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（由申请人提供）：伯内特柯克斯体是一种革兰氏阴性 g 变形菌，可引起 Q 热（一种使人衰弱的急性流感样疾病）。人们因农场动物或屠宰场和肉类包装厂的受污染材料而感染 Q 热。据估计，分娩时可排出多达 1010 个生物体。尽管 Q 热在大多数情况下具有自限性，但一小部分康复者可能会出现慢性、难以治疗的心内膜炎相关形式。荷兰的一个小型农业密集地区继续大规模爆发 Q 热，自 2007 年以来，该地区已有 4,000 多人感染该病。伯内特隐球菌在环境中极其稳定，因为它具有极强的抗干燥能力。此外，当伯氏念珠菌感染宿主时 在细胞中，它存在于细胞内液泡中，具有吞噬溶酶体的许多特性。事实上，该生物体仅在液泡内 pH 值为 4.5 时具有代谢活性，并且对成熟吞噬溶酶体中常见的抗菌防御具有抵抗力。人们对抵抗环境压力的分子机制知之甚少。 C.burnetii 无菌培养和新兴遗传技术的最新进展使得现在可以采用迄今为止不可能实现的实验方法。本提案中描述的工作旨在找出伯内氏衣藻极端干燥抵抗力的分子基础。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应激时应产生或积累的小分子量渗透保护剂相容性溶质。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。尚未针对 C. burnetii 探索过这种现象。我们将把 C.burnetii 暴露在高渗条件下，分离低分子量溶质并通过 13C-NMR 检查提取物的含量。第二个具体目标是鉴定受高渗条件、氧化损伤和干燥调节的伯氏梭菌基因。通常，即使细菌能够耐受高渗条件，它们仍然对干燥敏感。干燥敏感性被认为是由于细胞内高浓度 Fe 对蛋白质和 DNA 造成氧化损伤。 RNAseq 的转录组分析将用于鉴定伯内特伯氏菌基因，这些基因因伯内特伯内氏菌暴露于所有三种胁迫而上调。最后，第三个目标是使用遗传方法来鉴定 C. burnetii 的抗干燥性所需的基因。这将通过筛选转座子诱导的失去耐干燥能力的突变体来实现。拟议的工作将提供第一组有关 C. burnetii 如何抵抗渗透压和干燥的信息。