title: Identification and properties of plasma membrane azole efflux pumps from the pathogenic fungi Cryptococcus gattii and Cryptococcus neoformans.
abstract: OBJECTIVE
  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.
authors: Infectious Diseases Division, Department of Medicine, Oregon Health & Science University, Portland, OR, USA Department of Cellular and Molecular Biology and Pathogenic Bioagents, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil.
authors: Luiz R
authors: LR
authors: Basso
authors: Infectious Diseases Division, Department of Medicine, Oregon Health & Science University, Portland, OR, USA.
authors: Charles E
authors: CE
authors: Gast
authors: Infectious Diseases Division, Department of Medicine, Oregon Health & Science University, Portland, OR, USA.
authors: Igor
authors: I
authors: Bruzual
authors: Infectious Diseases Division, Department of Medicine, Oregon Health & Science University, Portland, OR, USA wongbri@ohsu.edu.
authors: Brian
authors: B
authors: Wong
mesh_terms: D000935:Antifungal Agents / Q000378:metabolism*; D001393:Azoles / Q000378:metabolism*; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D003453:Cryptococcosis / Q000382:microbiology; D056285:Cryptococcus gattii / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism; D003455:Cryptococcus neoformans / Q000187:drug effects / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D006801:Humans; D007553:Isotope Labeling; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D015205:Northwestern United States; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D000935:Antifungal Agents; D001393:Azoles; D026901:Membrane Transport Proteins
keywords: C. gattii; C. neoformans; azole resistance
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