【作者】 黄璐琳；

【导师】 夏庆友；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 在漫长的进化历程中,病原微生物与寄主之间形成了复杂的相互关系。黑胸败血芽孢杆菌(Bacillus bombyseptieus, Bb)是家蚕细菌性败血病的主要病原之一,1931年被分离鉴定。但迄今为止,Bb感染家蚕的分子病理、分子免疫等科学问题从未阐明。与之相似,典型的真菌病原球孢白僵菌(Beauveria bassiana)与家蚕相互作用的分子机制也尚不清楚；虽然BmNPV感染家蚕细胞的研究报道较多,但从个体水平系统分析BmNPV为代表的病毒感染家蚕引起的寄主应答机制也未阐明。本研究在家蚕基因组信息的基础上,利用Bb感染家蚕的生物模型,主要采用基因芯片技术,探索了Bb感染家蚕后引起的分子病理、免疫应答模式和致病机理。在此基础上,采用同样的方法,比较分析了白僵菌、BmNPV、Bb和E.coli感染家蚕引起的基因表达调控关系。获得的主要结果如下：(1)家蚕黑胸败血病病原-黑胸败血芽孢杆菌(Bacillus bombyseptieus, Bb),菌体大小为1-1.5μm×2.5-3gm,以单个、多个、短链或长链存在,能产生芽孢和伴孢晶体,添食感染能诱导家蚕死亡。克隆它的16S rRNA基因与NCBI己报道的同源序列比对和进化分析表明,Bb16S rRNA基因进化枝较长,这可能是由于它长期的独立进化所致。在进化关系上,它与蜡状芽孢杆菌(Bacillus cereus)和苏云金芽孢杆菌(Bacillus thuringiensis)的亲缘关系较近,而与炭疽芽孢杆菌(Bacillus anthracis)等种属的亲缘关系较远。(2)Bb添食感染家蚕能诱导家蚕强烈的寄主应答。在添食后的各个时间点(3h、6h、12h和24 h)，Bb都诱导了大量有功能活性的酶类等编码基因表达,全基因组应答在感染后24 h达到高峰。在Bb诱导家蚕的过程中,大约占基因组17%的基因(2,436个)被诱导表达。KEGG分析发现,Bb诱导了大量基本代谢通路相关基因,包含遗传物质的加工和转录(包括RNA聚合酶和基本转录因子)、核苷酸代谢(包括嘌呤和嘧啶代谢)、外来物质的生物降解(包括2,4-D降解、苯甲酸通过羟基化降解、苯乙烯降解)、氨基酸和氮代谢(包括色氨酸代谢、组氨酸代谢、缬氨酸、亮氨酸和异亮氨酸降解、尿素循环代谢和氨基酸代谢、氨基磷酸酯代谢、氮代谢),以及糖类代谢(包括戊糖和糖醛酸转换、三羧酸循环、丙酮酸代谢、磷酸戊糖途径、丁酸代谢)等多种基本代谢通路相关基因。参与这些基本代谢通路的基因,大部分上调表达,表明在Bb诱导家蚕后,寄主家蚕的物质和能量代谢加速进行,推测这是为了满足家蚕自身生存以及被迫为Bb提供物质和能量的结果。(3)无论是病毒病原BmNPV,还是真菌病原B.bassiana,都与革兰氏阳性细菌Bb一样,添食感染能引起家蚕强烈的寄主应答。比较这三种病原与非病原菌E.coli的诱导,病原菌的诱导强度更大,在4个时间点的感染过程中,分别有1804、2436和1743个基因被BmNPV、Bb及B.bassiana诱导表达,非病原菌E.coli诱导了912个基因表达,与病原比较相对较少。基因的表达总是由一系列的转录调控激活,调查在家蚕中鉴定的665个转录因子,Bb诱导的转录因子数量相对最多(68个),B.bassiana和BmNPV次之(分别为60个和56个),E.coli最少(27个),都包含了基本转录因子和其它转录因子。基因的诱导表达模式分析表明,从转录因子到其它基因表达,病毒BmNPV呈现了与众不同的诱导表达模式,主要表现在诱导的较早期(6h)强烈的寄主应答反应。而其它三种微生物的诱导表达模式比较相似,它们在诱导前中期(3 h-12h)的诱导反应较小,而在诱导后期24 h达到最高峰。比较四种微生物诱导的基因,它们之间有共通性,也有差异性。在发育相关的信号传导方面,JH、Wnt、MAPK、P53和cell cycle通路相关的生长因子、受体和必须复合物相关基因都在感染过程中不同程度被诱导表达。在基本代谢通路方面,包括-炭库叶酸、组氨酸代谢和色氨酸代谢相关基因被四种微生物都显著诱导,显示对微生物袭击较敏感。病原微生物具有诱导寄主致病的共性,共有7个基本代谢通路相关基因被病原特异诱导,它们是：异源物质的生物降解及代谢(羟基苯甲酸通过羟基化降解)、碳水化合物代谢(丙酮酸代谢)、核苷酸代谢(嘌呤代谢),能量代谢(氮代谢)、氨基酸代谢(尿素循环和氨基酸代谢)及辅因子和维生素代谢(泛酸和辅酶A合成、卟啉和叶绿素代谢),提示这些基本代谢通路对病原感染较敏感。在病理发生相关方面,包括细胞骨架、角质层相关蛋白、呼吸链、金属蛋白酶、蛋白水解酶家族等相关基因被诱导表达。在分子免疫方面,四种微生物不同程度的诱导了家蚕免疫信号识别、信号调节和效应因子等免疫相关基因上调表达,显示能激活家蚕的免疫应答。(4)对Bb引起的家蚕免疫相关基因分析表明,它可引起家蚕的细胞免疫、多酚氧化酶黑化级联,以及系统性免疫应答。在这些过程中,溶菌酶、凝集素、清道夫受体、铜／锌超氧化物歧化酶、免疫球蛋白超家族、肽聚糖识别蛋白、β-glucan识别蛋白、硫酯蛋白、CLIP丝氨酸蛋白酶、丝氨酸蛋白酶抑制剂(SPNs)、PPO、Toll信号通路JAK/STAT通路、抗菌肽Attacin、Enbocin、Gloverin、Lebocin和Moricin家族的部分基因都被诱导上调表达,提示Bb能引起家蚕较强烈的免疫应答反应。(5)对Bb感染家蚕的致病机制初步分析表明，Bb感染家蚕的致病性与Bb较相似。推测该模型如下：首先,Bb及其携带的毒素进入家蚕中肠,毒素释放,被寄主中肠的蛋白酶如丝氨酸蛋白酶等所消化激活,破坏家蚕的中肠围食膜,与寄主中肠上皮细胞微绒毛上的APN受体结合,由此进入细胞膜,在细胞上形成孔洞,破坏细胞。最后导致寄主家蚕的渗透平衡和细胞内外物质交换平衡受到破坏；细菌及毒素通过中肠上皮细胞的孔洞进入血淋巴,引起中毒性败血病,最终导致家蚕死亡。更多还原

【Abstract】 During the long evolutionary history, the relationships between pathogenic microorganisms and their hosts are very complex. Bacillus bombyseptieus (Bb) is one of the major pathogens of the silkworm, which can cause the silkworm bacterial septicemia by natural infection. Although Bb was identified in 1931, the scientific issues about Bb infection the silkworm such as the molecular pathology and molecular immunology of the silkworm host to Bb infection had never been investigated from now on. Similar conditions can be found about typical fungal pathogen Beauveria bassiana and typical virus pathogen BmNPV infection the silkworm. In order to explore the interaction relationships between pathogens and the silkworm, this study used the biological model of Bb oral infection the host silkworm to investigate the silkworm molecular pathology, its immune response patterns and its pathogenesis. Further, the biological models of silkworm pathogens Beauveria bassiana and BmNPV infection the host silkworm were also used to investigate the host-pathogen interactions at the host genome mRNA level. The main results obtained are showed as follows:(1) Silkworm black-breasted septicemia pathogen Bb is a typical Bacillus, which can produce parasporal crystal and kill the silkworm by oral infection. We cloned a 1.5-kb sequence of its 16S rRNA gene using universal primers. After comparison of the Bb 16S rRNA gene sequence with others in the NCBI database, the resulting phylogenetic tree clearly indicated that Bb belongs to Bacillus with a relatively long branch which might caused by a long-term of independent evolution. The phylogenetic analysis showed that Bb is with closest relationship to Bacillus cereus, close relationship to Bacillus thuringiensis, yet relatively far relationship to Bacillus anthracis and other microbes.(2) Bb oral infection the silkworm can cause the host strong responses which was involved in almost all silkworm tissues. During the infection (3 h,6 h,12 h and 24 h), totally 2,436 genes showed modulation, which is about 17% of all the silkworm genome genes. At the 24 h time point, the host silkworm showed the strongest response. Genome-wide survey of the host genes demonstrated many genes and pathways modulated after the infection. GO analysis of the induced genes indicated that their functions could be divided into 14 categories. KEGG pathway analysis identified that six types of basal metabolic pathway were regulated, including genetic information processing and transcription, carbohydrate metabolism, amino acid and nitrogen metabolism, nucleotide metabolism, metabolism of cofactors and vitamins, and xenobiotic biodegradation and metabolism.(3) Regardless of virus pathogen BmNPV or fungal pathogen B.bassiana are like Gram-positive bacteria Bb and they all can cause strong host responses. Totally 1804,2436 and 1743 genes showed regulation after BmNPV, Bb and B.bassiana infection respectively during the course of infection. For the silkworm non-pathogen E.coli infection, totally 912 genes showed regulation, which is relatively a smaller number. Searching the identified 665 transcription factors (TFs) of the silkworm genome, there are 68,60,56 and 27 genes were induced by Bb, B.bassiana, BmNPV and E.coli, respectively. Among the genes induced by four microbes, from TFs to total induced genes, BmNPV showed a distinctive induced expression pattern, which showed strong response at the "earlier" time of infection (6 h). However, for the other three microbes’infection, they showed strong responses at the "later" time point of infection (24 h).Notably, a lot of genes encode growth factors, receptors and essential components involved in development signaling pathways such as JH, Wnt, MAPK, P53 and cell cycle were modulated after microbes’infection. Major basic metabolic pathways, including genetic information processing and transcription, nucleic acid metabolism, metabolism of cofactors and vitamins, xenobiotics biodegradation and metabolism, amino acid metabolism and nitrogen metabolism, and carbohydrate metabolism illustrated their modulation. Among them three kinds of pathways that related to metabolism of cofactors and vitamins and amino acid metabolism were common induced after four microbes’infection, including one carbon pool by folate, histidine metabolism and tryptophan metabolism, indicating these pathways are very sensitive after microbes’infection. Totally seven pathways can be identified modulation by pathogens BmNPV, B.bombyseptieus and B.bassiana, including xenobiotics biodegradation and metabolism (benzoate degradation via hydroxylation), carbohydrate metabolism (pyruvate metabolism), nucleotide metabolism (purine metabolism), energy metabolism (nitrogen metabolism), amino acid metabolism (urea cycle and metabolism of amino groups), metabolism of Cofactors and vitamins (pantothenate and CoA biosynthesis and porphyrin and chlorophyll metabolism) indicating these pathways are very sensitive for pathogenic microbes’infection. Among the four induced microbes, BmNPV showed 14 specific pathways, which is much more than B.bombyseptieus (3 pathways), B.bassiana (5 pathways) and E.coli (2 pathways), indicating virus pathogen BmNPV leading to a broader level of host metabolic modulation. Gene expression associated with disease pathogenesis such as cytoskeleton, cuticle proteins, respiratory chain, metalloproteases, proteolytic enzymes coding genes were also modulated at different level. In the molecular immune response field, four kinds of microorganisms induced varying degrees of silkworm innate immune related genes up-regulation, such as signal recognition, signal modulation and effectors.(4) Bb can trigger the host immune responses, including cellular immune response and serine protease cascade melanization response. Real time PCR analysis showed that Bb can induce the silkworm systemic immune response, mainly by the Toll pathway. Anti-microorganism peptides (AMPs), including of Attacin, Lebocin, Enbocin, Gloverin and Moricin families, were upregulated at 24 hours post the infection.(5) Similar to Bacillus thuringiensis (Bt), Bb can also induce a silkworm poisoning-related response. This model has been speculated as follows:after oral infection, Bb interred the ingestion of silkworm. The parasporal crystal produced by Bb could be digested by silkworm midgut proteases. The digested toxins could pass through the peritrophic membrane to bind the aminopeptidase N receptors (APNs) of the midgut epithelial cells to damage them. Bb can go to the silkworm hemolymph from the damaged midgut and finally caused the silkworm toxic septicemia.更多还原