Structural and functional characterization of two Mycobacterium tuberculosis transporters

两种结核分枝杆菌转运蛋白的结构和功能表征

• 批准号: 2131125
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2131125
• 关键词: Structural; functional; characterization; two; Mycobacterium

Antimicrobial resistance poses a great threat in the modern world, by rendering current treatments ineffective. Mycobacterium tuberculosis is an ancient human pathogen that is acquiring resistance to an increasing number of antibiotics, making the discovery of alternative treatments an urgent need. Bacterial efflux pumps are membrane embedded proteins that mediate active transportation of molecules across the plasma membrane. They have been implicated in the development of resistance by reducing the intracellular concentration of antibiotics, as was demonstrated by resistance to bedaquiline. This signified that the rational design of a molecule that targeted such drug transporters and inhibited their function could be a promising way of preventing or reversing antimicrobial resistance or tolerance. The M. tuberculosis genome reveals two efflux pumps, Rv3239c and Rv3728, which have not yet been structurally or functionally characterised, that could potentially enable bacterial adaptation and survival. They belong in the Major Facilitator Superfamily (MFS) of bacterial transporters. Rv3239c and Rv3728 are two of ten putative cAMP-binding proteins of M. tuberculosis. cAMP is a universal second messenger that mediates downstream cascades through interactions with proteins. cAMP binds Rv3239c and Rv3728, inducing allosterically conformational changes in the membrane embedded region that result in the transport of a molecule across the membrane. The two genes are closely related and share 61.9 % sequence identity, which could suggest comparable structures and functions. The objective of this project is the characterization of Rv3239c and Rv3728 and their potential role in development of antibiotic resistance using a combination of structural biology, microbiology and metabolomics. The first part of the project entails production of the transporters in the non-pathogenic Mycobacterium smegmatis or another suitable expression system. Their successful expression will be followed by optimisation of a purification protocol to enable structural analysis using an appropriate technique, for example X-ray crystallography or cryo-electron microscopy. The structure, in combination with metabolomics and microbiology experiments will be used to characterize the mechanism and direction of transport and to identify the transported substrate. This is an essential step towards the potential design of drugs to interfere with or neutralize the development of resistance to antibiotics.

抗微生物药物耐药性使目前的治疗无效，在现代世界构成巨大威胁。结核分枝杆菌是一种古老的人类病原体，对越来越多的抗生素产生耐药性，因此迫切需要发现替代治疗方法。细菌外排泵是膜内嵌入的蛋白质，介导分子在质膜上的主动运输。它们与通过降低细胞内抗生素浓度而产生耐药性有关，如对贝达喹啉的耐药性所证明的那样。这表明，合理设计一种靶向此类药物转运体并抑制其功能的分子可能是预防或逆转抗菌素耐药性或耐受性的一种有希望的方法。结核分枝杆菌基因组揭示了两个外排泵，Rv3239c和Rv3728，它们尚未被结构或功能表征，可能使细菌适应和生存。它们属于细菌转运蛋白的主要促进者超家族（MFS）。Rv3239c和Rv3728是已知的结核分枝杆菌camp结合蛋白中的两种。cAMP是一种普遍的第二信使，通过与蛋白质的相互作用介导下游级联反应。cAMP结合Rv3239c和Rv3728，在膜嵌入区诱导变构构象变化，导致分子跨膜运输。这两个基因亲缘关系密切，序列同源性为61.9%，表明其结构和功能具有可比性。本项目的目的是利用结构生物学、微生物学和代谢组学相结合的方法，研究Rv3239c和Rv3728在抗生素耐药性发展中的潜在作用。该项目的第一部分需要在非致病性耻垢分枝杆菌或其他合适的表达系统中产生转运蛋白。它们的成功表达之后，将对纯化方案进行优化，以便使用适当的技术进行结构分析，例如x射线晶体学或冷冻电子显微镜。该结构将结合代谢组学和微生物学实验来表征转运的机制和方向，并确定转运的底物。这是对潜在的药物设计的重要一步，以干扰或中和对抗生素的耐药性的发展。