Role and function of Spx paralogs of Bacillus anthracis

描述(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方面发挥作用。休眠的孢子能够逃避免疫防御，被巨噬细胞吞噬，在巨噬细胞中萌发，产生活跃生长的营养细胞，使疾病永久化。炭疽杆菌在萌发时在巨噬细胞中被诱导的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发挥作用的基因。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素的表达有关。它通过蛋白水解酶ClpXP和底物结合接头蛋白YjbH的蛋白降解控制保持在低浓度。炭疽杆菌包含两种类似的SPX(SpxA1和SpxA2)形式，它们在生活史的不同阶段表达。该项目的目标是利用微阵列杂交分析来确定由两个Spx同源基因控制的调节子。利用枯草杆菌ICEBs1元件，将编码SpxA1和SpxA2蛋白酶抗性形式的突变版本的炭疽杆菌SPx基因通过接合导入炭疽杆菌。我们将通过RT-PCR和重建在微阵列分析中确定需要Spx进行诱导的基因的体外转录依赖于Spx的激活来验证在体内的微阵列结果。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。我们将通过用已知可诱导枯草杆菌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。关于氧化敏感性和Spx控制的基因表达，将检查SPx双突变体的两个SPx同源基因中零突变的表型。最后，我们将确定携带两个平行Spx蛋白的全酶是否识别一组不同于SpxA1和SpxA2全酶形式识别的启动子。这些发现将提供有关两个Spx Paralog在炭疽杆菌生活史和致病机制中的作用的信息。该项目还将开发和开发炭疽杆菌遗传操作的新工具，其突出特点是使用由枯草杆菌ICEBs1结合元件介导的种间结合系统。 公共卫生相关性：炭疽病的原因是芽胞形成细菌炭疽杆菌。一旦感染，炭疽孢子进入人类免疫防御细胞，并开始从孢子转变为生长的、有毒的细菌。这是通过激活细菌过程来实现的，这些细菌过程促进生长和防御人类免疫系统发起的有毒氧化攻击。Spx蛋白是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.