Identification and properties of plasma membrane azole efflux pumps from the pathogenic fungi Cryptococcus gattii and Cryptococcus neoformans.

OBJECTIVES: Cryptococcus gattii from the North American Northwest (NW) have higher azole MICs than do non-NW C. gattii or Cryptococcus neoformans. Since mechanisms of azole resistance in C. gattii are not known, we identified C. gattii and C. neoformans plasma membrane azole efflux pumps and characterized their properties. METHODS: The C. gattii R265 genome was searched for orthologues of known fungal azole efflux genes, expression of candidate genes was assessed by RT-PCR and the expressed genes' cDNAs were cloned and expressed in Saccharomyces cerevisiae. Azole MICs and intracellular [(3)H]fluconazole were measured in C. gattii and C. neoformans and in S. cerevisiae expressing each cDNA of interest, as was [(3)H]fluconazole uptake by post-Golgi vesicles (PGVs) isolated from S. cerevisiae sec6-4 mutants expressing each cDNA of interest. RESULTS: Intracellular [(3)H]fluconazole concentrations were inversely correlated with fluconazole MICs only in 25 NW C. gattii strains. S. cerevisiae expressing three C. gattii cDNAs (encoded by orthologues of C. neoformans AFR1 and MDR1 and the previously unstudied gene AFR2) and their C. neoformans counterparts had higher azole MICs and lower intracellular [(3)H]fluconazole concentrations than did empty-vector controls. PGVs from S. cerevisiae expressing all six Cryptococcus cDNAs also accumulated more [(3)H]fluconazole than did controls, and [(3)H]fluconazole transport by all six transporters of interest was ATP dependent and was inhibited by excess unlabelled fluconazole, voriconazole, itraconazole and posaconazole. CONCLUSIONS: We conclude that C. gattii and C. neoformans AFR1, MDR1 and AFR2 encode ABC transporters that pump multiple azoles out of S. cerevisiae cells, thereby causing azole resistance.

Azole resistance in Cryptococcus gattii from the Pacific Northwest: Investigation of the role of ERG11.

Cryptococcus gattii is responsible for an expanding epidemic of serious infections in Western Canada and the Northwestern United States (Pacific Northwest). Some patients with these infections respond poorly to azole antifungals, and high azole MICs have been reported in Pacific Northwest C. gattii. In this study, multiple azoles (but not amphotericin B) had higher MICs for 25 Pacific Northwest C. gattii than for 34 non-Pacific Northwest C. gattii or 20 Cryptococcus neoformans strains. We therefore examined the roles in azole resistance of overexpression of or mutations in the gene (ERG11) encoding the azole target enzyme. ERG11/ACT1 mRNA ratios were higher in C. gattii than in C. neoformans, but these ratios did not differ in Pacific Northwest and non-Pacific Northwest C. gattii strains, nor did they correlate with fluconazole MICs within any group. Three Pacific Northwest C. gattii strains with low azole MICs and 2 with high azole MICs had deduced Erg11p sequences that differed at one or more positions from that of the fully sequenced Pacific Northwest C. gattii strain R265. However, the azole MICs for conditional Saccharomyces cerevisiae erg11 mutants expressing the 5 variant ERG11s were within 2-fold of the azole MICs for S. cerevisiae expressing the ERG11 gene from C. gattii R265, non-Pacific Northwest C. gattii strain WM276, or C. neoformans strains H99 or JEC21. We conclude that neither ERG11 overexpression nor variations in ERG11 coding sequences was responsible for the high azole MICs observed for the Pacific Northwest C. gattii strains we studied.

Transformation of Candida albicans with a synthetic hygromycin B resistance gene.

Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2-containing plasmids or single-copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT-1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild-type C. albicans. We used PCR to fuse CaHygB or SAT-1 to approximately 1 kb of 5' and 3' noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild-type C. albicans strains. Homologous targeting frequencies were approximately 50-70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates.

Fluconazole transport into Candida albicans secretory vesicles by the membrane proteins Cdr1p, Cdr2p, and Mdr1p.

A major cause of azole resistance in Candida albicans is overexpression of CDR1, CDR2, and/or MDR1, which encode plasma membrane efflux pumps. To analyze the catalytic properties of these pumps, we used ACT1- and GAL1-regulated expression plasmids to overexpress CDR1, CDR2, or MDR1 in a C. albicans cdr1 cdr2 mdr1-null mutant. When the genes of interest were expressed, the resulting transformants were more resistant to multiple azole antifungals, and accumulated less [(3)H]fluconazole intracellularly, than empty-vector controls. Next, we used a GAL1-regulated dominant negative sec4 allele to cause cytoplasmic accumulation of post-Golgi secretory vesicles (PGVs), and we found that PGVs isolated from CDR1-, CDR2-, or MDR1-overexpressing cells accumulated much more [(3)H]fluconazole than did PGVs from empty-vector controls. The K(m)s (expressed in micromolar concentrations) and V(max)s (expressed in picomoles per milligram of protein per minute), respectively, for [(3)H]fluconazole transport were 0.8 and 0.91 for Cdr1p, 4.3 and 0.52 for Cdr2p, and 3.5 and 0.59 for Mdr1p. [(3)H]fluconazole transport by Cdr1p and Cdr2p required ATP and was unaffected by carbonyl cyanide 3-chlorophenylhydrazone (CCCP), whereas [(3)H]fluconazole transport by Mdr1p did not require ATP and was inhibited by CCCP. [(3)H]fluconazole uptake by all 3 pumps was inhibited by all other azoles tested, with 50% inhibitory concentrations (IC(50)s; expressed as proportions of the [(3)H]fluconazole concentration) of 0.2 to 5.6 for Cdr1p, 0.3 to 3.1 for Cdr2p, and 0.3 to 3.1 for Mdr1p. The methods used in this study may also be useful for studying other plasma membrane transporters in C. albicans and other medically important fungi.