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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.