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

Abstract

<i>Cryptococcus neoformans</i> and <i>Cryptococcus gattii</i> 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, <i>C. neoformans</i> H99 and <i>C. gattii</i> R265. Deletion of <i>AFR1</i> 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 <i>AFR2</i> or <i>MDR1</i> was deleted in both strains. However, when these genes were deleted in combination with <i>AFR1</i>, 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 <i>afr1Δ</i> mutants of both strains, but rhodamine 6G accumulation increased only in the <i>mdr1Δ</i> mutant of H99. Thus, the three efflux pumps play different roles in the two strains when exposed to different azoles and xenobiotics. <i>AFR1</i> and <i>AFR2</i> expression was upregulated in H99 and R265 when treated with fluconazole. However, <i>MDR1</i> 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.