Cryptococcus neoformans infections cause approximately 15% of AIDS-related deaths owing to a combination of limited antifungal therapies and drug resistance. A collection of clinical and environmental C. neoformans isolates were assayed for increased mutation rates via fluctuation analysis, and we identified two hypermutator C. neoformans clinical isolates with increased mutation rates when exposed to the combination of rapamycin and FK506. Sequencing of drug target genes found that Cnl1 transposon insertions conferred the majority of resistance to rapamycin and FK506 and could also independently cause resistance to 5-fluoroorotic acid and the clinically relevant antifungal 5-flucytosine. Whole-genome sequencing revealed both hypermutator genomes harbour a nonsense mutation in the RNA-interference component ZNF3 and hundreds of Cnl1 elements organized into massive subtelomeric arrays on each of the fourteen chromosomes. Quantitative trait locus mapping in 28 progeny derived from a cross between a hypermutator and wild-type identified a locus associated with hypermutation that included znf3. CRISPR editing of the znf3 nonsense mutation abolished hypermutation and restored small-interfering-RNA production. We conclude that hypermutation and drug resistance in these clinical isolates result from RNA-interference loss and accumulation of Cnl1 elements.

由于有限的抗真菌治疗和耐药性，新型隐球菌感染导致大约 15% 的艾滋病相关死亡。通过波动分析，对一系列临床和环境新型隐球菌分离株进行了突变率增加的检测，我们鉴定了两种超突变的新型隐球菌临床分离株，当暴露于雷帕霉素和 FK506 的组合时，其突变率增加。药物靶基因测序发现，Cnl1 转座子插入赋予了对雷帕霉素和 FK506 的大部分耐药性，并且还可以独立地引起对 5-氟乳清酸和临床相关抗真菌药物 5-氟胞嘧啶的耐药性。全基因组测序显示，两个超变基因组的 RNA 干扰成分ZNF3和数百个 Cnl1 元件均含有无义突变，这些元件在 14 条染色体的每一条上组织成大量亚端粒阵列。对衍生自超变基因和野生型之间的杂交的 28 个后代进行数量性状基因座作图，鉴定出与包括znf3在内的超变相关的基因座。 znf3无义突变的 CRISPR 编辑消除了超突变并恢复了小干扰 RNA 的产生。我们得出的结论是，这些临床分离株的超突变和耐药性是由 RNA 干扰丢失和 Cnl1 元件积累造成的。