Temperature-dependent transposon mobilization in Cryptococcus neoformans

新生隐球菌是一种普遍存在的环境真菌，是免疫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。这种真菌发病机制中的一个中心问题是它如何适应并在人类宿主的恶劣环境中繁殖。已经确定了三个主要的毒力因素：抗吞噬多糖胶囊、抗氧化黑色素和复杂的耐热性。初步结果表明，存在一个额外的毒力因素：人类宿主温度下的遗传不稳定性增加，这有助于快速适应。在培养的酵母细胞中观察到了依赖温度的不稳定性，在迄今检测的两个报告基因中，这是由于转座元件(TES)的插入失活所致。据估计，新生隐孢子虫含有约30种不同的TES，但这些成分尚未得到很好的表征。拟议实验的目的是确定在自由生活的酵母细胞中依赖温度的TE动员的程度/性质，并检查类似的动员是否在模型宿主系统的背景下发生。到目前为止，已经使用单一的选择性试剂检测到了DNA转座子和反转录转座子的运动。AIM 1将使用其他选择性系统来探索动员更多的TES。作为在全球范围内表征TE运动的第一步，AIM 2将使用PacBio测序产生的长读数来注释基因组中的所有元素。在目标3中，通过继代培养酵母细胞获得的转座子积累系将进行全基因组测序，以确定TE在全球范围内的移动程度。最后，Aim 4将使用三个宿主模型(大蜡蛾Galleria mellonella、培养的小鼠巨噬细胞和鼻部感染的小鼠)来证明在复杂的真菌-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为新生葡萄球菌如何快速适应人类宿主而导致疾病提供基本的见解，这反过来可能为新的治疗干预措施的发展提供信息。

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.