The Production of Reactive Oxygen Species Is a Universal Action Mechanism of Amphotericin B against Pathogenic Yeasts and Contributes to the Fungicidal Effect of This Drug

Abstract

e Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species (Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans, and Cryptococcus gattii). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.

Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host

“…The following C. tropicalis strains resistant to AmB were used in this work: ATCC 200956 and CL-6835 (from the yeast collection of the Mycology Reference Laboratory of the Spanish National Centre for Microbiology). The MIC values reported for these strains to AmB and fluconazole were 2 and Ͼ64 mg/liter, respectively (following the EUCAST antifungal susceptibility testing method [9,21]). …”

“…In this sense, AmB induces ergosterol sequestration, which causes alterations in the cell membrane that result in killing of the cells (3,4). In addition, AmB induces a significant accumulation of reactive oxygen species (ROS), which has been correlated with cell damage, apoptosis induction, and death (5)(6)(7)(8)(9).…”

Untitled

“…This complex functions as an ion channel, enhancing the leakage of intracellular K + and other ionic substances. AmBmediated lethality is alternatively attributed to the induction of oxidative stress via the enhanced generation of superoxide anions by this macrolide, as well as to its ability to extract ergosterol from the plasma membrane phospholipid bilayer [2,3]. In previous studies, for the first time, we found that AmB is incorporated across the plasma membrane of fungal cells, resulting in vacuolar membrane disruption with lethal effects [4][5][6].…”

Troponoids Can Inhibit Growth of the Human Fungal Pathogen Cryptococcus neoformans

“…The plates were incubated at 30°C for 2 days and checked for growth. The growth of cells treated with test compounds was compared with that of cells treated with FLC, which is known to be fungistatic, and with that of cells treated with AMB, which is known to be fungicidal (29,30).…”

“…284 for fungicidal versus fungistatic activities. This was done in a qualitative assay in parallel with FLC, which is known to be fungistatic (29) and AMB, which is known to be fungicidal (30). The MICs were 3 M (0.9 g/ml), 1.5 M (1.4 g/ml), 12 M, and 0.31 M for FLC, AMB, no.…”