Role and function of Spx paralogs of Bacillus anthracis

描述（由申请人提供）：炭疽杆菌的生命周期涉及两种细胞状态，这两种状态都在建立生产性感染中发挥作用。休眠的孢子能够逃脱免疫防御并被巨噬细胞吞噬，孢子在巨噬细胞中发芽，产生活跃生长的营养细胞，从而使疾病持续存在。发芽时在巨噬细胞中诱导的炭疽芽孢杆菌基因包括全局转录调节因子 Spx 的直系同源物，Spx 是 ArsC 蛋白家族成员，在革兰氏阳性细菌中激活在氧化应激反应中起作用的基因。 Spx 在革兰氏阳性细菌中高度保守，并且与李斯特菌和葡萄球菌中毒力决定簇的表达有关。通过蛋白酶 ClpXP 和底物结合接头蛋白 YjbH 发挥的蛋白水解控制，将其保持在低浓度。炭疽芽孢杆菌含有两种旁系同源（SpxA1 和 SpxA2）形式的 spx，它们在生命周期的不同阶段表达。拟议项目的目标是使用微阵列杂交分析来定义由两个 Spx 旁系同源物控制的调节子。编码蛋白酶抗性形式的 SpxA1 和 SpxA2 的炭疽芽孢杆菌 spx 基因的突变版本将通过使用枯草芽孢杆菌 ICEBs1 元件接合而引入炭疽芽孢杆菌中。我们将通过 RT-PCR 验证体内微阵列结果，并通过重建在微阵列分析中鉴定为需要 Spx 诱导的基因的体外转录的 Spx 依赖性激活。我们将确定 Spx 调节子在生命周期的哪个阶段（营养生长与孢子形成）表达。我们将通过用已知能在枯草芽孢杆菌中诱导 Spx 活性的氧化剂处理培养物来确定 Spx 激活的转录和 Spx 稳定性的控制是否受到氧化还原平衡变化的影响。将针对氧化剂敏感性和Spx控制的基因表达来检查spx双突变体的两个spx旁系同源物中无效突变的表型。最后，我们将确定带有两种旁系同源 Spx 蛋白的全酶是否识别一组不同的启动子，与 SpxA1 和 SpxA2 全酶形式所识别的启动子不同。这些发现将提供有关两种 Spx 旁系同源物在炭疽芽孢杆菌生命周期和发病机制中的作用的信息。该项目还将开发和利用炭疽芽孢杆菌遗传操作的新工具，重点是使用由枯草芽孢杆菌ICEBs1接合元件介导的种间接合系统。 公共卫生相关性：炭疽病的病因是形成芽孢的炭疽杆菌。感染后，炭疽孢子进入人体免疫防御细胞，并开​​始从孢子转变为正在生长的剧毒细菌。这是通过激活促进生长和防御人体免疫系统发起的有毒氧化攻击的细菌过程来实现的。 Spx 蛋白是一种已知的氧化应激反应调节因子，是入侵细菌产生的首批控制因子之一，了解其在炭疽杆菌基因表达中的作用将为了解感染期间活跃的毒力机制提供线索。

DESCRIPTION (provided by applicant): The life cycle of Bacillus anthracis involves two cellular states, both of which function in establishing a productive infection. The dormant spores are able to escape immune defenses and become engulfed by macrophages, where the spores germinate to generate actively growing vegetative cells that perpetuate the disease condition. The B. anthracis genes that are induced in the macrophage upon germination include the ortholog of the global transcriptional regulator, Spx, an ArsC protein family member, which in Gram-positive bacteria activates genes that function in the oxidative stress response. Spx is highly conserved in Gram-positive bacteria, and has been implicated in the expression of virulence determinants in Listeria and Staphylococcus.. It is kept at low concentrations by proteolytic control that is exerted by the protease ClpXP and a substrate-binding adaptor protein YjbH. B. anthracis contains two paralogous (SpxA1 and SpxA2) forms of spx that are expressed at different stages of the life cycle. The objective of the proposed project is to define the regulons controlled by both Spx paralogs using microarray hybridization analysis. Mutant versions of the B. anthracis spx genes encoding protease resistant forms of SpxA1 and SpxA2 will be introduced into B. anthracis by conjugation using the B. subtilis ICEBs1 element. We will verify the microarray results in vivo by RT-PCR and by reconstructing Spx-dependent activation of transcription in vitro of genes identified in microarray analysis as requiring Spx for induction. We will determine which stage of the life cycle (vegetative growth versus sporulation) the Spx regulons are expressed. We will determine if both Spx-activated transcription and control of Spx stability is affected by changes in redox balance by treating cultures with oxidants known to induce Spx activity in B. subtilis. The phenotype of null mutations in both spx paralogs, of the spx double mutant will be examined with respect to oxidant sensitivity and Spx-controlled gene expression. Lastly, we will determine if holoenzymes bearing both paralogous Spx proteins recognize a distinct set of promoters, different from those recognized by either SpxA1 and SpxA2 holoenzyme forms. The findings will provide information pertaining to the roles of the two Spx paralogs in the B. anthracis life cycle and pathogenesis. The project will also develop and exploit a new tool in the genetic manipulation of B. anthracis, highlighted by the use of the interspecies conjugation system mediated by the B. subtilis ICEBs1 conjugative element. PUBLIC HEALTH RELEVANCE: The cause of Anthrax is the spore-forming bacterium Bacillus anthracis. Upon infection, the anthrax spore enters the human immune defense cell and begins a transformation from spore into a growing, virulent bacterium. This is accomplished through activation of the bacterial processes that promote growth and defense against the toxic oxidative attack mounted by the human immune system. The Spx protein, a known regulator of the oxidative stress response, is one of the first control factors produced by the invading bacterium, and understanding its role in Bacillus anthracis gene expression will provide clues to the virulence mechanisms that are active during infection.