| RESEARCH SUMMARY:
AIDS has contributed to a significant increase in the population
of immunocompromised patients who are exquisitely vulnerable
to fungal infections. In particular, Cryptococcus neoformans
is an opportunistic fungal pathogen that causes meningitis and
is a leading cause of death in immunocompromised patients. Our
laboratory is interested in understanding fungal pathogenesis
and identifying novel virulence genes in C. neoformans. The
genome of C. neoformans has been sequenced, and we are using
post-genomic approaches to understanding the molecular basis
for pathogenesis and for discovery of new antifungal targets.
We are using proteomics to identify proteins that are regulated
during pathogenesis with an emphasis on oxidative and nitrosative
stresses. Proteins that are upregulated during pathogenesis
are selected for gene-specific knockouts. Using this approach,
we have shown that thiol-peroxidases have increased expression
at 37°C vs. 25°C and on exposure to peroxidases. Gene
deletion of one of the C. neoformans thiol peroxidase causes
a peroxide-sensitive, NO-sensitive, temperature-sensitive and
avirulent phenotype. We are currently other proteins from the
thioredoxin and glutathione pathways for their contribution
to protection from oxidative and nitrosative stresses and virulence.
In addition, our laboratory has initiated a systematic gene
deletion project for C. neoformans. We have begun deleting genes
involved in cell wall structure or biosynthesis and have developed
in vitro screens for cell wall integrity. Each gene deletion
is “tagged” with a unique DNA sequence that allows
us to follow the abundance of the mutant in a pool of mutants.
Using these tags we can test 48 mutants in a single mouse for
their ability to proliferate. We have identified several genes
that are clearly important for cell wall integrity and virulence.
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