ABSTRACT

Cryptococcus neoformans: (H99W) was serially passaged in the invertebrate wax moth Galleria mellonella fifteen times to study how fungal virulence evolves under selection and whether those adaptations affect virulence. The G. mellonella passaged strain (P15) and the pre-passage H99W strains were used to infect three different host models of C. neoformans: C. elegans, G. mellonella, and Balb/c mice. While there was no difference in survival in the invertebrate models, P15 killed mice faster than H99W through both intratracheal and intravenous routes of infection and mice infected intravenously with P15 showed higher fungal burden in the brain. Characterization of the major virulence factors of C. neoformans found that P15 had increased capsule size, GXM release, and melanization. Whole genome sequencing of P15 and H99W revealed two mutations in P15, an insertion in the promoter region of NADH dehydrogenase (CNAG_09000) and an insertion in the LMP1 gene (CNAG_06765). Both ATP production and metabolic rate were higher in P15 compared to H99W. Quantitative RT-PCR suggested that the increased ATP was due to increased RNA levels of NADH dehydrogenase. Thus, adaptation to growth in hemocytes resulted in increased production of ATP, increased metabolic rate, and increased virulence in mice. This was likely due to differential expression of virulence factors, which skewed the host immune response to a less efficient Th2 response, with higher levels of IL-4, IL-10, and TNF-α in the brain. Overall, serial passage experiments have increased our understanding of how this yeast evolves under innate immune selection pressure.

摘要

新型隐球菌：（H99W）在无脊椎动物蜡蛾被连续传代蜡螟十五倍研究下选择和是否适应那些致病真菌的演变如何影响毒性。在蜡螟传代株（P15）和所述预通道H99W菌株用于感染三种不同模型主机隐球菌：线虫，大蜡螟，和Balb / c小鼠。尽管在无脊椎动物模型中存活率没有差异，但通过气管内和静脉内感染途径，P15杀死小鼠的速度比H99W快，而静脉内感染P15的小鼠在大脑中的真菌负担更高。新型梭状芽胞杆菌主要毒力因子的表征发现P15具有增加的胶囊大小，GXM释放和黑色素化。P15和H99W的全基因组测序揭示了P15中的两个突变，即在NADH脱氢酶（CNAG_09000）的启动子区域中的插入和在LMP1中的插入。基因（CNAG_06765）。与H99W相比，P15中的ATP产量和代谢率均更高。定量RT-PCR表明ATP的增加是由于NADH脱氢酶RNA水平的增加。因此，对血细胞生长的适应导致增加的ATP产生，增加的代谢率和增加的小鼠毒性。这可能是由于毒力因子的差异表达所致，后者使宿主免疫反应偏向效率较低的Th2反应，大脑中的IL-4，IL-10和TNF-α含量较高。总的来说，连续传代实验增加了我们对酵母在先天免疫选择压力下如何进化的理解。