using BLAST software. There are three subtilisin-like serine proteases in N. bombycis genome. Comparative genomics analyses show that all microsporidia genomes contain two kinds of subtilisin proteases, one has an Inhibitor_I9domain and a Peptidase_S8catalytic domain denoted as SLP1, the other has Peptidase_S8catalytic domain and a transmembrane domain denoted as SLP2. Both of hem possess catalytic residues Asp192, His224and Ser413. By homology with other subtilases, NbSLP1contains a76-aa Inhibitor_I9domain (Asp28to Ala104, Nbslp1p) and a290-aa (about31kDa) Peptidase_S8catalytic domain (Met172to Asn461, Nbslp1c). At the N terminal, the forward30aa of NbSLPl sequence was predicted as the signal peptide. Five N-glycosylation sites and one calcium binding site in NbSLPl were predicted which suggest NbSLP1is a metal dependent protease. Multiple sequences alignment reflected the NbSLP1shares46%dentity with SLP1from N. ceranae. Syntenic distribution of sips among microsporidia genomes reveals the sips genes loci are conserved. Then three dimensional modeling of NbSLPlC exhibited a typical three-layer sandwich structure, suggested that the mechanism of proteolytic and feature of binding substrate were similar to subtilase from Bacillus subtilis. For the structure of NbSLP1, the Inhibitor_I9domain extended into the inner of catalytic domain which could block the catalytic region. It suggested NbSLP1need to be processed into mature enzyme NbSLPlC through the cleavage of Inhibitor_I9domain. The phylogenetic tree showed the subtilisins of microsporidia fall into two groups:slp1and slp1. The Nbslpl transcripts were detected from Id to5d p.i.. It seemed that NbSLP1played a key role during the process of spore development and proliferation.2. Hetero-expression and localization of the NbSLPl The fragments of Nbslpl sequence according to the predicted domains (full sequence denoted as Nbslpl, catalytic domain denoted as Nbslplc, inhibitor domain denoted as Nbslplp) were cloned into the heterologous expression vector pET30a and over-expressed in BL21(DE3). The recombinant proteins were purified using nickel-nitrilotriacetic superflow columns and were used as antigen to raise polyclonal antibodies (PAb) in rabbit and mouse, respectively. The proteolytic activity of the refolded rNbSLP1was58U/mL at30℃and pH8.0.For immunoblotting assay, the antiserum to NbSLPIC acknowledged four major spots in2-D PAGE. Based on their MW and pI we deduced that the～60kDa spot was NbSLP1, while the other three spots in group about～1kDa werethe mature enzyme forms of NbSLP1with different post-translational modification.Indirect immunofluorescence assay (IFA), immunoelectron microscopy (IEM) and immunohistochemical assay (IHC) were performed to determine the distribution pattern of NbSLP1in spores. IFA showed the mature enzyme were located at the apical of the spore, just the site of polar tube extrusion, suggested NbSLP1may carry out its activity during the germination process. Meanwhile, the signals were observed in the spores proliferated in both Sf9cells and silk gland tissues, revealed NbSLPl was probably secreted during the proliferation of spores.3. Functional analysis of NbSLPl in N. bombycis sporesBased on the apical localization of NbSLP1, the effects of serine protease inhibitors phenylmethanesulfonyl fluoride (PMSF) and antibody blocking on the germination and infection of N. bombycis were investigated. Our results indicated that PMSF significantly reduced the spore germination rate with20%, suggested that subtilisin-like proteases in N. bombycis may participate in spore germination process and behave a key role in the polar tube extrusion. However, antibody blocking assay showed the incubated spores with anti-NbSLP1did not cause reduction of adherence rate to Sf9cells.Co-immunoprecipitation (Co-IP) was used to obtain the N. bombycis proteins interacted with NbSLPl. The candidate band from the gel of co-immunoprecipitation was analyzed by linear ion trap quadrupole MS and the results showed mitochondrial glycerol-3-phosphate dehydrogenase, eukaryotic translation initiation factor2C, Polar tube protein2, M1family aminopeptidase1, Hypothetical spore wall protein7, Spore wall protein1, ATPase, transitional endoplasmic reticulum ATPase and heat shock 70kDa protein (HSP70) were precipitated. Among these, both two types of ATPase and HSP70were coincidence with the predicted substrate by motif scan. Notably, the cleavage site of ATPase was between two AAA domains. We presumed that NbSLP1may process ATPase to hydrolysis ATP for energy production.Most of subtilase are non-specific proteases, thus we also try to find the substrates of NbSLP1in the host. Firstly we expressed Nbslpl in Sf9cells by transfection. The morphology of transfected cells was irregular and the cells were suspended more easily.2D-PAGE was used to find different spots between the Nbslp1transfected cell line and the control, there are seven spots absent from the cell line of transfected Nbslpl. MALDI-TOF/MS analysis indicated N-acetyltransferase, cuticle protein and hemolymph protein maybe the substrates of NbSLPl. Secondly, by subtilase proteolytic motif scan, we found a leucine rich transmembrane protein (BmTIR) containing intracellular TIR domain, which shared highest identity with Toll like receptor, could be the candidate substrate of NbSLPl. Toll receptors play an important role in innate immune and are very conservative in both insects and vertebrates. In the co-immunoprecipitation of anti-NbSLPIC incubated with the total tissue proteins of the infected Bombyx mori, anti-BmTIR could recognize the precipitated protein band, indicated that the precipitated band contain BmTIR protein from B. mori. Proteolytic test in vitro demonstrated rBmTIR was degraded by the refolded rNbSLP1. The interaction between NbSLP1and BmTIR may interfere with the Toll mediated immunity pathway by hydrolyzing TIR domain.In conclusion, our study on the apical localization and functional analysis of NbSLP1enriched the research of SLP in microsporidia and offered some clues to investigate the NbSLP1’s substrate intensively.更多还原