Identification and characterization of a Cryptococcus neoformans ATP binding cassette (ABC) transporter-encoding gene, CnAFR1, involved in the resistance to fluconazole.
Resistance to fluconazole is a possible event during prolonged suppressive drug therapy for cryptococ-cal meningitis, the most frequently encountered life-threatening manifestation of cryptococcosis. The knowledge of this resistance at the molecular level is important for management of cryptococcosis. In order to identify genes involved in azole resistance in Cryptococcus neoformans, a cDNA subtraction library technique was chosen as a strategy. First, a fluconazole-resistant mutant BPY22.17 was obtained from a susceptible clinical isolate BPY22 by in vitro exposure to the drug. Then, a subtractive hybridization procedure was used to compare gene expression between the obtained strains. We identified a cDNA overexpressed in the fluconazole-resistant strain BPY22.17 that was used as a probe to isolate the entire gene in a C. neoformans genomic library. Sequence analysis of this gene identified an ATP Binding Cassette (ABC) transporter-encoding gene called C. neoformans AntiFungal Resistance 1 (CnAFR1). Disruption of CnAFR1 gene in the resistant isolate (BPY22.17) resulted in an enhanced susceptibility of the knock-out mutant cnafr1 against fluconazole, whereas reintroduction of the gene in cnafr1 resulted in restoration of the resistance phenotype, thus confirming that CnAFR1 is involved in fluconazole resistance of C. neoformans. Our findings therefore reveal that an active drug efflux mechanism can be involved in the development of azole resistance in this important human pathogen.
Posteraro
Brunella
B
Istituto Microbiologia, Università Cattolica del S. Cuore, L. go F. Vito, 1, 00168 Rome, Italy.
Sanguinetti
Maurizio
M
Sanglard
Dominique
D
La Sorda
Marilena
M
Boccia
Stefania
S
Romano
Lucio
L
Morace
Giulia
G
Fadda
Giovanni
G
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D003455:Cryptococcus neoformans / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development; D025141:Drug Resistance, Fungal / Q000235:genetics*; D015725:Fluconazole / Q000494:pharmacology*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015723:Gene Library; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods; D016415:Sequence Alignment
D018528:ATP-Binding Cassette Transporters; D000935:Antifungal Agents; D005656:Fungal Proteins; D015725:Fluconazole