GENETIC ANALYSIS OF CHLAMYDIAL VIRULENCE

我们的长期目标是通过研究这种专性细胞内病原体如何调节其不寻常的二态生命周期，并通过鉴定新的毒力基因和机制来研究沙眼衣原体的致病机制。本应用程序的目的是应用遗传技术来研究发育基因调控和衣原体发病机制。一个有待验证的假设是，在生命周期的关键阶段，基因诱导或抑制反映了对环境信号的反应，这些信号在真核细胞释放之前触发了营养网状体（RB）和代谢惰性但具有传染性的初级体（EB）之间的分化。此外，我们的目标是产生沙眼衣原体等位基因替代突变体，可以在合适的衣原体感染和疾病动物模型中进行研究。我们还打算测试沙眼衣原体菌株MoPn表达特定基因产物的假设，使其在生殖器感染的小鼠模型中具有独特的毒性。这项研究的基本原理是，沙眼原体基因操作技术的发展将导致检测体内毒力基因表达和调控的方法。为了实现这个应用程序的目标，我们将追求三个具体目标；(i)识别和描述体内暂时调节的启动子，特别关注那些在发育周期后期强烈上调的启动子；（ii）建立一种普遍适用的衣原体基因替代突变体的直接选择方法；（iii）通过在相对无毒的沙眼衣原体血清H中表达互补文库，然后在体内传代富集，从而区分对沙眼衣原体菌株MoPn毒力重要的基因，从而增强了转化体在小鼠中建立感染的能力。在这项研究完成后，我们希望能够确定参与RBs向EBs分化的基因。我们还期望推导出沙眼衣原体的位点特异性突变系统，并通过在基因中产生位点特异性突变来证明其有效性，这些基因被认为在衣原体病的发病机制中起作用。此外，我们预计鉴定至少一个MoPn基因有助于该菌株的毒力。

Our long-range goals are to investigate the pathogenic mechanisms of Chlamydia trachomatis by examining how this obligate intracellular pathogen regulates its unusual dimorphic life cycle, and by identifying novel virulence genes and mechanisms. The objective of this application is to apply genetic techniques to the investigation of developmental gene regulation and chlamydial pathogenesis. One hypothesis to be tested is that gene induction or repression at critical phases of the life cycle reflects a response to environmental signals which trigger differentiation between the vegetative reticulate body (RB) and the metabolically inert but infectious elementary body (EB) prior to release from the eukaryotic cell. In addition, we aim to generate allele-replacement mutants of C. trachomatis that can be studied in suitable animal models of chlamydial infection and disease. We also intend to test the hypothesis that C. trachomatis strain MoPn expresses specific gene products that render it uniquely virulent in the mouse model of genital infection. The rationale behind this research is that the development of techniques for the genetic manipulation of C. trachomatis will lead to methods of examining virulence gene expression and regulation in vivo. To accomplish the objectives of this application we will pursue three specific aims; (i) to identify and characterize temporally regulated promoters in vivo, focusing particularly on those that are strongly up regulated late in the developmental cycle; (ii) to develop a generally applicable method for the direct selection of gene replacement mutants in Chlamydia and iii) to distinguish genes important for the virulence of C. trachomatis strain MoPn by expressing a complementation library in the relatively avirulent C. trachomatis serovar H then enriching by passage in vivo for transformants enhanced ability to establish infection in mice. At the completion of this research we expect to have identified genes which are involved in differentiation of RBs to EBs. We also expect to have derived a system for site-specific mutagenesis of C. trachomatis and to have demonstrated its efficacy by generating site-specific mutations in genes which have been suggested to play a role in the pathogenesis of chlamydial disease. In addition, we anticipate identifying at least one MoPn gene that contributes to the virulence of this strain.