【作者】 刘威；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2014， 博士

【摘要】 支原体是一类缺少细胞壁的原核微生物,宿主范围十分广泛,具有高接触性、高传染性和高发病率的特性,而且对常用的作用于细胞壁的抗生素（如p-内酰胺酶类抗生素等）不敏感,因此临床控制十分困难。随着养殖业的快速发展,集约化程度越来越高,国际贸易日趋频繁,动物支原体的危害也逐渐显现,并且形势越来越严峻。由猪肺炎支原体（Mycoplasma hyopneumoniae,Mhp）引起的猪气喘病是严重危害全球养猪业的重大疫病。现已证实,猪肺炎支原体感染导致猪呼吸道纤毛损伤、脱落,从而引起肺炎。由于缺乏有效的遗传操作工具,人们对猪肺炎支原体致病机制方面的研究还相对滞后。目前,猪肺炎支原体毒力因子的研究主要集中在粘附因子方面,其中P97蛋白是最早被证实具有粘附作用的粘附因子,随后为数不多的粘附因子如P102、P146、P159和P216等相继报道,猪肺炎支原体是否存在其他与致病相关的毒力因子尚不明确。本研究以具有明晰进化历程的猪肺炎支原体168亲本株和疫苗株为切入点,通过系统的比较基因组学分析,从核苷酸碱基序列差异、整合性结合元件、引起基因组全长差异的因素、代谢途径差异以及氨基酸水平上的差异对粘附因子、被膜蛋白、蛋白分泌系统、免疫原性蛋白、转运蛋白的影响等方面,开展了猪肺炎支原体在实验室连续传代培养过程中毒力减弱的分子机制研究,为阐明猪肺炎支原体的致病机制提供理论依据。猪鼻支原体和猪肺炎支原体都被划分为猪支原体病的病原,但猪鼻支原体却表现出多样的寄生方式,具有侵染实验室多种细胞（来源于不同物种）的能力。猪肺炎支原体专性定植于呼吸道,而猪鼻支原体能定位于机体中多种组织,能在多种疾病的污染物中分离得到,而且有报道指出猪鼻支原体的长期感染能够诱导癌症的发生。相对于猪肺炎支原体,猪鼻支原体表现出了多样的生活方式,从基因组学层面剖析猪鼻支原体多样的生活方式会加深人们对猪鼻支原体的了解。本课题主要研究内容包括：1.猪肺炎支原体、猪鼻支原体的全基因组测序与生物信息学分析采用Solexa Genome Analyzer Ⅱx、Roche GS FLX和ABI3730测序系统相结合的优化方案,分别对猪肺炎支原体168亲本株和疫苗株、猪鼻支原体HUB-1株的全基因组序列进行了测定,并利用Multiplex-PCR和ABI Sequencing方法填补基因组序列空缺。在完成了全基因组序列的测定后,通过基因组ORFs的预测.编码区起点终点的校正、基因注释、功能基因分类、IS序列等分析,最终绘制了基因组完成图,其中HUB-1株的全基因组序列是国际上测定并公布在GenBank上的第一株猪鼻支原体全基因组序列。根据猪肺炎支原体的基因组信息,开展了基因组共线性分析、IS插入元件分析、膜相关蛋白分析和代谢分析,发掘了与病原菌生长、代谢相关的基因,重建了猪肺炎支原体的代谢通路,一共包括八大代谢门类,依次分别是核苷酸代谢、糖代谢、聚糖的生物合成和代谢、其它氨基酸的代谢、脂质代谢、辅因子和维生素代谢、氨基酸代谢和能量代谢。进一步分析发现猪肺炎支原体的碳源和能量主要来源于糖酵解途径,而在分析糖酵解途径时,猪肺炎支原体不能利用淀粉蔗糖代谢产生的α-D-Glucose-1P,其碳源几乎全部来源于外源葡萄糖的摄取。2.猪肺炎支原体亲本株、疫苗株的比较基因组学分析比较基因组学的分析主要从核苷酸碱基序列差异、整合性结合元件、引起基因组全长差异的因素、代谢途径差异和氨基酸水平上的差异对粘附因子、被膜蛋白、蛋白分泌系统、免疫原性蛋白、转运蛋白的影响等方面展开。猪肺炎支原体168亲本株和疫苗株基因组全长分别为925576bp和921093bp,两菌株基因组中基因的组成和顺序高度保守。通过对核苷酸碱基序列差异的系统分析,最终挖掘到了330个遗传变异位点（其中包括227个SNVs,60个插入和43个缺失）。有意义的是,目前国际上已报道的猪肺炎支原体毒力相关基因（如：P97, P102, P146, P159, P216等）和主要免疫原性基因（如：P36,P46,P65等）几乎全部包含在这330个遗传变异位点内。深入分析后发现,P97中的氨基酸突变发生在串联重复的短肽序列AAKPV/E中,而AAKPV/E功能域被证明与P97的粘附能力密切相关。P146中的谷氨酰胺的插入发生在[Q]n[（P/S）Q]m串联重复区,脯氨酸和谷氨酰胺富集区会形成polyproline Ⅱ helix的螺旋结构,这一螺旋结构在粘附过程中也十分重要。P216中四个氨基酸（poly Q）的缺失位于poly Q的重复区域,poly Q功能域在维持P216C端P85片段定位于细胞表面起着十分重要的作用,而P85具有粘附能力。P159中的错义突变发生在（S）（S）G（G）S重复区域中。进一步从氨基酸水平上分析了遗传变异对转运蛋白的影响。支原体拥有最低程度的新陈代谢以及最小的基因组冗余,为了适应寄生生活,支原体丢失了许多与代谢相关的基因,其营养物质的补给大部分来源于对外源物质的摄取。与此对应,支原体需要许多转运系统,猪肺炎支原体有两套转运系统：一种是磷酸转移酶系统（PTS）；一种是ABC transporter转运系统。磷酸转移酶系统在168亲本株传代过程中并没有受到影响。ABC转运系统中MHP168L₃94（ABC transporter permeaseprotein）和MHP168L₄13（ATP-binding protein）发生了突变,而它们在Mhp体内感染过程中的表达量相对于体外生长时是上调的,提示MHP168L₃94和MHP168L₄13在猪肺炎支原体感染的过程中发挥着某种作用,转运蛋白的突变很可能影响支原体在宿主细胞内的生存和增殖。对168亲本株和疫苗株的代谢途径进行了差异分析,经鉴定差异基因主要分布在7条代谢通路中,依次分别为糖酵解途径、嘌呤代谢途径、嘧啶代谢途径、甘油磷脂代谢途径、氨酰基tRNA生物合成途径以及磷酸戊糖途径。综上所述,似乎正是这些遗传变异在粘附、免疫、代谢等功能基因中不断地积累,造成了基因功能的改变,最终导致了菌株毒力的差异。3.支原体比较基因组学分析以支原体科20株支原体的完整基因组序列为研究材料,首先通过BLAST方法进行基因预测、Inparanoid程序寻找直系同源基因对,并采用Tribe MCL方法完成了20株支原体直系同源群的聚类化分析。随后进行了核心基因组学分析,确定了整个支原体科的核心基因。结合核心基因组与已报道的随机转座突变实验结果,系统分析了支原体赖以生存所必须的保守基因,指出在随机转座实验中干扰掉的wecA、 recU、uvrA、uvrB、mutM等基因可能在支原体野外的长期生存中是必须的,而它们在转座实验后短暂的生存能力可能是因为其对IS元件插入的暂时性耐受。通过对核心基因组和猪鼻支原体蛋白功能聚类的比较分析,发现支原体中与氨基酸合成、糖类运输和防御功能相关的蛋白急剧减少,指出了支原体在进化过程中功能基因丢失的趋势。将支原体的196个核心基因分别比对,并将比对结果串联,根据串联基因的比对结果,使用Tree-puzzle,在GTR+gamma+I模型下进行最大相似度拟合,计算支原体菌株间的距离矩阵,最终绘制了支原体科的超级进化树。在进化树的基础上,用Branch-Site方法评估了各分支上支原体在进化过程中自然选择压力的分布情况,发现猪鼻支原体的oriC（复制起始位点）区域受到了显著的自然选择,这很可能与猪鼻支原体拥有广泛的细胞侵染能力有着一定的联系。更多还原

【Abstract】 Mycoplasmas are widespread in nature as parasites of humans, mammals, reptiles, fish, arthropods, and plants. As conditional pathogenic organism, they associate with various diseases, including pneumonia, arthritis, meningitis and chronic urogenital tract disease. Since Mycoplasmas are total lack of a cell wall, they are not sensitive to β-lactamase drugs, which increases the difficulty of disease prevention and control. With the rapid development of breeding industry, the economic losses caused by Mycoplasmas are gradually revealed.Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia, which results in a mild, chronic pneumonia of swine. While progress has been made in understanding the molecular basis of some Mycoplasma diseases, advances in M. hyopneumoniae research have been hampered by its fastidious growth condition and the lack of genetic tools and transformation protocols. To date, few virulence determinants or virulence-associated determinants have been identified. Attachment to the respiratory epithelium is a prerequisite for host colonization and is mediated by the membrane protein P97. This protein is located on the outer membrane surface, and its role in adherence has been firmly established. Previous studies have revealed that P102, P146, P159and P216are also identified as virulence-associated determinants. Whether there is any other virulence factors contributed to M. hyopneumoniae pathogenesis remains largely unknown. In this study, we performed the first comprehensive analysis of M. hyopneumoniae strain168and its attenuated strain and made a preliminary survey of coding sequences （CDSs） that may be related to virulence. M. hyopneumoniae and M. hyorhinis are the causal agents of swine mycoplasmosis. The former causes a mild, chronic pneumonia of swine and results in deactivation of mucociliary functions. This agent is infective for a single host species. M. hyorhinis is generally considered a swine pathogen, yet is most commonly infect laboratory cell lines, implying that it can thrive among different species of cell lines. A strong link between M. hyorhinis and human cancer was reported recently. Interest has therefore shifted to questions of why M. hyorhinis exhibit high levels of functional diversity. The main researches are described as follows.1. Genome Sequencing and bioinformatics analysis of M. hyopneumoniae and M. hyorhinisWhole-genome sequencing was performed by combining GS FLX, ABI3730and Solexa paired-end sequencing technologies. Gaps were filled by local assembly of the Solexa/Roche454reads or sequencing PCR products using an ABI3730capillary sequencer. Open reading frames containing more than30amino acid residues were predicted using Glimmer3.0and verified by comparing with closely related genome sequences. This is the first complete genome sequence of M. hyorhinis, and its availability will provide a better defined genetic background for future studies of gene expression and regulation.We have constructed the metabolic network of M. hyopneumoniae, including eight metabolic pathways:Nucleotide Metabolism, Glycolysis, Glycan Biosynthesis and Metabolism, Lipid Metabolism, Metabolism of Cofactors and Vitamins, Amino Acid Metabolism, and Energy Metabolism。 Further analysis revealed that, the carbon source and energy are mainly produced by the glycolytic pathway. However, M. hyopneumoniae fails to utilize a-D-Glucose-1P, and almost all the carbon source comes from the uptake of exogenous glucose.2. Comparative genomic analyses of Mycoplasma hyopneumoniae pathogenic168strain and its high-passaged attenuated strainTo gain new insight into the components that contribute to virulence and the mechanisms by which M. yopneumoniae causes disease, we sequenced the genomes of strains168and168-L. The168-L genome has a highly similar gene content and order to that of168, but is4,483bp smaller because there are60insertions nd43deletions in168-L. Besides these indels,227single nucleotide variations （SNVs） were identified. We further investigated the variants affected CDSs, and compared them to reported virulence determinants. Notably, almost all the reported virulence determinants are included in these variants affected CDSs, including mycoplasma adhesins （P97, P102, P146, P159, P216, and LppT）, cell envelope roteins （P95）, cell surface antigens （P36）, secreted proteins and chaperone protein （DnaK）, mutations in genes elated to metabolism and growth. Furthermore, many mutations were located in the previously described repeat motif, which may be of primary importance for irulence.As Mycoplasmas are dependent on the exogenous supply of many nutrients, it has been predicted that they may need many transport systems. M. hyopneumoniae has two transport systems, including PTS transporter system and ABC transporter system. No mutations were identified in this PTS transporter family. However, five missense mutations and one synonymous substitution were identified in ABC transporter family. These included an ABC transporter permease protein （MHP168L₃94） and ABC transporter ATP-binding proteins （MHP168L₄13）. Interestingly, the expression of MHP168L₃94and MHP168L₄13was reported to be up-regulated in vivo during disease relative to in vitro-grown. The variability between strains168and168-L in multi-transport proteins indicates that they may affect growth and survival in different hosts or host tissues.3. Comparative genomics of Mycoplasma: analysis of conserved essential genes and diversity of the pan-GenomeMycoplasma, the smallest self-replicating organism with a minimal metabolism and little genomic redundancy, is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. This study employs comparative evolutionary analysis of twenty Mycoplasma genomes to gain an improved understanding of essential genes. By analyzing the core genome of mycoplasmas, we finally revealed the conserved essential genes set for mycoplasma survival. Further analysis showed that the core genome set has many characteristics in common with experimentally identified essential genes. Several key genes, which are related to DNA replication and repair and can be disrupted in transposon mutagenesis studies, may be critical for bacteria survival especially over long period natural selection. Phylogenomic reconstructions based on3,355homologous groups allowed robust estimation of phylogenetic relatedness among mycoplasma strains. To obtain deeper insight into the relative roles of molecular evolution in pathogen adaptation to their hosts, we also analyzed the positive selection pressures on particular sites and lineages. The oriC region was identified to be under positive selection in the HUB-1lineage. Previous studies have already demonstrated that replication may contribute to proliferation and efficiency of the colonization of hostile environments. Therefore, we suspected that selection pressure on oriC may be one of the reasons why M. hyorhinis can thrive among different species of cell lines.更多还原