Myriocin enhances the antifungal activity of fluconazole by blocking the membrane localization of the efflux pump Cdr1

Abstract

Introduction: Extrusion of azoles from the cell, mediated by an efflux pump Cdr1, is one of the most frequently used strategies for developing azole resistance in pathogenic fungi. The efflux pump Cdr1 is predominantly localized in lipid rafts within the plasma membrane, and its localization is sensitive to changes in the composition of lipid rafts. Our previous study found that the calcineurin signal pathway is important in transferring sphingolipids from the inner to the outer membrane.Methods: We investigated multiple factors that enhance the antifungal activity of fluconazole (FLC) using minimum inhibitory concentration (MIC) assays and disk diffusion assays. We studied the mechanism of action of myriocin through qRT-PCR analysis and confocal microscopy analysis. We tested whether myriocin enhanced the antifungal activity of FLC and held therapeutic potential using a mouse infection model.Results: We found that this signal pathway has no function in the activity of Cdr1. We found that inhibiting sphingolipid biosynthesis by myriocin remarkably increased the antifungal activity of FLC with a broad antifungal spectrum and held therapeutic potential. We further found that myriocin potently enhances the antifungal activity of FLC against C. albicans by blocking membrane localization of the Cdr1 rather than repressing the expression of Cdr1. In addition, we found that myriocin enhanced the antifungal activity of FLC and held therapeutic potential.Discussion: Our study demonstrated that blocking the membrane location and inactivating Cdr1 by inhibiting sphingolipids biogenesis is beneficial for enhancing the antifungal activity of azoles against azole-resistant C. albicans due to Cdr1 activation.

Small Molecules Restore Azole Activity Against Drug-Tolerant and Drug-ResistantCandidaIsolates

“…It is noteworthy that disruption of phospholipid homeostasis affects FLC susceptibility (69). The fact that both ergosterol and sphingolipids are essential for the correct localization of Cdr1 to lipid rafts in C. albicans adds further complexity to ascribing a role for Cdr1 in FLC resistance (70, 71). As drug combinations of FLC and potentiators alter lipid homeostasis, one could imagine that they could affect Cdr1 localization, thereby altering drug efflux (and therefore drug susceptibility).…”

Step-wise evolution of azole resistance through copy number variation followed byKSR1loss of heterozygosity inCandida albicans

“…Based on the observed reduction in sphingolipid species, 189R/R appears to reduce Ksr1p function relative to the progenitor that is heterozygous (189R/Q). Interestingly, inhibition of sphingolipid biosynthesis with myriocin, NPD827 or aureobasidin A, increases FLC sensitivity rather than increasing FLC resistance [33–35]; similarly, other loss of function mutations in sphingolipid genes tend to decrease, rather than increase FLC resistance [24,32]. This does not appear to be the case for KSR1 and suggests that the relationship between drug resistance and sphingolipid abundance phenotypes may be more complex than previously appreciated.…”

“…Conversely, the KSR1B/B strain had, on average, a significantly lower number of BODIPY stained lipid droplets, which was similar to the decrease in the number of lipid droplets seen when wild-type cells were treated with myriocin (S13 Fig A ,B). Since myriocin inhibits the step in sphingolipid biosynthesis immediately preceding the reaction catalyzed by KSR1 [33,34], this indicates that KSR1B/B shares a phenotype characteristic of sphingolipid pathway inhibition while the KSR1 LOH1 does not. Together, these results indicate that KSR1 LOH1 as well as strains with homozygous nonsense codon affect lipid homeostasis, membrane stress, and the formation of lipid droplets, but that these two types of mutations differ in their effect on lipid droplet properties.…”

“…Azole susceptibility in LCB4/lcb4 mutants can be ameliorated by the addition of the metabolic intermediate phytosphingosine-1-P (PhytoSph-1-P), which leads to upregulation of drug efflux pumps [32]. In addition, chemical inhibitors of sphingolipid synthesis including aureobasidin A, which targets AUR1, [33,34]; NPD827, which likely targets LCB1/LCB2 [35]; MMV688766 [36]; and myriocin, which inhibits 3ketosphinganine formation [34], all synergize with azoles by disrupting the localization or activation of drug efflux pumps. 3-ketosphinganine, which is inhibited by myriocin, is reduced by the 3-ketosphinganine reductase encoded by KSR1 (S1 Fig) . Despite its importance in the sphingolipid pathway [37], KSR1 has not been previously connected to resistance.…”

Management of Chronic Atrophic Candidiasis (Denture Stomatitis)—A Narrative Review

“…It is also worth mentioning that although fluconazole is one of the most popular systemic antifungals, it only has a fungistatic action against C. albicans , which may result in fluconazole resistance after prolonged use. [ 65 ] There is emerging evidence that the combination of fluconazole with other drugs, such as myriocin [ 66 ] (a fungus-derived antibiotic), may result in a synergistic effect that may potentiate the effects of fluconazole against C. albicans . However, such interventions need further evidence before routine clinical use.…”