Structural and functional characterization of two Mycobacterium tuberculosis transporters

抗生素耐药性在现代世界中造成了巨大的威胁，使目前的治疗无效。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，迫切需要发现替代治疗方法。细菌外排泵是膜包埋蛋白，介导分子主动转运穿过质膜。它们通过降低抗生素的细胞内浓度而与耐药性的发展有关，如对贝达喹啉的耐药性所证明的。这意味着，针对这些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有希望的方法。分枝结核病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，这可能使细菌适应和存活。它们属于细菌转运蛋白的主要易化因子超家族（MFS）。Rv3239c和Rv3728是M.结核cAMP是通过与蛋白质相互作用介导下游级联的通用第二信使。cAMP结合Rv3239 c和Rv3728，诱导膜包埋区域的变构构象变化，导致分子跨膜转运。这两个基因密切相关，并且具有61.9%的序列同一性，这可能表明具有可比的结构和功能。该项目的目的是使用结构生物学、微生物学和代谢组学的组合来表征Rv3239c和Rv3728及其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另一种合适的表达系统中生产转运蛋白。它们的成功表达将通过纯化方案的优化进行，以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。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，并确定运输的基板。这是朝着潜在药物设计的重要一步，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.