Factors Mediating Host Resistance to Chlamydia trachomatis

描述（由申请人提供）：沙眼衣原体感染是全球发病率最高的疾病。慢性生殖器感染沙眼衣原体可导致盆腔炎、不孕症和其他妇女并发症。我们在这项拨款中的总体目标是确定沙眼衣原体在感染期间如何与哺乳动物宿主相互作用和操纵。在之前的项目期间，我们专注于在小鼠中进行大规模的前向遗传筛选，以绘制影响沙眼衣原体抗性的变异等位基因。我们确定了一个免疫相关gtp酶（IRGs）家族，该家族负责小鼠对沙眼衣原体的抗性。然而，在人类中，IRGs与沙眼衣原体耐药性无关；相反，人类通过表达吲哚-2,3-双加氧酶（IDO）来抵抗感染。在ifng介导的耐药性方面，IRGs和IDO是小鼠和人类之间的关键差异，这使得我们现在提出建立一种模拟人类沙眼衣原体感染的小鼠模型。为了实现这一目标，我们将设计人源化小鼠菌株，其中小鼠特异性免疫反应驱动的IRGs被依赖于IDO表达的人类反应所取代。目前，没有一种小动物模型能够再现这种慢性疾病在人类身上的表现，特别是导致人类生殖道病变和不孕症的长期或反复感染。我们还表明，在感染期间，许多沙眼衣原体蛋白效应物被转运到宿主细胞质中，在那里它们切割或以其他方式改变宿主蛋白。由于宿主细胞蛋白稳定性的改变似乎是沙眼衣原体使用的一般策略，我们正在应用两种新的筛选方法，即“细胞培养中氨基酸稳定同位素标记”（SILAC）和“全球蛋白稳定性”（GPS）来分析全球范围内沙眼衣原体感染期间哪些宿主蛋白受到干扰。一旦我们确定了在沙眼衣原体感染期间稳定或不稳定的宿主蛋白，我们将测试减少或增加这些蛋白在细胞中的流行是否会损害沙眼衣原体的发展。虽然病原体诱导的宿主改变是发病机制的关键，但以前不可能同时评估感染对单个宿主蛋白的影响，而这些方法现在可能达到这种规模。在本应用中探索的方法首次允许对细菌感染如何全局调节宿主细胞蛋白和转录水平以外的途径进行欣赏。了解沙眼衣原体与其人类宿主的相互作用需要大规模的方法来编目沙眼衣原体在感染过程中诱导宿主蛋白的变化。一旦了解了这些蛋白及其作用途径，破坏这些衣原体诱导的操作的影响只能通过准确反映人类沙眼衣原体感染发病机制的小动物模型来了解。

DESCRIPTION (provided by applicant): Infection with Chlamydia trachomatis is responsible for significant morbidity throughout the world. Chronic genital infections with C. trachomatis can lead to pelvic inflammatory disease, infertility, and other complications in women. Our overall goal in this grant is to define how C. trachomatis interacts with and manipulates the mammalian host during infection. In the previous project period we focused on a large-scale forward genetic screen in mice to map variant alleles that affect resistance to C. trachomatis. We identified a family of Immunity-Related-GTPases (IRGs) as responsible for mouse resistance to C. trachomatis. However in humans, IRGs are not responsible for C. trachomatis resistance; instead humans resist infection through the expression of indole-2,3-dioxygenase (IDO). The identification of IRGs and IDO as the key differences between mice and humans with regard to IFNg-mediated resistance leads us now to propose the development of a mouse model that mimics human infection with C. trachomatis. To achieve this goal we will engineer strains of humanized mice in which the mouse-specific immune response driven IRGs is replaced with the human response that depends on IDO expression. Currently, no small animal model exists that recapitulates the chronic disease as it manifests itself in humans, particularly the prolonged or recurring infections that cause pathologies of the genital tract and infertility in humans. We have also shown that a number of C. trachomatis protein effectors are translocated into the host cell cytosol during infection where they cleave or otherwise alter host proteins. Because alteration of host cell protein stability appears to be a general strategy used by C. trachomatis, we are applying two novel screening methods called "stable isotope labeling with amino acids in cells culture" (SILAC) and "Global Protein Stability" (GPS) to analyze, on a global scale, which host proteins are perturbed during infection with C. trachomatis. Once we have identified host proteins stabilized or destabilized during C. trachomatis infection, we will test whether reducing or increasing the prevalence of these proteins in cells impairs C. trachomatis development. Although pathogen-induced alterations of the host are a key to pathogenesis, it has not previously been possible to simultaneously assess the impact of infection on individual host proteins at the scale now possible with these approaches. The methods explored in this application allow, for the first time, an appreciation of how bacterial infection globally regulates host cell proteins and pathways beyond the transcriptional level. Understanding the interaction of C. trachomatis with its human host requires a large-scale approach to catalog the changes C. trachomatis induces in host proteins during infection. Once these proteins and the pathways in which they act are understood, the impact of disrupting these Chlamydia-induced manipulations can only be appreciated using small animal models that accurately reflect the pathogenesis of human C. trachomatis infection.