Unmasking the Amphotericin B Resistance Mechanisms in Candida haemulonii Species Complex

The polyene amphotericin B (AMB) exerts a powerful and broad antifungal activity. AMB acts by (i) binding to ergosterol, leading to pore formation at the fungal plasma membrane with subsequent ion leakage, and (ii) inducing the intracellular accumulation of reactive oxygen species (ROS). Herein, we have deciphered the AMB resistance mechanisms in clinical isolates of Candida haemulonii complex (C. haemulonii, C. duobushaemulonii, C. haemulonii var. vulnera) in comparison to other clinically relevant non-albicans Candida species. Membrane gas chromatography-mass spectrometry analysis revealed that the vast majority of sterols were composed of ergosterol pathway intermediates, evidencing the absence of AMB target. Supporting this data, C. haemulonii species complex demonstrated poor membrane permeability after AMB treatment. Regarding the oxidative burst, AMB induced the formation of ROS in all species tested; however, this phenomenon was slightly seen in C. haemulonii complex isolates. Our results indicated that these isolates displayed altered respiratory status, as revealed by their poor growth in nonfermented carbon sources, low consumption of oxygen, and derisive mitochondrial membrane potential. The use of specific inhibitors of mitochondrial respiratory chain (complex I-IV) revealed no effects on the yeast growth, highlighting the metabolic shift to fermentative pathway in C. haemulonii strains. Also, C. haemulonii complex proved to be highly resistant to oxidative burst agents, which can be correlated with a high activity of antioxidant enzymes. Our data demonstrated primary evidence suggesting that ergosterol content, mitochondrial function, and fungal redox homeostasis are involved in AMB fungicidal effects and might explain the resistance presented in this multidrug-resistant, emergent, and opportunistic fungal complex.