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型生命周期，并通过确定新的毒力基因和机制，研究沙眼衣原体的致病机制。本申请的目的是将遗传技术应用于发育基因调控和衣原体发病机制的研究。 待检验的一个假设是，在生命周期的关键阶段的基因诱导或抑制反映了对环境信号的响应，所述环境信号在从真核细胞释放之前触发营养网状体（RB）和代谢惰性但具有感染性的基本体（EB）之间的分化。 此外，我们的目标是产生等位基因置换突变体的C。沙眼衣原体，可以在合适的衣原体感染和疾病的动物模型中进行研究。 我们还打算测试的假设，C。沙眼菌株MoPn表达特定的基因产物，使其在生殖器感染的小鼠模型中具有独特的毒性。 这项研究背后的基本原理是，C。沙眼衣原体的研究将导致研究体内毒力基因表达和调节的方法。 为了实现本申请的目的，我们将追求三个具体目标：（i）鉴定和表征体内时间调节的启动子，特别关注在发育周期后期强烈上调的那些启动子;（ii）开发用于直接选择衣原体中基因置换突变体的普遍适用的方法;和iii）区分对衣原体毒力重要的基因。沙眼衣原体菌株MoPn中表达互补文库。沙眼衣原体血清型H，然后通过体内传代富集转化体，增强了在小鼠中建立感染的能力。在这项研究完成后，我们希望已经确定了参与RB分化为EB的基因。 我们还希望能建立一个C.本发明涉及一种治疗沙眼衣原体的新方法，并且已经通过在基因中产生位点特异性突变来证明其功效，所述基因已经被认为在衣原体疾病的发病机制中起作用。 此外，我们预计确定至少一个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.