Replacing animal models in fungal pathogen research: An in vitro Titam virulence assay for the human fungal pathogen Cryptococcus neoformans

新生隐球菌每年导致100万人感染，死亡人数超过结核病。虽然大多数患者艾滋病毒呈阳性，但30%的患者没有潜在的缺陷。未经治疗的感染是致命的，需要两性霉素B和5-氟胞嘧啶联合诱导治疗和长期氟康唑治疗。穿透性感染很常见，67%的人对氟康唑有短暂的耐药性。传统上，AMR是由靶蛋白的可遗传突变引起的，然而真菌AMR通常是由药物暴露后的非整倍体引起的。新城疫杆菌泰坦细胞可能是这种基因组可塑性能力的最极端的例子。当5M单倍体酵母内复制DNA，成为大(&gt；15m)，高度多倍体(4C-360C)细胞时，泰坦就形成了。然后，泰坦会产生小的、非整倍体的女儿，这些女儿会增殖并返回单倍体。然而，与由核或染色体分离缺陷引起的药物诱导的非整倍体不同，我们的数据表明钛化是一种受调控的形态转变。因此，TITAN代表了一种宿主介导的、可诱导的非整倍体途径，它是真菌耐药性的基础，这是一个主要的新兴研究领域。尽管它们很重要，但目前还不存在体外钛化试验。泰坦是短暂的，只在肺中被观察到。目前，分析包括通过吸入酵母感染大量小鼠，并在7天后扑杀进行终点分析。在过去5年里，研究体内钛化作用的实验室数量增加了一倍多，而且还将继续增长。手稿通常记录100-200只小鼠，未发表的实验中的动物数量可能更高(300-500只)，这进一步推动了体外模型的发展。因此，这个项目将验证我的实验室开发的体外钛化试验，它将取代小鼠，并促进使用标准和新兴的组织培养技术对宿主环境进行体外建模。此外，该模型将通过允许在体外观察泰坦细胞生物学并与宿主免疫细胞相互作用来改进体内技术。

Cryptococcus neoformans causes 1 million infections each year and more deaths than TB. While most patients are HIV-positive, 30% have no underlying deficiency. Untreated infections are fatal, requiring combination Amphotericin B and 5-flucytosine induction therapy and long-term Fluconazole. Break-through infections are common, and 67% are transiently Fluconazole resistant. Classically, AMR is caused by heritable mutations in target proteins, however fungal AMR is frequently caused by aneuploidy after drug exposure. The C. neoformans Titan cell is perhaps the most extreme example of this capacity for genome plasticity. Titans form when 5m haploid yeast endoreduplicate DNA, becoming large (>15m), highly polyploid (4C-360C) cells. Titans then produce small, aneuploid daughters that proliferate and return to haploidy. However, unlike drug-induced aneuploidy caused by defects in nuclear or chromosome segregation, our data demonstrate that Titanisation is a regulated morphological transition. Titans therefore represent a host-mediated, inducible path to aneuploidy that underlies fungal drug resistance, a major emerging research area. Despite their importance, no in vitro Titanisation assay exists. Titans are transient and only observed in the lung. Currently, analysis involves infecting large numbers of mice via inhalation of yeast and culling after 7 days for end-point analysis. The number of labs studying Titanisation in vivo more than doubled in the last 5 years and will continue to grow. Manuscripts typically document 100-200 mice, and animal numbers in unpublished experiments are likely to be higher (300-500), further urging development of in vitro models. Therefore, this project will validate an in vitro Titanisation assay developed in my lab that will replace mice and facilitate in vitro modelling of the host environment using standard and emerging tissue culture techniques. Further, this model will improve on in vivo techniques by allowing in vitro observation of Titan cell biology and interaction with host immune cells.