Identification and properties of plasma membrane azole efflux pumps from the pathogenic fungi Cryptococcus gattii and Cryptococcus neoformans Luiz R. Basso Jr 1,2 , Charles E. Gast 1 , Igor Bruzual 1 and Brian Wong 1 * 1 Infectious Diseases Division, Department of Medicine, Oregon Health & Science University, Portland, OR, USA; 2 Department of Cellular and Molecular Biology and Pathogenic Bioagents, Faculty of Medicine of Ribeira ˜o Preto, University of Sa ˜o Paulo, Ribeira ˜o Preto, SP, Brazil *Corresponding author. Tel: +1-503-494-7735; Fax: +1-503-494-4264; E-mail: wongbri@ohsu.edu Received 14 January 2014; returned 24 March 2014; revised 1 December 2014; accepted 11 December 2014 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. Keywords: C. gattii, C. neoformans, azole resistance Introduction The environmental fungus Cryptococcus gattii causes pneumo- nias and CNS infections in both healthy and immunocomprom- ised people. 1 C. gattii was formerly believed to cause serious infections almost exclusively in tropical or subtropical regions of Africa, Asia and Australia, 2 but an outbreak of serious C. gattii infections on Vancouver Island, British Columbia was reported in 2002. 3 – 5 Since the occurrence of these outbreak cases, severe C. gattii infections have been frequently reported in western Canada and the northwestern USA. 1 C. gattii infections in the Pacific Northwest (NW) differ in several important respects from C. gattii infections described in other geographic regions. For example, among people with proven C. gattii infections, 41 of 124 from Canada 6 and 14 of 52 from Washington and Oregon 1 had serious underlying diseases, compared with 0 of 20 people from Australia. 7 Similarly, mortality in people with proven C. gattii infections was 15 of 124 in Canada 6 and 15 of 52 in Washington or Oregon, 1 compared with 0 of 20 in people in Australia. 7 Slow or incomplete response to antifungal therapy has been reported in some patients infected with C. gattii worldwide 7, 8 and in C. gattii patients from the NW treated with azoles. 1 There are likely multiple reasons for these poor responses to azole ther- apy, but the observations that MICs of several azole antifungals are higher in C. gattii than in Cryptococcus neoformans 1,9,10 and also are higher in C. gattii strains with genotypes prevalent in the NW than in C. gattii with other genotypes 10 – 12 suggest that resistance to azole antifungals may play an important role. The principal mechanisms by which fungi acquire resistance to azole antifungals are overexpression of the gene (ERG11) encod- ing the azole target enzyme lanosterol 14-a demethylase (Erg11p), 13 – 16 mutations in ERG11 that decrease the susceptibility # The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com J Antimicrob Chemother doi:10.1093/jac/dku554 1 of 12 Journal of Antimicrobial Chemotherapy Advance Access published January 27, 2015 at Universidade de S�o Paulo on February 4, 2015 http://jac.oxfordjournals.org/ Downloaded from