Osh2 mediates Candida resistance to miltefosine by regulating zymosterol transport

Miltefosine has been identified for its antifungal properties, yet the mechanisms underlying its action and the development of resistance remain unclear. In this study, we induced drug resistance in various Candida species through concentration gradient exposure, successfully obtaining a miltefosine-resistant strain of Candida glabrata. Whole-genome sequencing revealed a premature stop codon mutation in the OSH2 gene within the resistant strain. To further investigate the function of the Osh2, we deleted it in Candida albicans, observing a significant increase in miltefosine resistance in the knockout strain. RNA-seq and lipidomics analyses indicated that Osh2 influences the ergosterol biosynthetic pathway by regulating zymosterol transport, thereby mediating miltefosine resistance. Additionally, exogenous supplementation of zymosterol significantly enhanced the resistance of Candida to miltefosine. Further studies revealed that deletion of the ERG11 gene, a rate-limiting enzyme in the zymosterol synthesis pathway, rendered the strain highly sensitive to miltefosine, while deletion of the ERG6 gene, which catalyzes the conversion of zymosterol to fecosterol, led to miltefosine resistance. This study is the first to elucidate that the Osh2 gene mediates miltefosine resistance in Candida by regulating zymosterol transport and affecting the ergosterol biosynthetic pathway, providing new theoretical insights into the mechanism of action of miltefosine and strategies to overcome fungal resistance.

Miltefosine（米替福新）已被证实具备抗真菌活性，但其具体作用机制与耐药性产生的分子机理仍未明确。本研究通过浓度梯度暴露法在多种念珠菌（Candida species）中诱导药物耐药性，成功获得一株光滑念珠菌（Candida glabrata）的米替福新耐药菌株。全基因组测序（Whole-genome sequencing）结果显示，该耐药菌株的OSH2基因（OSH2 gene）存在一处提前终止密码子突变。为进一步探究Osh2蛋白的功能，我们在白色念珠菌（Candida albicans）中敲除了OSH2基因，观察到敲除菌株（knockout strain）对米替福新的耐药性显著升高。RNA测序（RNA-seq）与脂质组学分析表明，Osh2通过调控酵母固醇（zymosterol）的转运过程影响麦角固醇生物合成途径（ergosterol biosynthetic pathway），进而介导米替福新的耐药性。此外，外源补充酵母固醇可显著增强念珠菌对米替福新的耐药性。进一步研究发现，敲除酵母固醇合成途径的限速酶（rate-limiting enzyme）编码基因ERG11（ERG11 gene）会使菌株对米替福新高度敏感；而敲除催化酵母固醇转化为粪甾醇（fecosterol）的ERG6基因（ERG6 gene），则会导致菌株产生米替福新耐药性。本研究首次阐明了Osh2基因通过调控酵母固醇转运、影响麦角固醇生物合成途径，从而介导念珠菌对米替福新的耐药性，为揭示米替福新的作用机制及破解真菌耐药性提供了全新的理论依据与干预策略。