Role and function of Spx paralogs of Bacillus anthracis

炭疽杆菌 Spx 旁系同源物的作用和功能

基本信息

批准号：
8028620
负责人：
PETER ZUBER
金额：
$ 23.85万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-03 至 2012-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8028620
关键词：
Adaptor Signaling Protein Affect Affinity Alleles Anthrax disease Antibiotics Attention Bacillus anthracis Bacillus anthracis spore Bacillus subtilis Bacteria Binding Breathing Cells Cessation of life Column Chromatography Complex DNA-Directed RNA Polymerase Deletion Mutation Disease Disulfides Elements Equilibrium Family member Gene Expression Generations Genes Genetic Transcription Genome Genus staphylococcus Germination Gram-Positive Bacteria Growth Holoenzymes Human Immune Immune system In Vitro Infection Invaded Life Cycle Stages Listeria Mediating Microarray Analysis Nature Orthologous Gene Oxidants Oxidation-Reduction Oxidative Stress Pathogenesis Pathogenicity Peptide Hydrolases Phagocytes Phenotype Play Process Production Protein Family Proteins RNA RNA amplification Reducing Agents Regulon Reproduction spores Resistance Reverse Transcriptase Polymerase Chain Reaction Role Sepsis Specificity Sulfhydryl Compounds System Testing Thioredoxin Transcription Initiation Transcriptional Activation Virulence Virulent Zoonoses biological adaptation to stress gene function genetic manipulation in vivo macrophage member mutant null mutation paralogous gene promoter reconstitution response thioredoxin reductase tool transcriptomics weapons

项目摘要

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.

描述（由申请人提供）：炭疽芽孢杆菌的生命周期涉及两个细胞状态，两者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并被巨噬细胞吞没，巨噬细胞发芽以产生积极生长的营养细胞，使疾病永存。发芽时在巨噬细胞中诱导的炭疽基因基因包括全球转录调节剂SPX（ARSC蛋白家族成员SPX）的直系同源物，在革兰氏阳性细菌中激活了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的毒力决定簇的表达有关。它通过蛋白酶CLPXP和蛋白酶ClpxP和底物结合衔接蛋白YJBH施加的蛋白水解对照保持低浓度。 B.炭疽病包含在生命周期不同阶段表达的两种副型SPX的（SPXA1和SPXA2）形式。拟议项目的目的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。炭疽芽孢杆菌SPX基因的突变版本通过使用B. uttilis Icebs1元素结合将SPXA1和SPXA2抗蛋白酶抗性形式的SPXA1和SPXA2的抗性形式引入。我们将通过RT-PCR验证微阵列在体内验证体内，并通过重建在微阵列分析中鉴定的基因转录的SPX依赖性激活，因为它需要SPX进行诱导。我们将确定生命周期的哪个阶段（营养生长与孢子形成）SPX调节子表示。我们将确定SPX激活的转录和对SPX稳定性的控制是否受氧化还原平衡的变化影响，通过使用已知会在枯草芽孢杆菌中诱导SPX活性的氧化剂处理培养物。在两个SPX旁系同源物中，SPX双突变体的无效突变的表型将在氧化剂敏感性和SPX控制的基因表达方面进行检查。最后，我们将确定携带两种副SPX蛋白的全酶是否识别一组不同的启动子，与SPXA1和SPXA2全酶形式识别的启动子不同。这些发现将提供与炭疽芽孢杆菌生命周期和发病机理中两个SPX旁系同源物的作用有关的信息。该项目还将开发和利用一种新工具，用于炭疽芽孢杆菌的遗传操纵，这是通过使用由枯草芽孢杆菌ICEB1共轭元素介导的种间偶联系统强调的。公共卫生相关性：炭疽病的原因是形成孢子的细菌炭疽菌。感染后，炭疽孢子进入人类的免疫防御细胞，并开始从孢子转变为生长，有毒的细菌。这是通过激活细菌过程来实现的，这些过程促进了针对人类免疫系统安装的有毒氧化攻击的生长和防御。 SPX蛋白是氧化应激反应的已知调节剂，是入侵细菌产生的第一个控制因子之一，了解其在炭疽芽孢杆菌基因表达中的作用将为感染过程中活性的毒力机制提供线索。