Roles of Three Cryptococcus neoformans and Cryptococcus gattii Efflux Pump-Coding Genes in Response to Drug Treatment.

Cryptococcus neoformans and Cryptococcus gattii species complexes are the etiologic agents of cryptococcosis. We have deciphered the roles of three ABC transporters, Afr1, Afr2, and Mdr1, in the representative strains of the two species, C. neoformans H99 and C. gattii R265. Deletion of AFR1 in H99 and R265 drastically reduced the levels of resistance to three xenobiotics and three triazoles, suggesting that Afr1 is the major drug efflux pump in both strains. Fluconazole susceptibility was not affected when AFR2 or MDR1 was deleted in both strains. However, when these genes were deleted in combination with AFR1, a minor additive effect in susceptibility toward several drugs was observed. Deletion of all three genes in both strains caused further increases in susceptibility toward fluconazole and itraconazole, suggesting that Afr2 and Mdr1 augment Afr1 function in pumping these triazoles. Intracellular accumulation of Nile Red significantly increased in afr1Δ mutants of both strains, but rhodamine 6G accumulation increased only in the mdr1Δ mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. AFR1 and AFR2 expression was upregulated in H99 and R265 when treated with fluconazole. However, MDR1 expression was upregulated only in R265 under the same conditions. We screened a library of transcription factor mutants and identified several mutants that manifested either altered fluconazole sensitivity or an increase in the frequency of fluconazole heteroresistance. Gene expression analysis suggests that the three efflux pumps are regulated independently by different transcription factors in response to fluconazole exposure.

Identification of high-affinity copper transporters in Aspergillus fumigatus.

We investigated the copper metabolism of Aspergillus fumigatus, which has not been characterized well. We cloned the putative copper transporters ctrA2 and ctrC from A. fumigatus and investigated the functions of these transporters in copper metabolism. Four putative copper transporters were identified in the A. fumigatus genome; ctrA2 and ctrC complemented CTR1 functionally and localized to the plasma membrane in Saccharomyces cerevisiae. ctrA2 and ctrC single-deletion mutants and a double-deletion mutant of ctrA2 and ctrC were constructed in A. fumigatus. The ctrA2 and ctrC double-deletion mutant exhibited a growth defect on Aspergillus minimal medium (AMM) supplemented with bathocuproine disulfonic acid (BCS) and was sensitive to H2O2. Furthermore, the deletion of ctrA2 and ctrC reduced superoxide dismutase (SOD) activity, laccase activity, and intracellular copper contents. The activities of the ctrA2 and ctrC genes were up-regulated by BCS treatment. In addition, the deletion of ctrA2 up-regulated ctrC and vice versa. ctrA2 and ctrC were localized to the A. fumigatus plasma membrane. Although ctrA2 and ctrC failed to affect the mouse survival rate, these genes affected conidial killing activity. Taken together, these results indicate that ctrA2 and ctrC may function as membrane transporters and that the involvement of these genes in pathogenicity merits further investigation.