Analysis of the Genome and Transcriptome of Cryptococcus neoformans var. grubii Reveals Complex RNA Expression and Microevolution Leading to Virulence Attenuation

Abstract

Cryptococcus neoformans is a pathogenic basidiomycetous yeast responsible for more than 600,000 deaths each year. It occurs as two serotypes (A and D) representing two varieties (i.e. grubii and neoformans, respectively). Here, we sequenced the genome and performed an RNA-Seq-based analysis of the C. neoformans var. grubii transcriptome structure. We determined the chromosomal locations, analyzed the sequence/structural features of the centromeres, and identified origins of replication. The genome was annotated based on automated and manual curation. More than 40,000 introns populating more than 99% of the expressed genes were identified. Although most of these introns are located in the coding DNA sequences (CDS), over 2,000 introns in the untranslated regions (UTRs) were also identified. Poly(A)-containing reads were employed to locate the polyadenylation sites of more than 80% of the genes. Examination of the sequences around these sites revealed a new poly(A)-site-associated motif (AUGHAH). In addition, 1,197 miscRNAs were identified. These miscRNAs can be spliced and/or polyadenylated, but do not appear to have obvious coding capacities. Finally, this genome sequence enabled a comparative analysis of strain H99 variants obtained after laboratory passage. The spectrum of mutations identified provides insights into the genetics underlying the micro-evolution of a laboratory strain, and identifies mutations involved in stress responses, mating efficiency, and virulence.

Author Summary

Cryptococcus neoformans var. grubii is a major human pathogen responsible for deadly meningoencephalitis in immunocompromised patients. Here, we report the sequencing and annotation of its genome. Evidence for extensive intron splicing, antisense transcription, non-coding RNAs, and alternative polyadenylation indicates the potential for highly intricate regulation of gene expression in this opportunistic pathogen. In addition, detailed molecular, genetic, and genomic studies were performed to characterize structural features of the genome, including centromeres and origins of replication. Finally, the phenotypic and genome re-sequencing analysis of a collection of isolates of the reference H99 strain resulting from laboratory passage revealed that microevolutionary processes during in vitro culturing of pathogenic fungi can impact virulence.