Structure-Function Analysis of Chlamydia Secretion Chaperones

衣原体分泌伴侣的结构-功能分析

• 批准号: 9211281
• 负责人: Raphael H Valdivia
• 金额: $ 51.79万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9211281
• 关键词: Affect; Affinity; Bacteria; Binding; Biochemical; Calorimetry; Cell physiology; Cells; Chlamydia; Chlamydia Infections; Chlamydia trachomatis; Complex; Cytoplasm; Dimerization; Epithelial; Genetic; Genitourinary system; Impairment; In Vitro; Infection; Infertility; Inflammatory; Kinetics; Lead; MCHR1 gene; Maps; Mass Spectrum Analysis; Mediating; Membrane Proteins; Modeling; Molecular; Molecular Chaperones; Molecular Genetics; Morbidity - disease rate; Motor; Mutagenesis; Mutation; Pathogenesis; Pelvic Inflammatory Disease; Process; Proteins; Protons; Regulation; Reporting; Reproductive Health; Role; Salmonella; Serine Protease; Signal Transduction; Site; Stimulus; Structure; Surface; System; Testing; Therapeutic Intervention; Titrations; Trachoma; Triad Acrylic Resin; Variant; Woman; base; conjunctiva; dimer; experimental study; in vivo; insight; mutant; novel; pathogen; public health relevance; response; sensor; seryl-histidine; targeted treatment; tool

 DESCRIPTION (provided by applicant): The obligate intracellular bacterium Chlamydia trachomatis is a widely disseminated obligate pathogen that infects epithelial surfaces of the conjunctiva and urogenital tract, leading to severe sequela such as blinding trachoma, pelvic inflammatory disease, and infertility. Chlamydia modulates multiple host cellular functions by delivering a large number (>80) of Type III secreted (T3S) "effector" proteins into the target host cell, whose secretion is regulated by specialized T3S chaperones that stabilize the secretory cargo and enhance their delivery to host cells. We hypothesize that in addition to these canonical functions, Chlamydia T3S chaperones can act as sensors of intracellular stimuli and impart a hierarchy to the secretion of effector proteins. We propose to elucidate the molecular basis and functional role of environmental stimulus-triggered recognition and release of inclusion membrane proteins, a subset of T3S effectors, by the chaperone Mcsc. Preliminary structural and biochemical analysis indicates that pH is an important intracellular signal that modulates the interaction between Mcsc and its effectors. We will test a model wherein an influx of protons due to the proton motor force triggers the rapid release of effectors from Mcsc to facilitate secretion. We will use a combination of mutagenesis, isothermal titration calorimetry, H/D exchange mass spectrometry, NMR, and the newly developed molecular genetic tools to define the binding interfaces of the oligomeric form of Mcsc and its functional implication in Chlamydia. We also propose to elucidate the molecular function of Slc1, the most abundant T3S chaperone in Chlamydia. We will examine if the temporal regulation in the secretion of effectors early in infection is determined by the differential binding affinities of Slc1 towards its effecto cargo proteins. By performing domain swapping experiments and point mutagenesis in key residues determining binding affinities, we will determine the general rules that govern chaperone-mediated temporal control of secretion. In addition, we will explore the significance of Slc1 interactions with the T3S basal component CdsD to assess its role in establishing a hierarchy in the translocation of effectors early during infection. Efficient interactions between T3S chaperones and secretion cargoes are important for stabilizing effector proteins and for regulating their orderly secretion, but these interactions also present an energy barrier that impedes rapid cargo release. How T3S chaperones regulate this process in response to environmental stimuli is not known. Our proposed structure-function analysis of Mcsc and Slc1 will unveil exciting new features of T3S chaperones and will shed fresh insights on how these conserved proteins regulate a key step in bacterial pathogenesis.

 描述（申请人提供）：专性细胞内细菌沙眼衣原体是一种广泛传播的专性病原体，可感染结膜和泌尿生殖道的上皮表面，导致严重的后遗症，例如致盲性沙眼、盆腔炎和不孕症。通过提供大量 (>80) III 型细胞功能分泌（T3S）“效应”蛋白到目标宿主中 我们发现，除了这些典型功能外，衣原体 T3S 伴侣还可以充当细胞内刺激的传感器，并赋予分泌层次结构。我们建议通过分子伴侣阐明环境刺激触发的包涵膜蛋白（T3S 效应子的一个子集）的识别和释放的分子基础和功能作用。 Mcsc 的初步结构和生化分析表明，pH 是调节 Mcsc 与其效应器之间相互作用的重要细胞内信号，我们将测试一个模型，即质子动力引起的质子流入会触发效应器从 Mcsc 快速释放。我们将结合使用诱变、等温滴定量热法、H/D 交换质谱、NMR 和新开发的分子遗传工具来定义结合界面。 Mcsc 的寡聚形式及其在衣原体中的功能含义我们还建议阐明衣原体中最丰富的 T3S 伴侣 Slc1 的分子功能，我们将检查感染早期效应物分泌的时间调节是否由差异决定。 Slc1对其效应货物蛋白的结合亲和力 通过在确定结合亲和力的关键残基中进行结构域交换实验和点突变，我们将确定控制的一般规则。此外，我们将探讨 Slc1 与 T3S 基础成分 CdsD 相互作用的重要性，以评估其在感染早期建立效应器易位层次结构中的作用。对于稳定效应蛋白和调节其有序分泌非常重要，但这些相互作用也存在阻碍 T3S 分子伴侣如何调节这一过程的能量屏障。我们提出的 Mcsc 和 Slc1 结构功能分析将揭示 T3S 伴侣的令人兴奋的新特征，并将为这些保守蛋白如何调节细菌发病机制的关键步骤提供新的见解。