【作者】 张继瑜；

【导师】 阎继业； 金奇；

【作者基本信息】 中国人民解放军军需大学 ， 基础兽医学， 2002， 博士

【摘要】 志贺菌属是一类具有高度传染性和严重危害性的革兰氏阴性肠道致病菌，临床感染可以导致痢疾。每年世界上约有2亿人感染痢疾，其中死亡人数达到100万以上。尤其以儿童为主要发病人群。我国历史上曾经发生过痢疾的大流行，并呈现散在发生的趋势，平均每年都有上百万人感染痢疾，对公共卫生与健康造成了巨大威胁。在兽医临床上，志贺菌是灵长类动物痢疾的致病菌，细菌性痢疾是猕猴最常见的一种急性传染病，其发病率和死亡率在各类疾病中占首位。对痢疾的防治一向在世界各国受到高度重视，但是，由于痢疾独特的致病方式和它的急性发病过程，尤其对大多数抗生素与化学合成抗菌药具有强的耐药性，使对该病的治疗十分困难。 志贺菌属分四个种至少47个血清型，即痢疾志贺菌(Shigella dysenteriae，13个血清型)、福氏志贺菌(Shigella flexneri，15个血清型))、包氏志贺菌(Shigella boydii，18个血清型)和宋内氏志贺菌(Shigella sonnei，1个血清型)。在亚洲地区，福氏志贺菌是发展中国家痢疾流行的优势株，其中福氏2a为主要致病血清型。 志贺菌的致病过程主要包括细菌到达结肠粘膜，侵入粘膜上皮细胞并在细胞内繁殖，同时扩散到相邻细胞，引起程序性细胞死亡，最终造成肠粘膜水肿和坏死并脱落。志贺菌的致病性与其所携带的毒力大质粒有密切关系，该质粒约220kb大小，其基因可以编码一系列毒力因子，大质粒编码的蛋白IpaB、IpaC、IpaD、Mxi-Spa类和VirG等毒力因子构成志贺菌大质粒的全部表型。在大质粒DNA中，编码Ipa、VirG和Mxi-Spa类蛋白的基因紧密连接在一起，形成一个长31kb的区域，被称为“侵入区”，它包含约30多个基因。志贺菌大质粒的毒力同时受分布在大质粒DNA和细菌染色体上的调节基因的双重调控，同时与环境因子的存在密不可分。 本研究首次在世界上对志贺菌福氏2a的毒力大质粒和基因组进行了测定与研究。志贺菌作为人类与其它灵长类动物重要的病原菌，获得其完整的基因组信息对新药研究和疫苗开发具有重要意义。志贺菌毒力大质粒DNA全序列和基因组的研究，可对阐明志贺菌的致病机制和耐药机理奠定理论基础。远期对疫苗与药物的研制、耐药性问题的解决、疾病的防治与诊断具有重大意义。由于志贺菌毒力大质粒具有独特的细胞内侵入表型，将这种机制应用于人与动物细胞内的靶向给药系统研究具有广阔的应用前景，同时对“超级致病菌”的构建在军事上有着潜在应用价值。 全文共分三部分，第一部分为文献综述，对研究的意义、病原微生物基因组研究概况、志贺菌致病性的分子基础和细菌耐药性产生的分子机制进行了综述；第二部分对志贺氏菌福氏2a 301株毒力大质粒的核苷酸序列进行了测定，并对基因组成进行了比较分析。我们将该大质粒命名为pCP301；第三部分对志贺氏菌福氏2a 301株的基因组测定 进行了概述，确定了志贺氏菌福氏 Za 301 eie因组中与喷诺酮类药物耐药性有关的 yrA 和parC基因，并对这两种基因对环丙沙星的耐药性突变以及不同病原菌中的同原性进行 了研究，同时对基因组中与致病性有关的特殊结构进行了分析。 志贺菌福氏 Za 301株毒力大质粒核着酸序列测定结果表明，pCP301由 221，618个碱 基对组成，基因分析共确定了272个开放读码框架（ORF），经过在基因数据库中的同原 性比较，其中194个ORF与已知基因或编码产物具有高同源性，61个ORF同源性较低， 其余17个为新确定的未知功能的ORF。 PCP301中的基因主要包括：（l）与细菌毒力有关的基因，包括中一。－sPa、OsP和 iPh。；①与毒力调控功能有关的vr基因；（3）与质粒的维持、稳定和DNA代谢功能 有关的myP、sib、rep和par等基因；（4）转座酶编码基因；（5）未知功能的推定基因。 在pCP301 221kb的序列中，有68kb为插人序列，占去总长度的 30％，包括 18个插 入序列种类共计111个片段，其中5种括人序列为在pCP301中新发现的，我们将其分 别命名为ISSFpl－ISSFps。 我们将 pCP301与来源于志贺菌福氏 sa M90T株中的毒力大质粒 pRqZ100进行了比 较分析，结果表明，pCP301核着酸序列总长比卜汛100大8，124hp，pCP301与对流100 所具有的与毒力相关的基因完全一致，差异成分主要与插人序列有关；其次为外来细菌。 质粒以及志贺菌中的未知蛋白成分，其功能不清楚，另外有3个新的开放读码框架，其 推测产物在数据库中没有同原性。 志贺菌福氏 Za 301 ＄g因组测定表明，染色体核着酸序列全长 4，635，278hP，蛋白编 码区占整个基因组长度的86．23％，整个基因组的G＋C含量为50．88％。基因组中含有7 套 16s－235 IJ：’－nA，8套SS tA和 97套tRNA，总共确定了 4，576个开放读码框W ORF）， 平均长度为877’hp，其中3711个ORF在COG库中具有功能分类，占总数的81刀9％。其 余 865（18．gi％）个 ORF编码的基因功能无法确定或推测。 与大肠杆菌 K12的序列进行比较分析表明，志贺菌福氏 Za 301株序列存在?更多还原

【Abstract】 Bacteria of Shigella spp are high contagious, severe hazardous and gram-negative facutative intracellular pathogens, responsible for Shigellosis which characterized by fever, abdominal cramping and scanty, loose stools containing blood and mucus in clinic. Shigellasis remain a leading cause of infant mortality in the world. Shigella belong to the family Enterobacteriaceae and the group Escherichiaeae. They are divided into four species and at least 47 serotypes: Shigella dysenteriae (13 serotypes), Shigella flexneri (15 serotypes), Shigella boydii (18 serotypes), and Shigella sonnei (1 serotype). Shigella flexneri, the prevalent species in developing countries, kill more than 25,000 lives annually. In China, about 2 to 3 million cases of shigellosis are identified every year, accounting for more than 80% of the reported bacillary dysentery. Shigella flexneri 2a has been found to be responsible for the major epidemics and pandemics of shigellosis in China, accounting for 50-70% of the isolated strains.The mechanism of pathogenicity of Shigella flexneri is based on the capacity of itself to reach the colonic mucusa and to invade colonic epithelial cells, leading to intracellula bacterial multiplication, spread to adjacent cells, cell death, and eventually inflammation and destruction of the colonic mucosa . Enteroinvasive strain Shigella flexneri contains a 220kb large virulence plasmid which encode a series of factors required for virulence. Expression of several plasmid-encoded proteins, such as IpaB, IpaC, IpaD, Spa/Mxi and VirG etc., are nessesary for the complete plasmid phenotype of Shigella flexneri. The Ipa proteins act as the invasine, while the Spa/Mxi proteins make up a type III secretion apparatus which are involved in the presentation and transport of the Ipa proteins to and beyond the bacterial surface. In the virulence plasmid, the genes for proteins Ipa, Spa, Mxi and VirG are tightly located in a 31kb area, which is named as "invasion region", including about 30 genes. The expression of the virulence of the plasmid is regulated by several genes located both on the virulence plasmid and on the chromosome, as well as environmental stimuli.Determination of the complete DNA sequence of the virulence plasmid and the complete genome sequence of S. flexneri 2a 301 not only offer the opportunity to understand themechanism of pathogenicity and drug resistance of Shigella flexneri, but also provide comparisons of the virulence to those of different Shigella serotypes and closely related species. It is important for the recognition of the relationship of pathogenicity and evolution of 5. flexneri, shigellosis control, new drug discovery and vaccine development. Based on the special mechanism of invading into the host cell, it is potential to apply this mechanism in intracellular target drug delivery systems and construction of supper pathogen in military.We determined the complete DNA sequence of the large virulence plasmid pCP301 and the complete genome of 5. flexneri 2a strain 301, as well as analysis of genes on the large virulence plasmid and the structural genomic properties associated with bacterial virulence. The mutations related to the quinolone resistance and genetics and evolution of the QRAR sequences of gyrA and parC genes were also carried out.It resulted that the entire DNA sequence of pCP301 compose of 221,618 bp. Sequence analysis identified 272 open reading frames (ORFs), which include 194 corresponding to proteins described previously, 61 have low identity (<60%) to known proteins and 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include virulence associated genes, regulation associated genes and genes relating to plasmid maintenance, stability and DNA metabolism. Eighteen kind of insertion sequence (IS) elements, including 5 new IS elements were identified in pCP301, which account for 68 kb in length and 30 percent of the plasmid. It suggests that the multitime gene rearrangements have taken place in S. flexneri evolution hi更多还原