Rampant transposition following RNAi loss causes hypermutation and antifungal drug resistance in clinical isolates of a human fungal pathogen

Abstract

Microorganisms survive and compete within their environmental niches and avoid evolutionary stagnation by stochastically acquiring mutations that enhance fitness. Although increased mutation rates are often deleterious in multicellular organisms, hypermutation can be beneficial for microbes in the context of strong selective pressures. To explore how hypermutation arises in nature and elucidate its consequences, we employed a collection of 387 sequenced clinical and environmental isolates of Cryptococcus neoformans. This fungal pathogen is responsible for ~15% of annual AIDS-related deaths and is associated with high mortality rates, attributable to a dearth of antifungal drugs and increasing drug resistance. Isolates were screened for the ability to rapidly acquire antifungal drug resistance, and two robust hypermutators were identified. Insertion of the non-LTR Cnl1 retrotransposon was found to be responsible for the majority of drug-resistant isolates. Long-read whole-genome sequencing revealed both hypermutator genomes have two unique features: 1) hundreds of Cnl1 copies organized in subtelomeric arrays on both ends of almost all chromosomes, and 2) a nonsense mutation in the first exon of ZNF3, a gene encoding an RNAi component involved in silencing transposons. Quantitative trait locus mapping identified a significant genetic locus associated with hypermutation that includes the mutant znf3 allele, and CRISPR-mediated genome editing of the znf3 single-base pair nonsense mutation abolished the hypermutation phenotype and restored siRNA production. In sum, hypermutation and drug resistance in these isolates results from loss of RNAi combined with subsequent accumulation of a large genomic burden of a novel transposable element in C. neoformans.

Genome-wide analysis of heat stress-stimulated transposon mobility in the human fungal pathogenCryptococcus deneoformans

“…However, as the number of genomic Cnl1 copies increases, the likelihood that the element could insert into genes or in proximity to genes also increases. This was demonstrated in a recent study investigating two clinical hypermutator strains of C. neoformans (33). Due to a nonsense mutation in ZNF3 that inactivated RNAi suppression of Cnl1, the copy number in the telomeres of most chromosomes of these strains had reached ~100-150 full-length copies, with numerous additional truncated copies.…”

“…In XL280α, as in other C. deneoformans and C. neoformans genomes, the majority of retrotransposon sequences (Tcn1-6) are located in the centromeric regions of each chromosome (29), with the notable exception of the Cnl1 non-LTR retrotransposon, which is located in the subtelomeric regions (33,41). Transposons in the centromeres are thought to be inactive due to silencing of repetitive elements by RNAi, heterochromatin, and DNA methylation (29,34,42).…”

“…Active transposition during mitotic growth has been reported in several C. deneoformans strains and most recently in C. neoformans (30-33). Two clinical strains of C. neoformans from South Africa were identified as hypermutators due to loss of RNAi function, which enabled mobilization of the Cnl1 ( C. neoformans LINE-1) retrotransposon (33).…”

“…Transposons make up ~5 to 6% of the Cryptococcus genome and the majority are retrotransposon sequences located in the centromeres of each chromosome (27,28) Active transposition during mitotic growth has been reported in several C. deneoformans strains and most recently in C. neoformans (30)(31)(32)(33). Two clinical strains of C. neoformans from South Africa were identified as hypermutators due to loss of RNAi function, which enabled mobilization of the Cnl1 (C. neoformans LINE-1) retrotransposon (33). In addition to a massive accumulation of Cnl1 copies in the sub-telomeric regions of chromosomes, Cnl1 inserted into the FRR1 reporter gene (encoding FKBP1), resulting in rapamycin+FK506 drug resistance.…”

Genomic diversity across Candida auris clinical isolates shapes rapid development of antifungal resistance in vitro and in vivo

“…The K-P isolate described here is the first known example of a mutator phenotype in C. auris (Figure 5). Elevated mutation rates have been observed in other fungal pathogens including Cryptococcus species mutator alleles in DNA repair pathway genes and activation of transposase activity enhance within-host adaptation and evolution of antifungal resistance [64][65][66][67].…”