title: Fluconazole Susceptibility in Cryptococcus gattii Is Dependent on the ABC Transporter Pdr11.
abstract: Cryptococcus gattii isolates from the Pacific Northwest have exhibited higher fluconazole MICs than isolates from other sites. The mechanism of fluconazole resistance in C. gattii is unknown. We sought to determine the role of the efflux pumps Mdr1 and Pdr11 in fluconazole susceptibility. Using biolistic transformation of the parent isolate, we created a strain lacking Mdr1 (mdr1Δ) and another strain lacking Pdr11 (pdr11Δ). Phenotypic virulence factors were assessed by standard methods (capsule size, melanin production, growth at 30 and 37 °C). Survival was assessed in an intranasal murine model of cryptococcosis. Antifungal MICs were determined by the M27-A3 methodology. No differences in key virulence phenotypic components were identified. Fluconazole susceptibility was unchanged in the Mdr1 knockout or reconstituted isolates. However, fluconazole MICs decreased from 32 μg/ml for the wild-type isolate to <0.03 μg/ml for the pdr11Δ strain and reverted to 32 μg/ml for the reconstituted strain. In murine models, no difference in virulence was observed between wild-type, knockout, or reconstituted isolates. We conclude that Pdr11 plays an essential role in fluconazole susceptibility in C. gattii. Genomic and expression differences between resistant and susceptible C. gattii clinical isolates should be assessed further in order to identify other potential mechanisms of resistance.
authors.affiliation: Department of Medical Microbiology and Immunology, Coccidioidomycosis Serology Laboratory, University of California-Davis, Davis, California, USA.
authors.forename: Mai Lee
authors.initials: ML
authors.lastname: Yang
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, Genome and Biomedical Sciences Facility, Davis, California, USA.
authors.forename: John
authors.initials: J
authors.lastname: Uhrig
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, Genome and Biomedical Sciences Facility, Davis, California, USA.
authors.forename: Kiem
authors.initials: K
authors.lastname: Vu
authors.affiliation: Department of Medical Microbiology and Immunology, Coccidioidomycosis Serology Laboratory, University of California-Davis, Davis, California, USA.
authors.forename: Anil
authors.initials: A
authors.lastname: Singapuri
authors.affiliation: Department of Medical Microbiology and Immunology, Coccidioidomycosis Serology Laboratory, University of California-Davis, Davis, California, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Dennis
authors.affiliation: Department of Pharmacology, School of Medicine, University of California, Genome and Biomedical Sciences Facility, Davis, California, USA acgelli@ucdavis.edu grthompson@ucdavis.edu.
authors.forename: Angie
authors.identifier: http://orcid.org/0000-0003-3351-0683
authors.initials: A
authors.lastname: Gelli
authors.affiliation: Department of Medical Microbiology and Immunology, Coccidioidomycosis Serology Laboratory, University of California-Davis, Davis, California, USA Department of Internal Medicine, Division of Infectious Diseases, University of California-Davis Medical Center, University of California-Davis, Davis, California, USA acgelli@ucdavis.edu grthompson@ucdavis.edu.
authors.forename: George R
authors.initials: GR
authors.lastname: Thompson
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism; D000368:Aged; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D003453:Cryptococcosis / Q000188:drug therapy / Q000382:microbiology; D056285:Cryptococcus gattii / Q000187:drug effects* / Q000235:genetics / Q000472:pathogenicity; D025141:Drug Resistance, Fungal / Q000187:drug effects / Q000235:genetics*; D005260:Female; D015725:Fluconazole / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D008815:Mice, Inbred Strains; D008826:Microbial Sensitivity Tests
chemical_list: D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D005656:Fungal Proteins; C109678:MDR1 protein, Filobasidiella neoformans; D015725:Fluconazole