Inducible Gene Regulation in Mycobacteria

描述(申请人提供)：结核病(TB)是全球传染病中第二大致死原因。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类脂霉菌酸的生物合成来发挥作用，强调了外膜在毒力和生存中的重要性。然而，对异烟肼和其他一线疗法的广泛耐药性需要开发新的药物来对抗耐多药和广泛耐药的生物(MDR/XDR-TB)。通过联合两种或多种药物来抑制多靶点的新疗法是非常可取的，以提高治疗的速度和有效性，并减缓耐药的出现。因此，迫切需要确定同时抑制两个基因的效果。长期目标是阐明(1)外膜生物发生的机制和(2)这些途径之间在促进结核分枝杆菌毒力和生存方面的协同作用。这项应用的目的是通过开发针对分枝杆菌的新的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。提出这项研究的基本原理是，基于核糖开关的可诱导基因调控工具将首次能够在体外和体内对两个基因进行独立和同时的实验控制。因此，提出了以下具体目标：(1)优化可诱导核糖开关以开启或关闭基因表达；(2)展示核糖开关对内源性分枝杆菌基因的调节；以及(3)为效应分子在小鼠中确定合适的剂量方案。对不同效应分子作出反应的核糖开关已经在申请人实验室的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的文库来确定最佳的核糖开关。本申请中提出的研究具有创新性，因为它通过应用核糖开关对分枝杆菌基因调控的应用改变了现状，并将克服目前在系统评估双基因敲除表型方面的技术障碍。这一贡献是重要的，因为阐明双重敲除表型，无论是合成致命的还是抑制的，将有助于创建代谢和信号通路及其相互作用的图谱，也有助于对数百个未知功能的Mtb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.