Structural and functional characterization of two Mycobacterium tuberculosis transporters

抗菌素耐药性使目前的治疗方法无效，在现代世界构成了巨大的威胁。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抗药性，因此迫切需要找到替代治疗方法。细菌外排泵是一种膜包埋蛋白，可以调节分子在质膜上的主动运输。它们通过减少细胞内抗生素的浓度而与耐药性的形成有关，就像对贝达奎兰的耐药性所证明的那样。这表明，针对这种药物转运蛋白并抑制其功能的分子的合理设计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，这两个泵的结构或功能尚未确定，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是结核分枝杆菌cAMP结合蛋白中的两个。CAMP是一种通用的第二信使，通过与蛋白质的相互作用调节下游的级联反应。CAMP结合Rv3239c和Rv3728，在膜嵌入区引起变构构象变化，导致分子跨膜运输。这两个基因关系密切，有61.9%的序列同源性，提示它们的结构和功能具有可比性。本项目的目标是利用结构生物学、微生物学和代谢组学相结合的方法来表征Rv3239c和Rv3728，以及它们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污垢分枝杆菌或其他合适的表达系统中生产转运蛋白。在成功表达它们之后，将优化纯化方案，以便能够使用适当的技术进行结构分析，例如X射线结晶学或冷冻电子显微镜。这种结构，结合代谢组学和微生物学实验，将被用来表征运输的机制和方向，并鉴定运输的底物。这是朝着潜在的药物设计迈出的重要一步，以干扰或中和抗生素耐药性的发展。

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.