Role of three Cryptococcus neoformans and C. gattii efflux pump coding genes in response to drug treatment [PublishAheadOfPrint]

Cryptococcus neoformans and C. 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 resistance levels 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 displayed 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 afr1 of both strains but Rhodamine 6G accumulation increased only in mdr1 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 were upregulated in H99 and R265 when treated with fluconazole. However, MDR1 expression was only upregulated in R265 under the same conditions. We screened a library of transcriptional 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.

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