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Project Information

Sequencing Cryptococcus neoformans Serotype A

The Cryptococcus neoformans Serotype A sequencing project reflects a collaboration between the Fungal Genome Initiative at the Broad Institute and Fred Dietrich at the Duke Center for Genome Technology. The goal for the FGI is to release a high-quality genome assembly with 10X coverage for C. neoformans serotype A, strain H99.

Our specific aims are to:

  1. Generate and assemble sequence reads yielding 10X coverage of the Cryptococcus neoformans genome through whole-genome shotgun sequencing.
  2. Generate and incorporate BAC and Fosmid end sequences into the genome assembly to provide a paired-end long-link information.
  3. Integrate the genomic sequence with existing fingerprint map information.
  4. Perform automated annotation of the sequence assembly.
  5. Distribute the sequence assembly and results of our annotation and analysis through a freely accessible, public web server at the Broad Institute and by deposition of the sequence assembly in GenBank.

The Center for Genome Technology at Duke University Medical Center contributed ~2X coverage in plasmid reads to this assembly. The Broad Institute of MIT and Harvard produced additional whole-genome shotgun sequence from 4 kb & 10 kb plasmids, 40 kb Fosmids and 110 kb BACs. The H99 BAC library was constructed by Klaus Lengeler and Joseph Heitman at Duke University, finger printed and end sequenced by Jim Kronstad and the Vancouver Genome Sequence Centre, and provided by these investigators to the Broad Institute. Genomic DNA for the H99 libraries was provided by James Fraser in the Heitman lab at Duke University. All the reads were assembled with Arachne, the Whole Genome Assembler.

The cDNA was provided by Dr. Doris Kupfer at University of Oklahoma.

What is Cryptococcus neoformans?

Significance

Cryptococcus neoformans is an encapsulated fungal pathogen causing fatal meningitis in humans. The infection, initiated by inhalation into the lungs, occurs mainly in immunocompromised individuals, but can also occur in healthy individuals. Cryptococcus neoformans is usually found in tissues in the yeast form. Infection of the brain and meninges is the most common clinical manifestation. In immunocompetent individuals, the initial infection is usually controlled and asymptomatic and the organism remains dormant in a lymph node complex, much like tuberculosis. Reactivation occurs in immunocompromised hosts where the fungus can spread via the blood to infect the central nervous system. Once C. neoformans reaches this stage it can cause meningitis that is uniformly fatal if untreated. Few antifungal agents exist and drug-resistant strains are emerging.

There are four serotypes of C. neoformans. The serotype D was the first serotype chosen for sequencing studies at The Institute for Genome Research and Stanford Genome Technology Center because of its advanced genetic tools. However, more than 90% of clinical isolates and more than 99% of isolates from AIDS patients are of the more divergent serotype A strains. Sequencing a serotype A strain will increase our understanding of this disease and comparative studies between the serotypes A and D will help us define the genome structure and the development of pathogenicity.

General Description

Cryptococcus neoformans is unique among the most common human fungal pathogens in that it is a basidiomycete, thus it is evolutionarily divergent from the more common pathogenic ascomycetes (e.g., Candida albicans) and more closely related to wood rotting fungi (e.g., Phanerochaete chrysosporium), mushrooms (e.g., Coprinus cinereus), and plant pathogens (e.g., Ustilago maydis). Cryptococcus neoformans elaborates two specialized virulence factors, the polysaccharide capsule, which inhibits phagocytosis, and melanin, which serves as an antioxidant. The typical vegetative form of C. neoformans is the yeast form. The organism can also undergo sexual reproduction and form basidiospores. Sexual reproduction appears to occur much less frequently in nature than asexual or vegetative reproduction.

Genome Facts

Most isolates of C. neoformans are haploid. The size of the genome is approximately 24 Mb with 12 chromosomes. Cryptococcus neoformans has a defined sexual cycle involving mating between cells of the MATalpha and MATa types. Thus, classical genetic approaches can be applied to study this organism.

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