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（由申请方提供）：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，感染结膜和泌尿生殖道的上皮表面，导致严重后遗症，如致盲性沙眼、盆腔炎和不孕症。衣原体通过将大量（>80）III型分泌的（T3 S）“效应”蛋白递送到靶宿主中来调节多种宿主细胞功能 细胞，其分泌由专门的T3 S分子伴侣调节，所述分子伴侣稳定分泌货物并增强其向宿主细胞的递送。我们假设，除了这些典型的功能，衣原体T3 S分子伴侣可以作为传感器的细胞内刺激，并赋予层次的效应蛋白的分泌。我们建议阐明的分子基础和功能作用的环境刺激触发的识别和释放的包涵体膜蛋白，T3 S效应子的一个子集，由分子伴侣Mcsc。初步的结构和生化分析表明，pH值是一个重要的细胞内信号，调节Mcsc和它的效应器之间的相互作用。我们将测试一个模型，其中由于质子马达力引起的质子流入触发效应物从Mcsc快速释放以促进分泌。我们将使用诱变，等温滴定量热法，H/D交换质谱，NMR和新开发的分子遗传学工具的组合，以确定结合界面的寡聚体形式的Mcsc和其功能的含义在衣原体。我们还建议阐明Slc 1的分子功能，最丰富的T3 S伴侣衣原体。我们将研究是否在感染早期的效应分泌的时间调节是由Slc 1对它的效应货物蛋白的差异结合亲和力。通过进行结构域交换实验和点突变的关键残基确定结合亲和力，我们将确定的一般规则，管理伴侣介导的时间控制分泌。此外，我们将探讨Slc 1与T3 S基础组分CdsD相互作用的意义，以评估其在感染早期效应子易位中建立层次结构的作用。T3 S分子伴侣和分泌货物之间的有效相互作用对于稳定效应蛋白和调节其有序分泌是重要的，但这些相互作用也存在阻碍快速货物释放的能量屏障。T3 S分子伴侣如何响应环境刺激调节这一过程尚不清楚。我们提出的Mcsc和Slc 1的结构-功能分析将揭示T3 S分子伴侣的令人兴奋的新功能，并将对这些保守蛋白如何调节细菌发病机制中的关键步骤提供新的见解。