VIRULENCE FACTORS OF CHLAMYDIAE

生殖器沙眼衣原体 D-K 血清型是流行的罪魁祸首 在美国，性传播感染影响了超过 400 万男性， 每年妇女和婴儿。 在女性中，如果不进行治疗，衣原体可能会感染 从子宫颈管呈小管状扩散至子宫内膜 （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 疤痕和/或卵子运输受损、宫外孕和 可能会发生不孕症。 显然，衣原体疾病 重大的初级、二级和三级卫生保健问题，其中 妇女承受着特殊的负担，因为她们遭受不利影响的风险增加 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、激素响应的人类生殖器上皮细胞模型中 系统，以便更多地了解原发性和 持续感染。 在具体目标1中，我们将分析 沙眼衣原体中的 3 种衣原体基因产物 - OrfA、GrpE 和 DnaK 通过 (i) 评估工程化亚克隆的附着性 确定哪些 ORF 负责附着表型，以及 (ii) 结合和衣原体竞争/抑制分析，使用 通过非变性方法纯化的重组蛋白和单特异性 针对这些蛋白质的抗体。 检验基因的真实性 表达及其与功能依从性的关系需要 将克隆的衣原体 DNA 重新引入沙眼衣原体。 在 具体目标 2，将评估 orfA-grpE-dnaK 的启动子区域 用于我们测试的穿梭载体。 此外，我们还将调查 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，用于启动子检测和 电穿孔后衣原体中的转座事件。 具体来说 目标3，传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“右侧朝外”顶端 膜囊泡，由长春花素/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 结合的衣原体的 pH 值 早期内体 - 未暴露与哇巴因和巴弗洛霉素暴露的比较 受感染的宿主细胞 - 将通过双通道扫描确定 共焦荧光显微镜。

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.