Table2_Myriocin enhances the antifungal activity of fluconazole by blocking the membrane localization of the efflux pump Cdr1.XLSX

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.

引言：由细胞内排泵Cdr1介导的唑类化合物外排是病原性真菌产生唑类化合物抗药性的最常用策略之一。外排泵Cdr1主要定位于质膜内的脂筏中，其定位对脂筏组成的改变敏感。我们前期研究发现，钙调神经磷酸酶信号通路在将鞘脂从内膜转移到外膜过程中起着重要作用。
方法：我们通过最小抑菌浓度（MIC）试验和纸片扩散试验研究了多种增强氟康唑（FLC）抗真菌活性的因素。我们通过qRT-PCR分析和共聚焦显微镜分析研究了myriocin的作用机制。我们通过小鼠感染模型测试了myriocin是否增强了FLC的抗真菌活性并具有治疗潜力。
结果：我们发现该信号通路对Cdr1的活性无功能。我们发现通过myriocin抑制鞘脂生物合成显著增加了FLC的广谱抗真菌活性，并具有治疗潜力。我们进一步发现，myriocin通过阻断Cdr1的膜定位而非抑制Cdr1的表达，有效地增强了FLC对白念珠菌的抗真菌活性。此外，我们还发现myriocin增强了FLC的抗真菌活性，并具有治疗潜力。
讨论：我们的研究证实，通过抑制鞘脂的生物合成来阻断Cdr1的膜定位和失活，对于增强唑类化合物对唑类抗药性白念珠菌的抗真菌活性具有益处，这归因于Cdr1的激活。