VIRULENCE FACTORS OF CHLAMYDIAE

衣原体的毒力因子

• 批准号: 2059974
• 负责人: Priscilla B. Wyrick
• 金额: $ 25.2万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2059974
• 关键词: Chlamydia trachomatis; SDS polyacrylamide gel electrophoresis; adhesin; antibacterial antibody; apical membrane; cell membrane; chlamydial disease; confocal scanning microscopy; disease /disorder model; epithelium; genetic promoter element; host organism interaction; human tissue; laboratory rabbit; molecular cloning; receptor; tissue /cell culture; transfection; transposon /insertion element; vinblastine; virulence; western blottings

Genital Chlamydia trachomatis serovars D-K are responsible for epidemic sexually transmitted infections in the USA, affecting over 4 million men, women and infants per year. In women, without treatment, chlamydiae may spread canalicularly from the endocervical canal to the endometrium (endometritis), and the fallopian tubes (salpingitis) - the constellation of which is defined as pelvic inflammatory disease. As a result of tubal scarring and/or impaired ovum transportation, ectopic pregnancy and infertility can occur. Clearly, chlamydial diseases constitute significant primary, secondary and tertiary health care concerns in which women bear a special burden because of their increased risk of adverse reproductive consequences. Our ultimate goal is to understand the basic biology of chlamydial growth in a polarized, hormone-responsive human genital epithelial cell model system in order to learn more about the mechanism of primary and persistent infection. In Specific Aim 1, we shall analyze the function of 3 chlamydial gene products - OrfA, GrpE, and DnaK - in C. trachomatis adherence by (i) assessment of attachment of engineered subclones to determine which ORF(s) are responsible for the attachment phenotype, and (ii) binding and chlamydial competition/inhibition analyses using the recombinant proteins, purified by non-denaturing methods, and monospecific antibodies against these proteins. To test the authenticity of gene expression and its relationship to functional adherence will require reintroduction of the cloned chlamydial DNA into C. trachomatis. In Specific Aim 2, the promoter region(s) of orfA-grpE-dnaK will be evaluated for use in our tested shuttle vector. In addition, we shall investigate a derivative of the OMEGON transposon, containing a promoterless CAT::luciferase reporter gene fusion, for promoter detection and transposition events in chlamydiae following electroporation. In Specific Aim 3, infectious chlamydiae and recombinant proteins, reconstituted into liposomes, will be bound to isolated, radiolabeled "right-side-out" apical membrane vesicles, generated by vinblastin/cytochalasin D or phenylarsine oxide; the complex will be immunoprecipitated, subjected to SDS-PAGE, autoradiography and electroblot for preliminary identification of epithelial receptors. Finally, the pH of SNAFL-conjugated chlamydiae in early endosomes - in unexposed versus ouabain- and bafilomycin-exposed infected host cells - will be determined with dual channel scanning confocal fluorescence microscopy.

生殖道沙眼衣原体血清型D-K是导致流行的原因 性传播感染在美国，影响超过400万男性， 妇女和婴儿每年。 在女性中，如果不进行治疗，衣原体可能 从子宫颈管向子宫内膜呈管状扩散 （输卵管炎）和输卵管（输卵管炎）-星座 其中一种被定义为盆腔炎。 由于输卵管 疤痕和/或卵子运输受损，异位妊娠和 可能发生不孕症。 很明显，衣原体疾病 重要的初级、二级和三级卫生保健问题， 妇女承受着特殊的负担，因为她们的不良风险增加， 生殖后果。 我们的最终目标是了解衣原体生长的基本生物学 在极化的、对精子应答的人类生殖器上皮细胞模型中， 系统，以了解更多有关的机制， 持续感染 在具体目标1中，我们将分析 3种衣原体基因产物：OrfA、GrpE和DnaK。沙眼衣原体 （i）评估工程化亚克隆与 确定哪个ORF负责附着表型，和 (ii)结合和衣原体竞争/抑制分析， 重组蛋白，通过非变性方法纯化，和单特异性 针对这些蛋白质的抗体。 为了检验基因的真实性 表达及其与功能粘附的关系将需要 将克隆的衣原体DNA重新导入C.沙眼 在 具体目标2，将评价orfA-grpE-dnaK的启动子区 用于我们测试的穿梭载体 此外，我们将调查 OMEGON转座子的衍生物，含有无启动子 CAT：：荧光素酶报告基因融合体，用于启动子检测， 电穿孔后衣原体中的转座事件。 在特定 目的3，感染性衣原体和重组蛋白，重组成 脂质体，将结合到分离的，放射性标记的“右侧外”顶端 膜囊泡，由长春碱/细胞松弛素D或苯胂产生 氧化物;复合物将被免疫沉淀，进行SDS-PAGE， 放射自显影和电印迹初步鉴定 上皮受体 最后，SNAFL结合的衣原体蛋白的pH值 早期内涵体-未暴露与哇巴因-和巴非霉素暴露 感染的宿主细胞-将用双通道扫描确定 共聚焦荧光显微镜。