Role and function of Spx paralogs of Bacillus anthracis

描述（由申请人提供）：炭疽芽孢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起作用。休眠的孢子能够逃脱免疫防御，被巨噬细胞吞噬，孢子在巨噬细胞中发芽，产生积极生长的营养细胞，使疾病持续下去。巨噬细胞萌发时诱导的炭疽芽胞杆菌基因包括全球转录调节因子Spx的同源基因，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在革兰氏阳性菌中高度保守，并与李斯特菌和葡萄球菌的毒力决定因子表达有关。通过蛋白酶ClpXP和底物结合接头蛋白YjbH施加的蛋白水解控制，使其保持在低浓度。炭疽芽孢杆菌含有两种相似的（SpxA1和SpxA2） spx形式，它们在生命周期的不同阶段表达。该项目的目标是使用微阵列杂交分析来定义由两个Spx类群控制的规则。编码SpxA1和SpxA2蛋白酶抗性形式的炭疽芽孢杆菌spx基因突变体将通过结合枯草芽孢杆菌ICEBs1元件引入炭疽芽孢杆菌。我们将通过RT-PCR在体内验证微阵列结果，并在体外重建在微阵列分析中鉴定的需要Spx诱导的基因的Spx依赖性转录激活。我们将确定在生命周期的哪个阶段（营养生长与孢子形成）表达Spx调控。我们将通过用已知能诱导枯草芽孢杆菌中Spx活性的氧化剂处理培养物来确定Spx激活的转录和控制Spx稳定性是否受到氧化还原平衡变化的影响。在spx双突变体的两个spx类群中，null突变的表型将根据氧化敏感性和spx控制的基因表达进行检查。最后，我们将确定携带两个相似的Spx蛋白的全酶是否识别一组不同的启动子，不同于SpxA1和SpxA2全酶形式所识别的启动子。该发现将提供有关两个Spx类似物在炭疽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一种新的工具来进行炭疽芽孢杆菌的遗传操作，重点是利用由枯草芽孢杆菌ICEBs1偶联元件介导的种间偶联系统。

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.