Increasing the Fungicidal Action of Amphotericin B by Inhibiting the Nitric Oxide‐Dependent Tolerance Pathway

Abstract

Amphotericin B (AmB) induces oxidative and nitrosative stresses, characterized by production of reactive oxygen and nitrogen species, in fungi. Yet, how these toxic species contribute to AmB-induced fungal cell death is unclear. We investigated the role of superoxide and nitric oxide radicals in AmB's fungicidal activity in Saccharomyces cerevisiae, using a digital microfluidic platform, which enabled monitoring individual cells at a spatiotemporal resolution, and plating assays. The nitric oxide synthase inhibitor L-NAME was used to interfere with nitric oxide radical production. L-NAME increased and accelerated AmB-induced accumulation of superoxide radicals, membrane permeabilization, and loss of proliferative capacity in S. cerevisiae. In contrast, the nitric oxide donor S-nitrosoglutathione inhibited AmB's action. Hence, superoxide radicals were important for AmB's fungicidal action, whereas nitric oxide radicals mediated tolerance towards AmB. Finally, also the human pathogens Candida albicans and Candida glabrata were more susceptible to AmB in the presence of L-NAME, pointing to the potential of AmB-L-NAME combination therapy to treat fungal infections.

Tuning the Surface Interactions between Single Cells and an OSTE+ Microwell Array for Enhanced Single Cell Manipulation

“…However, PEG 2000 has a methoxy terminal group, which inhibits bonding of PDMS. Although methacrylated PEG of molecular weight 2000 g/mol with a hydroxy terminal group is also commercially available, there are no cost-effective options, , and hence, it has not been taken into account for this study.…”

Multiplex Analysis to Unravel the Mode of Antifungal Activity of the Plant Defensin HsAFP1 in Single Yeast Cells

“…Non-genetic heterogeneity in yeast cells is also observed during the real-time monitoring of single cell responses upon antifungal treatment. Indeed, different yeast subpopulations were, for example, detected with respect to amphotericin B (AmB)-induced superoxide radical production, based on their intracellular levels and timing [6,7]. This non-genetic heterogeneity can give rise to non-responsive subpopulations upon treatment, which can cause treatment failure and the recurrence of infections, pointing out the importance of single cell analyses.…”

“…Hereof, flow cytometry is considered as a standard single cell technique due to its high-throughput and the ability to analyze multiple parameters simultaneously [8]. However, it is also associated with a number of drawbacks, as it does not allow to monitor cellular growth and interactions, secreted cellular products or kinetic responses [7,[9][10][11]. Therefore, we previously optimized a poly(ethylene glycol) (PEG)-grafted off-stoichiometry thiol-ene epoxy (OSTE+) microwell array, integrated with a polydimethylsiloxane (PDMS) microfluidic channel that enables automated and effective single yeast cell seeding and washing of non-seeded yeast cells [12].…”

Membrane-Interacting Antifungal Peptides

“…Similar to certain antimycotics (e.g., amphotericin B), numerous AMPs, such as the plant defensin ApDef1, cecropin A, coprisin, histatin 5, HsAFP1, human lactoferrin, the plant defensin LpDef1, NaD1, OefDef1.1, the honeybee venom peptide melittin, PAF, the Penicillium chrysogenum antifungal protein C (PAFC), PvD1, RsAFP2 and the centipede peptide scolopendin induce the production of ROS (Figure 2; Leiter et al, 2005;Andrés et al, 2008;Park and Lee, 2010;Aerts et al, 2011;Mello et al, 2011;Lee et al, 2012Lee et al, , 2016Galgóczy et al, 2013;Choi et al, 2014;Yun and Lee, 2016;Soares et al, 2017;Vriens et al, 2017;Holzknecht et al, 2020). Excessive cellular levels of ROS can cause oxidative damage to proteins, nucleic acids, lipids, membranes and organelles, which in turn can lead to activation of cell death processes such as programmed cell death (Redza-Dutordoir and Averill-Bates, 2016).…”