TLR2 ligands of chlamydiae

描述(申请人提供)：沙眼衣原体感染是妇女非自愿性不孕症和异位妊娠的主要原因。尽管抗生素治疗可以治愈感染，但它并不能改善导致疾病发展的炎症过程。我们先前已经证明，Toll样受体2(TLR2)缺陷小鼠在衣原体感染后未能发生输卵管病变，表明TLR2信号直接参与疾病的发展。识别在衣原体感染反应中诱导TLR2依赖信号的细菌产物对于推进我们的最终目标至关重要，即确定驱动衣原体诱导的免疫病理学发展的分子机制。我们已经获得了新型的、缺乏质粒的鼠疫杆菌突变体，它们保留了感染小鼠生殖道的能力，但不会出现典型的上尿路疤痕和衣原体疾病的病理，因为它们无法在细胞培养和体内刺激TLR2依赖的信号转导。我们假设从衣原体质粒转录的基因产物或调控元件影响致病配体(S)的表达或修饰。在这项应用中，我们提出了仔细的、系统的蛋白质组学和生化方法来鉴定和表征驱动上尿路疾病发展的衣原体成分。具体地说，我们将：(1)使用二维差异凝胶电泳(2D-DGE)来解决与其亲本相比的表达谱的差异，并使用TLR2-Fc融合构建物作为鉴定候选TLR2配体和生化表征配体-受体复合体的一种方式；(2)检测不再由质粒缺陷的衣原体合成的衣原体糖原的免疫调节特性；以及(3)确定衣原体脂蛋白在诱导依赖TLR2的细胞因子表达中的作用。缺乏质粒的衣原体正常表达MIP，这是最近发现的衣原体TLR2候选配体，这表明天然的衣原体脂蛋白与生殖道疾病的发生无关。鉴定致病的TLR2配体(S)将有几个目的。我们将能够评估质粒缺陷菌株对TLR2依赖配体的表达。参与配体表达的基因或途径可能成为限制感染引起的组织损伤的治疗的药物靶点。它可能有助于识别内源性TLR2激活配体，这些配体可能有助于输卵管瘢痕形成的发展。最后，沙眼衣原体的TLR2配体可能作为一种免疫原，引起保护性反应，阻断或下调依赖于TLR2的信号转导，以应对衣原体感染，并提供一种新的方法来保护上生殖道免受有害后遗症的影响。公共卫生相关性：沙眼衣原体感染是妇女非自愿性不孕症和异位妊娠的主要原因。上生殖道的组织损伤和疤痕形成是由细菌制造的一种未知分子或配体引发的免疫反应的结果。本研究的目的是利用沙眼衣原体不能刺激这一信号通路的新型突变体作为工具来鉴定这种配体，并检测由衣原体制造的碳水化合物聚合物和脂蛋白的刺激特性，以确定它们是否也参与了这一破坏性的信号通路。

DESCRIPTION (provided by applicant): Chlamydia trachomatis infections are the leading cause of involuntary infertility and ectopic pregnancy in women. Although antibiotic treatment cures infection, it does not ameliorate the inflammatory process that leads to the development of disease. We have previously demonstrated that Toll-like receptor 2 (TLR2)-deficient mice fail to develop oviduct pathology after chlamydial infection, demonstrating that TLR2 signaling is directly involved in disease development. Identification of the bacterial product responsible for induction of TLR2-dependent signaling in response to chlamydial infection is essential to advance our ultimate goal of determining the molecular mechanisms that drive the development of chlamydia-induced immunopathology. We have derived novel, plasmid-deficient C. muridarum mutants that retain the ability to infect the murine genital tract but do not develop the characteristic upper tract scarring and pathology of chlamydial disease because they fail to stimulate TLR2-dependent signaling in cell culture and in vivo. We hypothesize that a gene product or regulatory element transcribed from the chlamydial plasmid influences expression or modification of pathogenic TLR2 ligand(s). In this application we propose careful, systematic proteomic and biochemical approaches to identify and characterize the chlamydial component that drives the development of upper tract disease. Specifically we will: (1) employ two-dimensional difference gel electrophoresis (2D-DIGE) to resolve differences in the expression profile of the plasmid-deficient strain when compared to its parent, and the use of a TLR2-Fc fusion construct as a way to identify candidate TLR2 ligands and to characterize ligand-receptor complexes biochemically; (2) examine the immunomodulatory properties of chlamydial glycogen which is no longer synthesized by plasmid-deficient chlamydiae and (3) determine the contribution of chlamydial lipoproteins to the induction of TLR2-dependent cytokine expression. Plasmid-deficient chlamydiae express Mip, a recently-identified candidate chlamydial TLR2 ligand, normally, suggesting that native chlamydial lipoproteins do not contribute to the development of reproductive tract disease. Identification of the pathogenic TLR2 ligand(s) will serve several purposes. We will be able to evaluate expression of the TLR2-dependent ligands by the plasmid-deficient strains. The genes or pathways involved in the expression of the ligand may serve as drug targets for therapies that limit tissue damage arising from infection. It may aid in the identification of endogenous TLR2 activating ligands that might contribute to the development of tubal scarring. Finally, it is possible that the TLR2 ligand of C. trachomatis could serve as an immunogen, eliciting protective responses that block or down-regulate TLR2-dependent signaling in response to chlamydial infection and provide a novel approach to protecting the upper reproductive tract from deleterious sequelae. PUBLIC HEALTH RELEVANCE: Chlamydia trachomatis infections are the leading cause of involuntary infertility and ectopic pregnancy in women. Tissue damage and scarring of the upper reproductive tract that occurs is the result of an immune response triggered by an as yet unknown molecule, or ligand, made by the bacteria. This application is to use novel mutants of C. trachomatis that cannot stimulate this signaling process as tools to identify this ligand and to examine the stimulatory properties of a carbohydrate polymer and a lipoprotein made by chlamydiae to determine if they also contribute to this damaging signal pathway.