Inducible Gene Regulation in Mycobacteria

描述（由申请人提供）：结核病（TB）是全球传染病中的第二大死亡原因。目前的一线结核病药物异烟肼通过抑制结核分枝杆菌外膜脂质分枝菌酸的生物合成来发挥作用，这强调了外膜在毒力和存活中的重要性。然而，对异烟肼和其他一线疗法的广泛耐药性要求开发新的药物来对抗多重耐药和广泛耐药微生物（MDR/XDR-TB）。通过组合两种或更多种药剂来抑制多个靶点的新型疗法对于增加治疗的快速性和功效以及减缓耐药性的出现是非常期望的。因此，迫切需要确定同时抑制两个基因的影响。长期目标是阐明（1）外膜生物发生的机制和（2）这些途径在促进结核分枝杆菌毒力和存活方面的协同作用。本申请的目的是通过开发分枝杆菌的新基因调控系统来促进这些目标。总体策略是优化核糖开关，以实现高效、可诱导的基因表达和抑制。提出的研究的理由是，基于核糖开关的诱导型基因调控工具将首次实现两个基因在体外和体内的独立和同时的实验控制。因此，提出了以下具体目标：（1）优化诱导型核糖开关以开启或关闭基因表达;（2）证明核糖开关对内源性分枝杆菌基因的调节;以及（3）确定小鼠中效应分子的适当给药方案。应答不同效应分子的核糖开关已经在申请人的实验室中在体外培养和巨噬细胞感染模型中得到验证。该方法是通过筛选随机序列变体文库来确定最佳核糖开关。本申请中提出的研究具有创新性，因为它通过将核糖开关应用于分枝杆菌基因调控而脱离了现状，并将克服当前系统评价双敲除表型的技术障碍。这一贡献是重要的，因为双敲除表型的阐明，无论是合成致死或抑制，将有助于创建代谢和信号传导途径及其相互作用的地图，也有利于数百个未知功能的结核分枝杆菌基因的功能分配。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is the second leading cause of death among communicable diseases worldwide. The current front-line TB drug isoniazid acts by inhibiting the biosynthesis of an Mtb outer membrane lipid, mycolic acid, underscoring the importance of the outer membrane in virulence and survival. However, widespread resistance to isoniazid and other front-line therapies requires the development of new drugs to combat multidrug- and extensively drug-resistant organisms (MDR/XDR-TB). Novel therapies that inhibit multiple targets by combining two or more agents are highly desirable to increase the rapidity and efficacy of treatment and slow the emergence of drug resistance. Therefore, there is an urgent need to identify the effects of repressing two genes simultaneously. The long-term goals are to elucidate (1) the mechanisms of outer membrane biogenesis and (2) the synergism between these pathways in promoting Mtb virulence and survival. The objective of this application is to facilitate these goals through the development of novel gene regulation systems for mycobacteria. The overall strategy is to optimize riboswitches for efficient, inducible gene expression and repression. The rationale for the proposed research is that inducible gene regulation tools based on riboswitches will enable, for the first time, the independent and simultaneous experimental control of two genes both in vitro and in vivo. Thus, the following specific aims are proposed: (1) To optimize inducible riboswitches to turn gene expression on or off; (2) to demonstrate riboswitch regulation of endogenous mycobacterial genes; and (3) to determine an appropriate dosing regimen for the effector molecule in mice. Riboswitches that respond to different effector molecules have been validated in in vitro culture and macrophage infection models in the applicant's laboratory. The approach is to identify optimal riboswitches by screening libraries of randomized sequence variants. The research proposed in this application is innovative because it departs from the status quo via the application of riboswitches to mycobacterial gene regulation and will overcome a current technical hurdle in the systematic evaluation of dual knockout phenotypes. This contribution is significant because the elucidation of dual knockout phenotypes, whether synthetic lethal or suppressive, will help create maps of metabolic and signaling pathways and their interactions, and also facilitate the functional assignment of the hundreds of Mtb genes of unknown function.