GENETIC ANALYSIS OF CHLAMYDIAL VIRULENCE

我们的长期目标是通过检测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，并通过识别新的毒力基因和机制来研究沙眼衣原体的致病机制。这一应用的目的是将基因技术应用于发育基因调控和衣原体致病机制的研究。一个需要检验的假说是，在生命周期的关键阶段，基因的诱导或抑制反映了对环境信号的反应，环境信号在真核细胞释放之前触发了营养网状小体(RB)和代谢惰性但具有感染性的基本体(EB)之间的分化。此外，我们的目标是产生沙眼衣原体的等位基因替换突变株，这些突变株可以在合适的衣原体感染和疾病动物模型中进行研究。我们还打算测试这样的假设，即沙眼衣原体MoPn株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力。这项研究背后的理论基础是，沙眼衣原体遗传操作技术的发展将导致检测毒力基因表达和体内调控的方法。为了实现这一应用的目标，我们将追求三个特定目标：(I)在体内鉴定和鉴定受时间调控的启动子，尤其是那些在发育周期后期上调的启动子；(Ii)建立一种通用的方法来直接选择衣原体基因替换突变株；以及(Iii)通过在沙眼衣原体相对毒力较强的H株中表达互补文库，来区分与沙眼衣原体毒力有关的重要基因，然后通过体内传代丰富转化子以提高建立小鼠感染的能力。在这项研究完成时，我们预计已经确定了与RBS向EBS分化有关的基因。我们还希望建立沙眼衣原体定点突变的系统，并通过在衣原体疾病发病机制中产生定点突变基因来证明其有效性。此外，我们预计将鉴定至少一个与该毒株毒力有关的MoPn基因。

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.