Candidiasis is the most common cause of deep fungal infections. Oropharyngeal candidiasis occurs in nearly all patients infected with the human immunodeficiency-1 virus. Treatment of candidiasis with azoles has provided good efficacy, low toxicity and ease of oral administration. Increasing resistance of Candida species to all azoles has complicated management and prompted a search for newer agents. Our work has focused on defining the molecular mechanisms by which azole resistance is occurring, evaluating different strategies for prevention of candidiasis and trying new drugs for patients failing azole therapy. We have been working with a series of C. glabrata isolates that showed increasing azole resistance during therapy of an HIV-1 infected patient. We found that the more resistant isolates had upregulation of a previously undescribed gene (PDH1), which we cloned and sequenced. The gene is an ATP-binding cassette (ABC) transporter based on homology and gene interruption experiments. Although the gene is most homologous to the Saccharomyces cerevisiae pleiotropic drug resistance gene, PDR5, gene interruption produces a phenotype more consistent with YOR1: increased susceptibility to cadmium and oligomycin, together with increased intracellular concentrations of rhodamine 123. Unlike pdr5 mutants, the pdh1 mutants do not change susceptibility to cycloheximide, fluconazole and fluphenazine. The arrangement of YOR1 differs from PDH1 in that the transmembrane portion preceeds, rather than follows,the nucleotide binding fold, although both genes exist as homologous halves and produce the same size transcript. Based on YOR1, we expect that Pdh1p causes efflux of toxic compounds, including Cadmium-S-glutathione compounds, at the Candida vacuolar and cytoplasmic membranes. PDH1 has two upstream nucleotide sequences identical to PDR1-3 response elements of PDR5. We predict that this nucleotide sequence binds transcriptional regulatory proteins, similar to the zinc-cluster regulatory proteins, Pdr1-3p. Based on homology to YOR1, we predict that regulatory factors such as yAP-1 and CAD1 will also influence transcription. The fact that fluconazole increases PDH1 transcript and that the pdh1 phenotype is not expressed in all strains is likely a result of an extensive cross-regulatory pathway of efflux pumps such as occurs in S. cerevisiae. We are finishing our studies of the azole resistant Darlington strain of Candida albicans. We found a Y132H change in deduced amino acid sequence in ERG11. This gene codes for the azole target enzyme, sterol 14-demethylase. This mutation was associated with increased azole resistance in a C. albicans strain studied by Dominque Sanglard. We overexpressed the Darlington ERG11 in S. cerevisiae using the GAL1 promoter and found that all five transformants studied were extremely azole resistant (MIC<256 mcg/ml) whereas the 5 S. cerevisiae transformants receiving the wild type ERG11 were susceptible (MIC 1.5-4.0). Integrative transformation of Darlington ERG11 into the wild type C. albicans is being done to confirm the result in a homologous host. However, changes in Darlington?s ERG11 now appear to explain the marked azole resistance in that strain. We have finished our work on the Darlington ERG3, which codes for a sterol C5,6 reductase, and submitted the work for publication. Since the last annual report we have expressed the Dar-1 ERG3 homologue in a S. cerevisiae erg3 mutant, using a multicopy plasmid and uracil selection. Darlington, which we have shown is an erg3 mutant, has two different ERG3 homologs. Dar-1 has a stop codon in the open reading frame. Dar-1 has changes in two deduced amino acids in areas that are conserved but outside the the three copper binding sites. The Dar-1 homolog transformants produced no ergosterol whereas transformants receiving the wild type ERG3 in the same construct produced 38.6-64.5% ergosterol. This supports our conclusion that two changes in conserved amino acids are responsible for lack of function in the gene product. Studies on the laccase of Cryptococcus neoformans, important for virulence of that fungus, have continued by producing rabbit antibody against recombinant laccase and oligopeptides from the amino-terminus. Affinity purified antibody is being sent to a collaborator for intracellular localization by immunoelectron microscopy.
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