Role of WhiB3 in M. tuberculosis virulence

WhiB3 在结核分枝杆菌毒力中的作用

• 批准号: 7425429
• 负责人: ADRIE JC STEYN
• 金额: $ 33.72万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-15 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7425429
• 关键词: Affect; Alleles; Attenuated; Bacillus (bacterium); Bacteria; Binding; Biochemical; Biological Assay; Cause of Death; Cavia; Cessation of life; Class; Communication; DNA; DNA Microarray Chip; DNA Microarray format; DNA-Binding Proteins; Doctor of Philosophy; Electron Spin Resonance Spectroscopy; Elements; Equilibrium; Event; Family; Gene Cluster; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Transcription; Goals; Growth; Homologous Gene; Human; Hybrids; Immune response; Immune system; In Vitro; Infection; Intervention; Iron; Knock-out; Life; Lung; Measures; Mediating; Molecular; Mus; Mutagenesis; Mycobacterium bovis; Mycobacterium tuberculosis; Nitrogen; Not Defined; Oxidation-Reduction; Pathology; Pathway interactions; Phase; Play; Point Mutation; Polymerase Chain Reaction; Production; Proteins; Public Health; Reaction; Regulation; Regulon; Role; Series; Sigma Factor; Signal Pathway; Signal Transduction; Spectrometry; Starvation; Streptomyces; Stress; Sum; Superoxides; Testing; Time; Transcription Coactivator; Virulence; Virulent; Work; Yeasts; genetic regulatory protein; immunopathology; in vitro Model; in vivo; insight; member; mortality; mutant; mycobacterial; null mutation; pathogen; polyketide synthase; promoter; reactive oxygen intermediate; research study; response; yeast two hybrid system

DESCRIPTION (provided by applicant): M. tuberculosis (Mtb) is one of the leading causes of death worldwide and claims millions of lives annually. Approximately 1.7 billion people worldwide are asymptomatically infected with the tubercle bacillus and constitute a major impediment to worldwide public health control measures. Previous work had shown that a point mutation (Arg515->His) in the 4.2 domain of RpoV, the principal sigma factor in Mycobacterium bovis, is attenuating. Using the yeast two-hybrid system, we have established that the 4.2 domain of virulent Mtb specifically interacts with a regulatory protein WhiB3. In contrast, the attenuated RpoV allele containing the single point mutation was unable to interact with WhiB3. We constructed a Mtb whiB3 mutant (deltawhiB3) and showed that it behaved identical to the wild-type strain with respect to its ability to replicate in mice and guinea pigs in vivo. Mice infected with AwhiB3 showed significantly longer survival times than mice infected with the wild type Mtb. In addition, the lungs of AwhiB3-infected mice appeared much less adversely affected. It is notable that this virulence gene would not have been detected using conventional screens such as signature tagged mutagenesis, which screens for mutants primarily defective in growth, and not virulence. Furthermore, we have shown that a whiB3 mutant of virulent M. bovis, in contrast to AwhiB3, was completely attenuated for growth in guinea pigs. Mtb contain seven WhiB homologues that show strong homology to proteins that are critical for sporulation in Streptomyces spp. We hypothesize that WhiB3 regulates the expression of mycobacterial components that modulate the host immune system. To better understand the mechanism of whiB3 in Mtb virulence, we will use electron paramagnetic resonance spectroscopy (EPR) to biochemically characterize the WhiB3 Fe-S cluster genes, identify genes under WhiB3 control, and characterize proteins that interact with the WhiB family. We will also demonstrate that WhiB3 is a DNA binding protein capable of activating transcription of specific target genes. We will study the in vivo expression of the whiB family and their role in virulence. These studies will characterize the WhiB family as potential targets for interventions that may abolish virulence, but not growth. These studies will also provide insight into understanding whether TB is an anomalous immunological reaction in response to the persistent bacilli, whether the bacilli themselves induce lethal immunopathology, or if it is a combination of both.

描述（由申请人提供）：结核分枝杆菌（Mtb）是全世界死亡的主要原因之一，每年夺去数百万人的生命。全球约有 17 亿人无症状感染结核杆菌，构成全球公共卫生控制措施的主要障碍。先前的研究表明，牛分枝杆菌的主要西格玛因子 RpoV 4.2 结构域中的点突变 (Arg515->His) 正在减弱。使用酵母双杂交系统，我们已经确定毒力 Mtb 的 4.2 结构域与调节蛋白 WhiB3 特异性相互作用。相反，含有单点突变的减毒RpoV等位基因无法与WhiB3相互作用。我们构建了结核分枝杆菌whiB3突变体（deltawhiB3），并表明其在小鼠和豚鼠体内复制的能力与野生型菌株相同。感染 AwhiB3 的小鼠比感染野生型 Mtb 的小鼠表现出显着更长的存活时间。此外，AwhiB3 感染小鼠的肺部受到的不利影响似乎要小得多。值得注意的是，使用传统的筛选（例如特征标记诱变）无法检测到这种毒力基因，这种筛选主要筛选生长缺陷的突变体，而不是毒力缺陷的突变体。此外，我们还发现，与 AwhiB3 相比，强毒力牛支原体的 whiB3 突变体在豚鼠体内的生长完全减弱。 Mtb 含有 7 个 WhiB 同源物，与链霉菌属中对孢子形成至关重要的蛋白质具有很强的同源性。我们假设 WhiB3 调节分枝杆菌成分的表达，从而调节宿主免疫系统。为了更好地了解 WhiB3 在 Mtb 毒力中的机制，我们将使用电子顺磁共振波谱 (EPR) 对 WhiB3 Fe-S 簇基因进行生化表征，识别 WhiB3 控制下的基因，并表征与 WhiB 家族相互作用的蛋白质。我们还将证明 WhiB3 是一种 DNA 结合蛋白，能够激活特定靶基因的转录。我们将研究whB家族的体内表达及其在毒力中的作用。这些研究将 WhiB 家族描述为可能消除毒力但不能消除生长的干预措施的潜在目标。这些研究还将深入了解结核病是否是针对持久性杆菌的异常免疫反应，杆菌本身是否会引起致命的免疫病理学，或者是否是两者的组合。