Regulation of VapC toxins by Ser/Thr phosphorylation of VapB antitoxins in M. tuberculosis

项目总结 这项研究的长期目标是了解结核分枝杆菌毒素-抗毒素 系统受到调节，以促进结核病的发病。这项R21研究的具体目标 建议调查我们的假设，即VAPB抗毒素的Ser/Thr通过受体类型进行磷酸化 Ser/Thr蛋白激酶代表了一种新的机制，通过它可以调节VapC毒素对 环境信号。VapC毒素是结核分枝杆菌所有TA毒素的主要成分，一直以来 与蛋白质组重塑、生长停滞、抗生素耐受性和在一系列压力下生存的能力有关 与结核病发病机制相关的基因。VAPB抗毒素在隔离中的既定作用 同源VapC毒素蛋白和自动调节VapBC基因表达，以及我们最近的发现 在特异性激酶耗尽的情况下，几种VAPB蛋白的磷酸化水平显著降低， 为本研究提供前提。 这项研究提案有两个目的。在目标1中，我们将确定两个蛋白磷酸化的影响 VAPB抗毒素及其同源物对结核分枝杆菌生长的影响 VapC毒素，以及II)这些VAPB毒素与其同源VapC蛋白及其相互作用的分子相互作用 同族推动者。在目标2中，我们将研究这些VAPB抗毒素的磷酸化对 VapC酶活性。 这项研究将检验我们的假设，即VAPB Ser/Thr磷酸化可能是一种调节 VapC的活性，并将开始描述这种调节发生的分子机制(S)。 本研究的结果将为进一步研究丝氨酸/苏氨酸的磷酸化过程提供基础 VAPB抗毒素的功能是传递信号，控制VapC毒素的活性，这些毒素与 结核分枝杆菌生长控制、应激耐受与结核病发病机制。

PROJECT SUMMARY The long-term goal of this research is to understand how Mycobacterium tuberculosis toxin-antitoxin systems are regulated to contribute to tuberculosis pathogenesis. The specific goal of this R21 research proposal is to investigate our hypothesis that Ser/Thr phosphorylation of VapB antitoxins by receptor-type Ser/Thr protein kinases represents a novel mechanism by which VapC toxins can be regulated in response to environmental signals. VapC toxins are the majority of all TA toxins in M. tuberculosis and have been implicated in proteome remodeling, growth arrest, antibiotic tolerance and ability to survive a range of stresses that are relevant for tuberculosis pathogenesis. The established role of VapB antitoxins in sequestering cognate VapC toxin proteins and autoregulating vapBC gene expression, together with our recent finding of significantly decreased phosphorylation of several VapB proteins in the setting of specific kinase depletion, provide the premises for this research. This research proposal has two Aims. In Aim 1 we will determine the effects of phosphorylation of two VapB antitoxins on i) the growth of M. tuberculosis expressing these antitoxins together with their cognate VapC toxins, and ii) the molecular interactions of these VapB toxins with their cognate VapC proteins and their cognate promoters. In Aim 2 we will investigate the effects of phosphorylation of these VapB antitoxins on VapC enzyme activity. This research will test our hypothesis that VapB Ser/Thr phosphorylation may be a means to regulate VapC activity and will begin to characterize the molecular mechanism(s) by which this regulation occurs. Results from this research will provide the foundation for further investigation into how Ser/Thr phosphorylation of VapB antitoxins can function to transduce signals to control the activity of VapC toxins that are relevant for M. tuberculosis growth control, stress tolerance and tuberculosis pathogenesis.