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.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

Summary Microsporidia are obligate, intracellular eukaryotic pathogens that infect animal cells, including humans [1]. Previous studies suggested the microsporidia might share a common ancestor with fungi [2–7]. However, the exact nature of this phylogenetic relationship is unclear due to the unusual features of microsporidial genomes, which are compact with a reduced number of highly divergent genes [8]. As a consequence, it is unclear whether microsporidia evolved from a specific fungal lineage, the identity of that lineage, or whether microsporidia are a sister group to all fungi. Here we present evidence to address this controversial question that is independent of sequence-based phylogenetic reconstruction, but rather based on genome structure. In the zygomycete basal fungal lineage, the sex locus is a syntenic gene cluster that governs sexual reproduction in which a high mobility group (HMG) transcription factor gene is flanked by a triose phosphate transporter (TPT) and an RNA helicase gene [9]. Strikingly, microsporidian genomes harbor a sex-related locus with the same genes in the same order (TPT, HMG, RNA helicase). Genome-wide analysis of synteny reveals multiple other loci where microsporidia and zygomycetes are conserved to the exclusion of all other fungal lineages with sequenced genomes. These findings support the hypothesis that microsporidia are true fungi that descended from a zygomycete ancestor, and suggest the microsporidia may have a genetically controlled sexual cycle.