Supporting information Abou-Kandil et al.

The polyene antimycotica amphotericin B (AmB) and its liposomal formulation AmBisome belong to the treatment options of invasive fungal infections, particularly of invasive aspergillosis caused by <i>Aspergillus fumigatus</i>. However, increasing resistance to AmB in clinical isolates of <i>Aspergillus </i>species is a growing concern. Despite this, the mechanisms of AmB resistance in <i>A. fumigatus</i> remain unclear. In this study, we conducted a proteomic analysis of <i>A. fumigatus</i> exposed to sublethal concentrations of AmB and AmBisome. Both antifungals induced significantly increased levels of proteins involved in aromatic acid metabolism, transmembrane transport and secondary metabolite biosynthesis. One of the most upregulated proteins was RtaA, a member of the RTA-like protein family, which includes conserved fungal membrane proteins with putative functions as transporters or translocases. Accordingly, we found that RtaA is mainly located in the cytoplasmic membrane and to a minor extent in vacuolar-like structures. Deletion of <i>rtaA</i> led to increased polyene sensitivity and its overexpression resulted in modest resistance. Interestingly, <i>rtaA</i> expression was only induced by exposure to the polyenes AmB and nystatin, but not by other antifungals such as itraconazole and caspofungin. Deletion of <i>rtaA</i> did not significantly change the ergosterol content of <i>A. fumigatus</i>, but decreased fluorescence intensity of the sterol-binding stain filipin. This suggests that RtaA is involved in sterol and lipid trafficking, possibly by transporting the target ergosterol to or from lipid droplets. These findings reveal the contribution of RtaA to polyene resistance in <i>A. fumigatus</i>, and thus provide a new putative target for antifungal drug development.