VIRULENCE FACTORS OF CHLAMYDIAE

生殖器沙眼衣原体D-K血清型与流行的关系 在美国，性传播感染影响了400多万男性， 妇女和婴儿每年。在女性中，如果不接受治疗，衣原体可能会 从宫颈管内扩散到子宫内膜 (子宫内膜炎)和输卵管(输卵管炎)--星座 其中被定义为盆腔炎。由于输卵管 疤痕形成和/或卵子运输受损，异位妊娠和 可能会发生不孕不育。显然，衣原体疾病构成了 初级、二级和三级卫生保健方面的重大关切 女性承担着特殊的负担，因为她们患上不良反应的风险增加了。 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。 在极化的激素反应的人类生殖器上皮细胞模型中 系统，以了解更多关于小学和 持续性感染。在具体目标1中，我们将分析 沙眼衣原体的3种衣原体基因产物-orfa、grpe和dna-k 依从性：(I)评估工程亚克隆与 确定哪些ORF(S)负责依恋表型，以及 (2)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，单特异性 针对这些蛋白质的抗体。检验基因的真实性 表达及其与功能坚持的关系将需要 将克隆的衣原体DNA重新导入沙眼衣原体。在……里面 特异靶2，评估orfa-grpe-dNAK的启动子区域(S) 在我们测试的航天飞机上使用。此外，我们还将调查 Omegon转座子的一种衍生物，含有无启动子的 CAT：：荧光素酶报告基因融合，用于启动子检测和 电穿孔后衣原体中的转位事件。具体而言 目的3，感染性衣原体和重组蛋白，重组为 脂质体，将结合在隔离的、放射性标记的“右侧向外”根尖上 由长春新碱/细胞松弛素D或苯砷产生的膜小泡 氧化物；络合物将被免疫沉淀，经过十二烷基硫酸钠-PAGE， 放射自显影和电印迹初步鉴定 上皮性受体。SNAFL偶联衣原体的pH在 未暴露的早期内体与哇巴因和巴菲霉素暴露的对照 感染的宿主细胞-将通过双通道扫描确定 共聚焦荧光显微镜。

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.