Desiccation resistance in Coxiella burnetii

伯内氏立克次体的干燥抗性

• 批准号: 8700034
• 负责人: HOWARD A SHUMAN
• 金额: $ 19.19万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-05 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8700034
• 关键词: Abattoirs; Acute; Aerosols; Agriculture; Alleles; Animal Housing; Antibiotic Therapy; Antimicrobial Resistance; Bacteria; Betaine; Biological Products; Biology; Birth; Breathing; Cattle; Cell physiology; Cells; Cellular biology; Centers for Disease Control and Prevention (U.S.); Chronic; Collection; Contracts; Coxiella; Coxiella burnetii; DNA; Deinococcus radiodurans; Desiccation; Development; Disease; Disease Outbreaks; Dose; Doxycycline; Endocarditis; Environment; Epidemic; Epithelial Cells; Farming environment; Gene Expression Profile; Genes; Genetic; Genetic Techniques; Goat; Growth; Human; Human Volunteers; Individual; Infection; Life; Livestock; Measurement; Meat; Metabolism; Methods; Military Personnel; Milk; Molecular; Molecular Weight; Mutate; Netherlands; Organism; Osmolar Concentration; Oxygen; Phagolysosome; Physiology; Plants; Prevalence; Proline; Property; Proteins; Q Fever; Radiation; Reporting; Research; Resistance; Sampling; Sheep; Stress; Time; Trehalose; Vaccines; Vacuole; Work; antimicrobial; axenic culture; base; cell type; design; flu; macrophage; mutant; oxidative damage; pathogen; preconditioning; programs; public health relevance; radiation resistance; research study; resistance mechanism; response; screening; solute; tissue culture; trafficking; trait; transmission process

DESCRIPTION (provided by applicant): Coxiella burnetii is a gram-negative g-proteobacterial species that causes Q fever, an acute debilitating flu-like illness. People contract Q fever from contaminated material from farm animals or from material in slaughterhouses and meat packing plants. It is estimated that as many as 1010 organisms can be shed at parturition. Although Q fever is self-limiting in most cases, a chronic, difficult to treat endocarditis - associated form f the disease can develop in a small percentage of recovered individuals. A large outbreak of Q fever continues in a small heavily agricultural region in the Netherlands, where more than 4,000 individuals have contracted the disease since 2007. C. burnetii is extremely stable in the environment as it is extremely resistant to desiccation. In addition, when C. burnetii infects host cells, it resides in an intracellular vacuole that has many properties of phagolysosomes. Indeed, the organism is only metabolically active at the intravacuolar pH of 4.5 and is resistant to the antimicrobial defenses usually found in mature phagolysosomes. The molecular mechanisms of resistance to environmental stresses are poorly understood. Recent advances in axenic culture of C. burnetii and emerging genetic techniques make it now possible to pursue experimental approaches that were not possible up until now. The work described in this proposal is aimed at finding out the molecular basis of the extreme desiccation resistance of C. burnetii. In order to do this three specific aims are proposed. The first specific aim is to identify small molecular weight osmo-protectant compatible solutes that should be produced or accumulated upon osmotic stress. Upon desiccation, the bacteria must first adapt to increased osmolarity usually by synthesis or accumulation of solutes such as trehalose, glycine betaine or proline. This phenomenon has not been explored for C. burnetii. We will expose C. burnetii to hyperosmotic conditions, isolate low molecular weight solutes and examine the contents of the extracts by 13C-NMR. The second specific aim is to identify C. burnetii genes that are regulated by hyperosmotic conditions, oxidative damage, and by desiccation. Usually even if bacteria are tolerant of hyperosmotic conditions, they remain sensitive to desiccation. Desiccation sensitivity is thought to be due to oxidative damage to proteins and DNA from high intracellular concentrations of Fe. Transcriptome analysis by RNAseq will be used to identify C. burnetii genes that are upregulated by exposure of C. burnetii to all three stresses. Finally the third aim is to use genetic approaches to identify genes required for desiccation resistance in C. burnetii. This will be accomplished by screening for transposon-induced mutants that have lost the ability to tolerate desiccation. The proposed work will provide the first set of information about how C. burnetii resists osmotic stress and desiccation.

描述(申请人提供)：伯氏柯克斯体是一种革兰氏阴性G蛋白细菌，可引起Q热，一种急性衰弱的流感样疾病。人们从农场动物或屠宰场和肉类加工厂的受污染材料中感染Q热。据估计，在分娩时可能会有多达1010个生物体脱落。虽然Q热在大多数情况下是自限性的，但一种慢性的、难以治疗的心内膜炎相关形式的疾病可以在一小部分康复的个人中发展。在荷兰一个以农业为主的小地区，Q热的大规模爆发仍在继续，自2007年以来，那里已有4000多人感染了这种疾病。伯氏梭菌在环境中非常稳定，因为它对干燥具有极强的抗性。此外，当伯氏梭菌感染宿主时 细胞，它驻留在细胞内的空泡中，具有许多吞噬溶酶体的特性。事实上，只有在液泡内pH值为4.5时，该微生物才能代谢活跃，并且对成熟吞噬菌体中常见的抗菌防御系统具有抵抗力。对环境胁迫抗性的分子机制知之甚少。伯氏梭菌无菌培养的最新进展和新兴的基因技术使人们有可能进行到目前为止还不可能的实验方法。本提案中所描述的工作旨在找出伯氏拟青霉极端耐脱水的分子基础。为了做到这一点，提出了三个具体目标。第一个具体目标是确定在渗透胁迫下应该产生或积累的小分子量渗透保护剂相容溶质。干燥后，细菌必须首先通过合成或积累海藻糖、甘氨酸甜菜碱或脯氨酸等溶质来适应渗透压的增加。这一现象在伯内提毛虫中还没有被探索过。我们将把伯氏梭菌暴露在高渗透条件下，分离低分子量的溶质，并用13C-核磁共振检测提取物的含量。第二个特定的目标是识别受高渗条件、氧化损伤和干燥调节的伯氏隐孢子虫基因。通常情况下，即使细菌对高渗透条件有耐受性，它们仍然对干燥很敏感。脱水敏感性被认为是由于细胞内高浓度的铁对蛋白质和DNA造成的氧化损伤。RNAseq的转录组分析将被用来识别因伯氏隐孢子虫暴露于所有三种压力而上调的基因。最后，第三个目标是使用遗传方法来鉴定布氏梭菌耐脱水所需的基因。这将通过筛选已经失去耐脱水能力的转座子诱导的突变体来实现。这项拟议的工作将提供关于布氏梭菌如何抵抗渗透胁迫和干燥的第一组信息。