Scientists Decipher Genome of a Fungus That Causes Life-Threatening Infections in Persons With Impaired Immunity

Cryptococcus Study Sheds Light On How Fungi Cause Disease

January 13, 2005

Rockville, MD — In a project that already has benefited an important field of biomedical research, scientists have deciphered the genomes of two closely related strains of Cryptococcus neoformans, a fungus whose importance as a human pathogen has risen in parallel with the HIV/AIDS worldwide epidemic and the increased use of immunosuppressive therapies.

The study, posted online January 13 in Science Express, revealed differences in the virulence strategies used by C. neoformans compared to other pathogenic fungi. Researchers also examined the genetic determinants of its pathogenicity by comparing the genomes of two closely related strains of significantly different virulence.

"Not only have we established a genomic platform for the further study of this increasingly important pathogen, but the data from the two strains may provide insight into what determines virulence," says Brendan Loftus, a scientist at The Institute for Genomic Research (TIGR) who is the first author of the Science paper. "Although the two Cryptococcus strains we examined differ significantly in virulence, we found surprisingly little difference in their gene content."

TIGR scientists led by primary investigator Claire M. Fraser, the President of TIGR, deciphered the genome of one strain of C. neoformans while researchers led by Richard Hyman at Stanford University's Genome Technology Center in Palo Alto, CA, sequenced the second strain. The project was funded by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health. Numerous collaborators helped to interpret and analyze the genome sequence data.

C. neoformans, an oval-shaped yeast, is an opportunistic human pathogen of global importance that is used by researchers as a model for fungal pathogenesis. Since the 1980s, the number of Cryptococcus infections has increased sharply — mainly among people with impaired immunity, including those who have HIV/AIDS or who receive cancer chemotherapy, steroid treatments, or therapy to prevent rejection of transplanted organs. One study indicated that as many as 13 percent of AIDS patients suffer a life-threatening cryptococcal infection at some point during the course of their HIV disease. The disease caused by the fungus, cryptococcosis, sometimes involves a fatal brain inflammation.

"In developing countries, cryptococcosis has emerged as one of the most common opportunistic infections, and a leading cause of meningitis and bloodstream infection," says Joseph Heitman, a senior community collaborator on the project who is James B. Duke Professor of Molecular Genetics and Microbiology at the Duke University Medical Center and an investigator at the Howard Hughes Medical Institute. "In Africa, where both HIV and concomitant cryptococcal infection are common, survival without therapy is as short as 7 to 10 days following diagnosis."

The major virulence factor of C. neoformans is its extensive polysaccharide capsule, an elaborate and dynamic structure surrounding the cell wall that is unique among fungi that affect humans. The Science study identified greater than 30 new genes likely involved in capsule biosynthesis, including a family containing 7 members of the capsule associated (CAP64) gene.

Researchers compared two closely related genomes of C. neoformans that differ markedly in their virulence properties. The results indicate that the differences likely involve other factors than the absence or presence of individual genes in the two isolates. A combination of other factors — perhaps including the cumulative impact of small (single-nucleotide) DNA differences and differences in when the genes are expressed (turned on or off) — may account for the disparity in virulence.

Among the surprising findings of the study were the complex gene structures discovered in C. neoformans that are unlike those found in previously sequenced yeasts and are reminiscent of the genomes of more complex organisms. The study also catalogued, for the first time on a genome-wide scale in fungi, examples of "alternative splicing" and "antisense" transcripts, in which the gene on one strand of the DNA differs from the counterpart gene on the opposite strand. Researchers also identified a high percentage of transposons (moveable elements) within the genome, which may account for the genome rearrangements often observed between different Cryptococcus isolates.

TIGR's Fraser says the Cryptococcus genome sequence will benefit a wide range of biomedical research into the increasingly significant human pathogen. Stanford's Hyman says, "The two new Cryoptococcus genome sequences and their analysis provide many targets for vaccine development and drug discovery."

Scientists in the C. neoformans research community — including Heitman at Duke and Jennifer Lodge at Saint Louis University (SLU) School of Medicine — say the project already has given impetus to biomedical research into fungal infections.

"The genome project has already had a major impact on basic research of Cryptococcus and will continue to be an enormous factor in the pace of discovery and in the scope of questions that can be asked about the basic biology of Cryptococcus as well as the disease process," says Lodge, who is SLU's Associate Dean for Research and who helped coordinate the C. neoformans genome project.

Heitman says, "The genomic sequence and transcriptome analysis are already proving to be invaluable to researchers working to understand and cure this infectious disease." He says the genome sequence has enabled more rapid gene identification, fueled genome wide insertional mutagenesis approaches, and provided the foundation for the development and implementation of a genetic map for quantitative genetics and population analysis. In addition, the accuracy of the gene annotation makes it feasible to use Cryptococcus as a model to study how fungi cause disease. Using the genomic sequence data, the Cryptococcus research community already has designed a microarray and has formed a consortium to further investigate the function of C. neoformans genes.

The sequence data also provides the foundation for a broader genome project that aims to define the molecular basis for the C. neoformans species cluster, which includes three related yet diverged species which differ in virulence attributes, environmental distribution and which have been diverging over millions of years of evolution.

This species cluster includes the serotype D strains analyzed in the Science study, the serotype A variety that is the predominant pathogenic form of the organism worldwide, and the serotype B Cryptococcus gattii species that is a primary pathogen and is currently causing an outbreak on Vancouver Island in British Columbia. Comparison of the genomes of the three divergent species promises to reveal in detail the molecular determinants of virulence.