Desiccation resistance in Coxiella burnetii

描述（由申请方提供）：贝氏柯克斯体是一种革兰氏阴性g-变形菌属，可引起Q热，一种急性衰弱性流感样疾病。人们从农场动物或屠宰场和肉类加工厂的污染材料中感染Q热。据估计，多达1010个生物体可以在分娩时脱落。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者中发展。Q热的大规模爆发继续在荷兰的一个小的重农业地区，那里自2007年以来已有4,000多人感染了这种疾病。C.贝氏藻在环境中非常稳定，因为它非常耐干燥。此外，当C.贝氏体感染宿主 在细胞中，它存在于具有吞噬溶酶体的许多特性的细胞内空泡中。事实上，该生物体仅在液泡内pH为4.5时具有代谢活性，并且对通常在成熟吞噬溶酶体中发现的抗菌防御具有抗性。对环境胁迫抗性的分子机制知之甚少。无菌培养的研究进展。伯内特氏菌和新兴的遗传技术使我们有可能采用迄今为止不可能的实验方法。本研究的目的是探讨C.伯内特氏菌为此，提出了三个具体目标。第一个具体目标是鉴定在渗透胁迫下应该产生或积累的小分子量的植物保护剂相容性溶质。在干燥时，细菌必须首先适应增加的渗透压，通常通过合成或积累溶质，如海藻糖，甘氨酸甜菜碱或脯氨酸。这一现象在C.伯内特氏菌我们会暴露C。将burnetii的提取物置于高渗条件下，分离低分子量溶质，并通过13 C-NMR检测提取物的含量。第二个具体目标是确定C。burnetii基因受高渗条件、氧化损伤和干燥调节。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的Fe对蛋白质和DNA的氧化损伤。通过RNAseq的转录组分析将用于鉴定C.暴露于C.伯内特对所有三种压力。第三个目标是利用遗传学方法鉴定柑橘抗脱水性所需的基因。伯内特氏菌这将通过筛选转座子诱导的突变体来实现，这些突变体已经失去了耐受干燥的能力。这项工作将提供第一组关于C. burnetii抗渗透胁迫和干燥。

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.