TLR2 ligands of chlamydiae

描述（由申请人提供）：沙眼衣原体感染是女性非自愿不孕和异位妊娠的主要原因。虽然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。我们之前已经证明toll样受体2 （TLR2）缺陷小鼠在衣原体感染后不能发生输卵管病理，这表明TLR2信号直接参与疾病的发展。鉴定在衣原体感染应答中负责诱导tlr2依赖信号的细菌产物对于确定驱动衣原体诱导免疫病理发展的分子机制至关重要。我们已经获得了新的、质粒缺乏的C. muridarum突变体，这些突变体保留了感染小鼠生殖道的能力，但不会产生典型的上生殖道疤痕和衣原体疾病的病理，因为它们在细胞培养和体内都不能刺激tlr2依赖性信号。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性TLR2配体的表达或修饰。在这个应用中，我们提出仔细的，系统的蛋白质组学和生化方法来识别和表征驱动上呼吸道疾病发展的衣原体成分。具体而言，我们将：(1)采用二维差异凝胶电泳（2D-DIGE）来解决质粒缺陷菌株与其亲本相比表达谱的差异，并使用TLR2- fc融合构建体作为鉴定候选TLR2配体和生物化学表征配体-受体复合物的方法；(2)检查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；(3)确定衣原体脂蛋白对诱导tlr2依赖性细胞因子表达的贡献。质粒缺陷衣原体通常表达Mip，这是最近发现的候选衣原体TLR2配体，这表明天然衣原体脂蛋白不会促进生殖道疾病的发展。鉴定致病性TLR2配体将有几个目的。我们将能够评估tlr2依赖性配体在质粒缺陷菌株中的表达。参与配体表达的基因或途径可以作为限制感染引起的组织损伤治疗的药物靶点。这可能有助于鉴定内源性TLR2激活配体，这些配体可能有助于输卵管瘢痕的形成。最后，沙眼衣原体的TLR2配体可能作为一种免疫原，在衣原体感染时引发保护性反应，阻断或下调TLR2依赖性信号，为保护上生殖道免受有害后遗症的影响提供了一种新的途径。公共卫生相关性：沙眼衣原体感染是妇女非自愿不孕和异位妊娠的主要原因。上生殖道的组织损伤和瘢痕形成是由细菌产生的一种未知分子或配体引发的免疫反应的结果。这项应用是利用不能刺激信号传导过程的沙眼衣原体的新突变体作为工具来识别这种配体，并检查由衣原体产生的碳水化合物聚合物和脂蛋白的刺激特性，以确定它们是否也有助于这种破坏性的信号传导途径。

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.