Copper Availability Modulates Early Membrane-Associated Events in Polyethyleneimine Antifungal Activity in Aspergillus nidulans

In healthy humans, a very low percentage of fungal species are capable of causing severe infections. On the contrary, in individuals with weak or compromised immune system, opportunistic fungal infections result in up to 3.8 million deaths annually. Drug resistance is the major problem for all of the currently available antifungals. Polyethyleneimine (PEI), a cationic polymer, exhibits potent antifungal activity against Aspergillus nidulans, yet the influence of copper ions on its mechanism of activity remains unclear. Here, we investigated how free exogenous Cu2+, influences PEI antifungal activity and the membrane-associated events underlying this effect. We demonstrate that (i) exogenous excess of homeostatic Cu2+ concentrations protects A. nidulans from PEI toxicity in a concentration-dependent and developmentally regulated manner; (ii) Cu2+ is effective only when present during the earliest stages of PEI action, identifying the plasma membrane as the primary site of Cu2+ protection; (iii) PEI, like Amphotericin B, induces translocation of ergosterol from the plasma membrane to intracellular compartments while additionally producing a distinct polarized enrichment of ergosterol at the hyphal tip; and (iv) Cu2+-mediated protection is independent of eisosome assembly. Together, these findings provide new mechanistic insights into the antifungal activity of PEI and the role of Cu+2 on PEI-induced cellular damage.