Temperature-dependent transposon mobilization in Cryptococcus neoformans

新型隐球菌中温度依赖性转座子动员

• 批准号: 9487877
• 负责人: SUE JINKS-ROBERTSON
• 金额: $ 23.85万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-25 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9487877
• 关键词: AIDS population; AIDS/HIV problem; Acids; Affect; Antioxidants; Biological Assay; Blood Circulation; Canavanine; Cells; Complex; Cryptococcus; Cryptococcus neoformans; DNA Transposable Elements; DNA Transposons; Development; Disease; Elements; Environment; FK506; Gene Expression; Gene Targeting; Generations; Genes; Genetic; Genetic Variation; Genome; Goals; Growth; Heat Stress Disorders; Human; Immune system; Immunocompromised Host; Infection; Inhalation; Invaded; Knowledge; Laboratories; Lung; Melanins; Meningoencephalitis; Modeling; Molecular Analysis; Monitor; Moths; Movement; Mus; Mutation; Nature; Neuraxis; Nose; Organism; Pathogenesis; Phagocytes; Phagocytosis; Pigments; Polysaccharides; Proliferating; Publishing; Reactive Oxygen Species; Reporter; Reporter Genes; Resistance; Retrotransposon; Sirolimus; Site; Southern Blotting; Specificity; Stress; System; Temperature; Virulence Factors; Waxes; Yeasts; burden of illness; capsule; experimental study; fungus; genome sequencing; genome-wide; innovation; insight; macrophage; next generation sequencing; novel; novel therapeutic intervention; pathogen; single molecule real time sequencing; therapeutic target; trait; whole genome

Cryptococcus neoformans is a ubiquitous environmental fungus that is a major cause of disease in immunocompromised patients. Following inhalation, there is propensity for yeast cells to invade the central nervous system, which can result in meningoencephalitis. A central question in the pathogenesis of this fungus is how it adapts to and proliferates in the harsh environment of the human host. Three major virulence factors have been identified: an anti-­phagocytic polysaccharide capsule, antioxidant melanin pigments, and the complex trait of thermotolerance. Preliminary results suggest the existence of an additional virulence factor: increased genetic instability at the human host temperature, which can contribute to rapid adaptation. Temperature-­dependent instability has been observed with cultured yeast cells and, at the two reporter genes examined to date, is due to insertional inactivation by transposable elements (TEs). C. neoformans is estimated to contain ~30 distinct TEs, but these elements have not been well characterized. The goal of the proposed experiments is to define the extent/nature of temperature-­dependent TE mobilization in free-­living yeast cells, and to examine whether similar mobilization occurs in the context of model host systems. Thus far, movement of both a DNA transposon and a retrotransposon has been detected using a single selective agent. Aim 1 will use other selective systems to probe for the mobilization of additional TEs. As the first step in characterizing TE movement on a global scale, Aim 2 will use the long reads produced by PacBio sequencing to annotate all elements in the genome. In Aim 3, transposon-­accumulation lines obtained by sub-­culturing yeast cells will be subjected to whole genome sequencing in order to define the extent of TE movement globally. Finally, Aim 4 will employ three host models (the greater wax moth Galleria mellonella, cultured murine macrophages, and nasally-­infected mice) to document similar TE mobilization in the context of complex fungal-­host interactions. Results obtained from these analyses will provide basic insight into how C. neoformans rapidly adapts to the human host to cause disease, which may in turn inform the development of novel therapeutic interventions.

新型隐球菌是一种普遍存在的环境真菌，是免疫功能低下患者疾病的主要原因。吸入后，酵母细胞有侵入中枢神经系统的倾向，这可能导致脑膜脑炎。这种真菌发病机制的一个核心问题是它如何适应人类宿主的恶劣环境并在其中增殖。已鉴定出三个主要毒力因子：抗细菌吞噬细胞多糖荚膜、抗氧化黑色素和耐热性的复杂特性。初步结果表明，存在一个额外的毒力因子：在人类宿主温度下遗传不稳定性增加，这可能有助于快速适应。在培养的酵母细胞中观察到了温度依赖性的不稳定性，并且在迄今为止检查的两个报告基因处，是由于转座因子（TE）的插入失活。C.据估计，neoformans包含约30种不同的TE，但这些元素尚未得到很好的表征。所提出的实验的目标是定义在自由酵母活酵母细胞中温度依赖性TE动员的程度/性质，并检查在模型宿主系统的情况下是否发生类似的动员。到目前为止，DNA转座子和反转录转座子的运动已经使用单一的选择剂检测。目标1将使用其他选择性系统来探测额外TE的动员。作为在全球范围内表征TE运动的第一步，Aim 2将使用PacBio测序产生的长读段来注释基因组中的所有元素。在目的3中，将通过亚细胞培养酵母细胞获得的转座子-β积累系进行全基因组测序，以全面定义TE移动的程度。最后，目标4将采用三种宿主模型（大蜡螟大蜡螟、培养的鼠巨噬细胞和鼻粘膜感染的小鼠），以记录在复杂的真菌-真菌宿主相互作用的背景下类似的TE动员。从这些分析中获得的结果将提供基本的洞察如何C。新型人迅速适应人类宿主而引起疾病，这反过来又可能为开发新的治疗干预提供信息。