Sigma factor networks of M. tuberculosis

描述(申请人提供)：细菌应激反应的一个关键方面是通过改变决定启动子特异性的亚基--西格玛因子，对RNA聚合酶(RNAP)进行重新编程。在RNAP全酶中用一种西格玛因子替代另一种西格玛因子会导致迄今为止沉默的细胞功能的表达。这些新表达的功能导致了细菌适应新环境条件和生存所需的代谢、形态和生理变化。人类病原体结核分枝杆菌建立和传播感染的能力与广泛的基因表达重新编程密切相关，这种重新编程使这种微生物在感染期间在复制(生长)和非复制(休眠)状态之间切换。对复杂基因调控的需要反映在结核分枝杆菌专性病原体中副西格玛因子/基因组大小的最高比率上。我们工作的前提是理解结核分枝杆菌的应激反应需要阐明这种微生物的西格玛因子的生物学意义。Sigma因子生物学的核心是Sigma因子之间的调控相互作用，这些相互作用是转录和转录后的。在目前的R21应用中，我们建议重建sigma因子转录网络的结构，并测试网络对压力条件的响应。实验方法是围绕三个目标建立的，这三个目标的特点是被测网络的复杂性不断增加。这些是：(1)鉴定在大肠杆菌中表达的结核分枝杆菌sigma因子之间的直接调控相互作用；(2)评估Sigma因子基因的应激反应非依赖性激活对结核分枝杆菌下游Sigma因子基因表达的影响；(3)表征暴露于抑菌应激源后的Sigma网络反应。AIMS的成功完成将使我们能够确定sigma因子之间的所有直接和间接转录相互作用，并为未来重建一个更大的、多尺度的调控网络产生假设，其中将包括对sigma因子活动的转录后控制，以及其他类别的应激反应和调节因子的参与。对西格玛因子网络的全面了解有望确定西格玛因子，这些因子是生长和休眠之间发育转换的一部分。这些西格玛因子(以及控制其表达和活性的调控途径)成为抗结核干预的潜在靶点。 公共卫生相关性：这项建议旨在重建人类病原体结核分枝杆菌中介导细菌对环境的适应和对压力反应的西格玛因子的转录调控网络，结核分枝杆菌是结核病的病原体。确定这个网络的特征将有助于确定关于如何改变病原体对压力的反应和在各种环境中生存的能力的假设。

DESCRIPTION (provided by applicant): A critical aspect of the bacterial stress response is the reprogramming of RNA polymerase (RNAP) by changing the sigma factor, a subunit that determines promoter specificity. Substituting one sigma factor with another in the RNAP holoenzyme results in the expression of hitherto silent cellular functions. These newly expressed functions lead to the metabolic, morphological, and physiological changes that are required for bacterial adaptation to the new environmental conditions and survival. The ability of the human pathogen Mycobacterium tuberculosis to establish and transmit infection is tightly associated with the extensive gene expression reprogramming that allows this microorganism to switch between replicating (growth) and nonreplicating (dormancy) states during infection. The need for complex gene regulation is reflected in the highest accessory sigma factor/genome size ratio seen in M. tuberculosis among obligate pathogens. The premise of our work is that understanding the stress response of M. tuberculosis requires elucidating the biology of this microorganism's sigma factors. Central to sigma factor biology are the regulatory interactions among sigma factors, which are transcriptional and post-transcriptional. In the present R21 application, we propose to reconstruct the architecture of the sigma factor transcriptional network and to test the network response to stress conditions. The experimental approach is built around three aims, which are characterized by the increasing complexity of the network tested. These are: (1) to identify direct regulatory interactions between M. tuberculosis sigma factors expressed in E. coli, (2) to assess the effect of stress-response independent activation of sigma factor genes on the expression of downstream sigma factor genes in M. tuberculosis, and (3) to characterize the sigma network response following exposure to bacteriostatic stressors. Successful completion of the aims will enable us to identify all the direct and indirect transcriptional interactions between sigma factors and to generate hypotheses toward future reconstruction of a larger, multi-scale regulatory network that will include post-transcriptional control of sigma factor activity and the involvement of additional classes of stress-sensing and regulatory factors. Full knowledge of the sigma factor network is expected to identify sigma factors that are part of the developmental switch between growth and dormancy. These sigma factors (and the regulatory pathways controlling their expression and activity) become potential targets for anti-tuberculosis intervention. PUBLIC HEALTH RELEVANCE: This proposal aims to reconstruct the transcriptional regulatory network of sigma factors, which mediate bacterial adaptation to the environment and response to stress, in the human pathogen M. tuberculosis, the causative agent of tuberculosis. Characterizing this network will help define hypotheses about ways to alter the pathogen's ability to respond to stress and survive in a variety of environments.