Multidrug efflux pumps and innate azole resistance of Mucor lusitanicus.

Objectives: This study aims to characterize the possible contribution of eight pleiotropic drug resistance (PDR) transporters to the azole resistance phenotype of Mucor lusitanicus.

Methods: Gene expression analysis (RNA-sequencing and RT-qPCR) was performed on M. lusitanicus CBS277.49 cells exposed to three different types of azoles (4.0 mg/L). C-terminally GFP-tagged M. lusitanicus PDR transporters were overexpressed in the hypersensitive model host, Saccharomyces cerevisiae ADΔΔ. Their efflux pump functions were evaluated by determining the azole susceptibilities of the PDR transporter overexpressing cells and measuring their plasma membrane ATPase activities.

Results: M. lusitanicus PDR transporters separated into two phylogenetic clusters: A (pdr1, pdr6-8) and B (pdr2-5). RNA-sequencing and RT-qPCR revealed strong up-regulation of pdr1 and pdr6, but down-regulation of pdr7 and pdr8 in response to 80 min exposures of 4.0 mg/L voriconazole, isavuconazole or posaconazole. The expression of Pdr6 and Pdr7 in S. cerevisiae ADΔΔ increased its resistance to short- and mid-length tailed azoles. Pdr1 and Pdr8 expression, however, conferred pan-azole resistance including long-tailed azoles such as itraconazole and posaconazole. No efflux pump function and ATPase activity were detected for Pdr3 and Pdr5. The ATPase activities of Pdr1, Pdr6, Pdr7 and Pdr8 were comparable to Candida albicans Cdr1 expressed in ADΔΔ.

Conclusions: All Mucor cluster A PDR transporters are multidrug efflux pumps, but Pdr1 and Pdr6 are possibly the major contributors to the innate azole resistance phenotype of M. lusitanicus.