Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
J Bacteriol. 2019 Nov 5;201(23). doi: 10.1128/JB.00569-19. Print 2019 Dec 1.
Anthrax toxin activator (AtxA) is the master virulence gene regulator of It regulates genes on the chromosome as well as the pXO1 and pXO2 plasmids. It is not clear how AtxA regulates these genes, and direct binding of AtxA to its targets has not been shown. It has been previously suggested that AtxA and other proteins in the Mga/AtxA global transcriptional regulators family bind to the curvature of their DNA targets, although this has never been experimentally proven. Using electrophoretic mobility shift assays, we demonstrate that AtxA binds directly to the promoter region of upstream of the RNA polymerase binding site. We also demonstrate that CO appears to have no role in AtxA binding. However, phosphomimetic and phosphoablative substitutions in the hosphotransferase system (PTS) egulation omains (PRDs) do appear to influence AtxA binding and regulation. and analyses demonstrate that one of two hypothesized stem-loops located upstream of the RNA polymerase binding site in the promoter region is important for AtxA binding and regulation Our study clarifies the mechanism by which AtxA interacts with one of its targets. Anthrax toxin activator (AtxA) regulates the major virulence genes in The bacterium produces the anthrax toxins, and understanding the mechanism of toxin production may facilitate the development of therapeutics for infection. Since the discovery of AtxA 25 years ago, the mechanism by which it regulates its targets has largely remained a mystery. Here, we provide evidence that AtxA binds to the promoter region of the gene encoding the main central protective antigen (PA) component of the anthrax toxin. These data suggest that AtxA binding plays a direct role in gene regulation. Our work also assists in clarifying the role of CO in AtxA's gene regulation and provides more evidence for the role of AtxA phosphorylation in virulence gene regulation.
炭疽毒素激活蛋白（AtxA）是炭疽杆菌主要毒力基因的调控因子，它既能调控染色体上的基因，也能调控 pXO1 和 pXO2 质粒上的基因。目前尚不清楚 AtxA 是如何调控这些基因的，也没有直接证据表明 AtxA 与它的靶标结合。先前曾有人提出，AtxA 及其在 Mga/AtxA 全局转录调控因子家族中的其他蛋白与它们 DNA 靶标的弯曲部分结合，尽管这从未在实验中得到证实。我们通过电泳迁移率变动分析实验证明，AtxA 直接与 RNA 聚合酶结合位点上游的 基因启动子区结合。我们还证明，CO 似乎对 AtxA 结合没有作用。然而，磷酸转移酶系统（PTS）调节域（PRD）中的磷酸模拟和磷酸失活突变似乎确实会影响 AtxA 结合和 基因的调控。点突变和染色质免疫沉淀分析表明，位于 RNA 聚合酶结合位点上游的 启动子区中两个假设的茎环结构之一对于 AtxA 结合和 基因的调控很重要。我们的研究阐明了 AtxA 与其中一个靶标相互作用的机制。炭疽毒素激活蛋白（AtxA）调控炭疽杆菌中的主要毒力基因。该细菌产生炭疽毒素，了解毒素产生的机制可能有助于开发炭疽感染的治疗方法。自 25 年前发现 AtxA 以来，其调控靶标的机制在很大程度上仍是一个谜。在这里，我们提供了证据表明 AtxA 结合到编码炭疽毒素主要中央保护性抗原（PA）成分的 基因的启动子区。这些数据表明 AtxA 结合在基因调控中发挥直接作用。我们的工作还有助于澄清 CO 在 AtxA 基因调控中的作用，并为 AtxA 磷酸化在毒力基因调控中的作用提供更多证据。
