An Inducible Expression System for M. tuberculosis

描述（由申请人提供）：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细菌。在过去的十年中，在了解结核病的分子发病机制方面取得了许多重要进展。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。虽然几年前开发了分枝杆菌中调控基因表达的系统，但其相对高水平的基础表达限制了其在许多实验中的使用。定义和表征必需基因是定义M中新型药物靶点的一个关键方面。结核该RO 3小额赠款申请的目标是适应诱导型表达系统，即在没有诱导的情况下具有最小基础表达的玫瑰色红球菌的正调控nitA启动子，用于分枝杆菌。该目标通过目标1来实现，其中a）将该系统的组分转移到一系列利用几种选择标记的复制和整合分枝杆菌载体中，B）将分析分枝杆菌中来自该启动子的未诱导的表达，并且如果需要，将该系统修饰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，该系统将用于子目标d）以确定感兴趣的基因（例如潜在的药物靶标）是否是必需的。该系统也将在子目的e）中进行测试，以确定其是否可用于在分枝杆菌宿主中，例如在分枝杆菌中表达全长可溶性重组分枝杆菌蛋白。恶臭

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.