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.

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans

“…Moreover, the nucleus also seemed to be visibly enlarged and dispersed in some cells (marked by arrows), hinting at nuclear fragmentation. To validate further the intracellular localization of the peptide, we prepared spheroplasts of C. neoformans by removing the cell wall using enzymatic digestion (45). When this spheroplast preparation was treated with 1 mM concentration of the FITC-peptide, the peptide showed a similar localization pattern as seen in intact cells 8 h posttreatment (Fig.…”

GMP Synthase Is Required for Virulence Factor Production and Infection by Cryptococcus neoformans

“…A single hit was observed, indicating that the gene is present in single copy. Located on chromosome 11, this locus was designated as CNAG_018770 in the published H99 genome (47); subsequently employing CNAG_018770 as the query sequence in a BLAST search of the S. cerevisiae genome identified GUA1 as the only hit in that species. The gene CNAG_018770 has therefore been named GUA1 after the S. cerevisiae ortholog, whose predicted product is 65% identical at the amino acid level.…”

“…Bioinformatic Analyses-The C. neoformans type strain H99 genome sequence was reported by Janbon et al (47). The GMP synthase-encoding gene was identified in the C. neoformans genome via reciprocal best hit BLAST analysis querying with the S. cerevisiae Gua1 protein.…”

Cryptococcosis: a model for the understanding of infectious diseases

“…We have identified and linked many virulence phenotypes of the yeast, such as high-temperature growth, melanin production, and capsule formation, to their controlling genes, and we continue to delve into the molecular details of how Cryptococcus produces disease in the genome-sequencing era. Whole-genome studies are beginning to be used to identify microevolutionary events that determine strain-specific pathogenicity (10,11) and to understand the plasticity of the Cryptococcus genome under stress (12). Furthermore, we can even capture how the yeast molecularly responds to the human CNS (13).…”