An Inducible Expression System for M. tuberculosis

描述(申请人提供)：结核病仍然是艾滋病毒/艾滋病患者死亡的主要原因。需要新的治疗方法来缩短治疗时间，对抗耐药性，并杀死潜伏的细菌。在过去的十年里，在理解结核病的分子发病机制方面取得了许多重要的进展。这一进展的一个主要因素是分子遗传工具的发展，使研究人员能够操纵结核分枝杆菌。虽然分枝杆菌基因表达调控系统是在几年前开发出来的，但其相对较高的基础表达水平限制了它在许多实验中的使用。定义和表征基本基因是确定结核分枝杆菌新药物靶点的关键方面。RO 3小额拨款申请的目标是使一种可诱导的表达系统用于分枝杆菌，该系统是红色红球菌的正调控NITA启动子，在没有诱导的情况下具有最低的基础表达。这一目标是通过目标1实现的，其中a)利用几个可选择的标记将该系统的组件转移到一系列复制和整合的分枝杆菌载体中，b)将分析该启动子在分枝杆菌中的未诱导表达，并在需要时对系统进行修改，以使基础表达最小化，以及c)将确定表达对诱导剂浓度的剂量响应。然后，这个系统将被用于子目标，以确定一个感兴趣的基因，如潜在的药物靶点，是否是必需的。该系统还将在子目标中进行测试，以确定它是否有助于在分枝杆菌宿主中表达全长可溶重组分枝杆菌蛋白，例如在耻垢分枝杆菌中。

DESCRIPTION (provided by applicant): Tuberculosis remains the leading cause of mortality among persons with HIV/AIDS. New approaches to therapy are needed to shorten the duration of therapy, combat drug resistance and kill latent bacteria. Many important advances in understanding the molecular pathogenesis of tuberculosis have been made in the past decade. A major factor in this progress has been the development of molecular genetic tools that allow investigators to manipulate Mycobacterium tuberculosis. While a system for regulated gene expression in mycobacteria was developed several years ago, its relatively high level of basal expression limits its use in many experiments. Defining and characterizing essential genes is a key aspect of defining novel drug targets in M. tuberculosis. The goal of this RO3 small grant application is to adapt an inducible expression system, the positively regulated nitA promoter of Rhodococcus rhodochrous that has minimal basal expression in the absence of induction, for use in mycobacteria. This goal is addressed through Aim 1, in which a) the components of this system will be transferred to a series of replicating and integrating mycobacterial vectors utilizing several selectable markers, b) un-induced expression from this promoter in mycobacteria will be analyzed and the system modified, if needed, to minimize basal expression, and c) the dose response of expression to inducer concentration will be determined. This system will then be used in sub-aim d) to determine whether a gene of interest, such as a potential drug target, is essential. This system will also be tested in sub-aim e) to determine whether it is useful for the expression of full length soluble recombinant mycobacterial proteins in a mycobacterial host, e.g. in M. smegmatis.