The limited availability of antifungal drugs
makes the treatment of fungal infections problematic. However, the development
of novel drugs is slow and very expensive. In recent years, increasing
attention has been given to the use of combination therapy to treat fungal
infection. The use of a secondary compound that exerts stress on the fungal
cell can increase the overall efficacy of treatment. Understanding the response
of the fungal cell to an antifungal drug is a necessary first step in selecting
possible targets for secondary compounds. To this end, we undertook a study of
the proteomic response of the basidiomycetous fungal pathogen Cryptococcus gattii to the antifungal fluconazole.
We identified a previously uncharacterised protein (designated HARP) that was
47.5-fold upregulated in a fluconazole tolerant strain (MIC = 64 ug/mL), 7.8
and 3.9 fold upregulated in two strains with intermediate fluconazole
sensitivity (32 ug/mL) and uninduced in a fluconazole sensitive strain (16
ug/mL) when the cells were treated with the drug. BLAST analysis of the HARP
protein sequence against the NCBI nr database yielded no significant
homologues. However, PFAM and DELTA-BLAST searches identified domain homology with
the Shwachman-Bodian-Diamond Syndrome domain. In humans and Saccharomyces cerevisae, proteins
containing this domain have a wide range of functions including ribosome maturation,
telomere capping and response to osmotic stress. Structural modelling of HARP,
and comparison to solved structures of known SBDS domain proteins, revealed
very similar tertiary structures. This analysis gives us confidence that HARP
may be a homologue of the S. cerevisae
protein RTC3 that has been shown to have a role in stress response. Deletion
studies are currently being conducted in C.
gattii strains. Analysis of the role of this protein may lead to insights
into more effective treatment of difficult or antifungal tolerant cryptococcal
strains.