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

• 批准号: 8204494
• 负责人: PETER ZUBER
• 金额: $ 19.34万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-12-03 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8204494
• 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; public health relevance; reconstitution; response; thioredoxin reductase; tool; transcriptomics; weapons

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（由申请方提供）：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，在那里孢子发芽产生活跃生长的营养细胞，使疾病状态永久化。B。在巨噬细胞中萌发时被诱导的炭疽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，其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，并且已经涉及李斯特菌和葡萄球菌中毒力决定簇的表达。它通过蛋白酶ClpXP和底物结合衔接蛋白YjbH发挥的蛋白水解控制保持在低浓度。B。炭疽菌含有两种旁系同源（SpxA 1和SpxA 2）形式的spx，其在生命周期的不同阶段表达。该项目的目的是利用微阵列杂交分析来确定由两个Spx旁系同源物控制的调节子。B基因的突变体。将编码蛋白酶抗性形式SpxA 1和SpxA 2的炭疽spx基因导入B。炭疽杆菌通过使用B结合。subtilis ICEBs 1元件。我们将通过RT-PCR在体内验证微阵列结果，并通过在微阵列分析中鉴定为需要Spx诱导的基因的体外转录重建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，通过用已知诱导B中Spx活性的氧化剂处理培养物。枯草杆菌。将针对氧化剂敏感性和Spx控制的基因表达检查spx双突变体的两种spx旁系同源物中的无效突变的表型。最后，我们将确定如果全酶轴承旁系同源Spx蛋白识别一组不同的启动子，从那些由SpxA 1和SpxA 2全酶形式。这些发现将提供有关两个Spx旁系同源物在B中的作用的信息。炭疽的生活史和致病机理。该项目还将开发和利用B基因操作的新工具。炭疽菌，通过使用由B介导的种间接合系统突出显示。subtilis ICEBs 1接合元件。 公共卫生相关性：炭疽病的病因是孢子形成细菌炭疽杆菌。感染后，炭疽孢子进入人体免疫防御细胞，并开始从孢子转化为生长中的有毒细菌。这是通过激活细菌过程来实现的，这些细菌过程促进生长并防御由人类免疫系统发起的有毒氧化攻击。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期间活跃的毒力机制提供线索。