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Osh2 mediates Candida resistance to miltefosine by regulating zymosterol transport

ABSTRACT: 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.

INSTRUMENT(S): Liquid Chromatography MS - positive - hilic, Liquid Chromatography MS - negative - hilic

PROVIDER: MTBLS12270 | MetaboLights | 2025-07-03

REPOSITORIES: MetaboLights

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Osh2 mediates Candida species resistance to miltefosine by regulating zymosterol accumulation.

Wu Yongqin Y Dai Yuanyuan Y Lu Huaiwei H Jiang Xiaohua X Wang Yuanyuan Y

Antimicrobial agents and chemotherapy 20250723 9

Invasive candidiasis poses a growing threat to global public health, compounded by the scarcity of effective antifungal treatments. Miltefosine exhibits broad-spectrum antifungal activity, yet its mechanisms of antifungal action and the development of resistance remain poorly understood. Here, we first generated miltefosine-resistant strains of Candida glabrata through stepwise exposure to increasing drug concentrations. Whole-genome sequencing revealed that nonsense mutations in the O ...[more]

PMID: 40698822

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Project description:Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of L. donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme is a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.

Project description:The monoxenous trypanosomatid Strigomonas culicis inhabits several mosquito species, including Aedes aegypti. Previously, our group described that the infection of A. aegypti females by this trypanosomatid triggers the insect's immune response, reduces its reproductive fitness, and could potentially influence the vector competence. S. culicis maintains a mutualistic relationship with an endosymbiotic bacterium, which enables the protozoa to survive without heme supplementation. Here, the impact of H2O2 resistance and symbiont elimination on intracellular heme and Fe2+ availability was analyzed, comparing both the wild-type H2O2-resistant (WTR) and aposymbiotic (Apo) with the wild-type (WT) strain. Relative quantification through label-free PRM targeted mass spectrometry approach pointed out that H2O2 resistance induction does not influence the abundance of heme biosynthetic pathway; however, symbiont elimination increases the abundance of all components of de novo heme synthesis, except for protoporphyrinogen oxidase (PPOX). Confirming the limited role of PPOX in this biossynthetic pathway, the addition of protoporphyrin IX (PPIX) to heme-decreased medium (HDM) leads to higher growth rates and enhances intracellular heme content in Apo strain. A putative ferrous iron transporter homologous to LIT1 and TcIT from Leishmania major and Trypanosoma cruzi, was also detected for the first time. Targeted mass spectrometry approach indicated that H2O2 resistance and symbiont elimination increased S. culicis iron transporter (ScIT) abundance by 1.6- and 16.4-fold in comparison to WT. Accordingly, antibody-mediated blockage of ScIT decreased 26.7 and 41.9% intracellular Fe2+ concentration in both WTR and Apo strains, whereas no effect was detected in WT. Gene expression analysis showed that Apo growth in HDM increases 1.8-fold ScIT transcript levels, comparing to protozoa maintained in standard medium. Also, addition of 10 µM hemin to HDM significantly decreased intracellular Fe2+ concentration and ferric iron reduction in Apo strain. Together, our data indicate molecular mechanisms of S. culicis to deal with symbiont elimination and reduced heme availability. Since heme and Fe2+ are essential cofactors in many metabolic pathways, such as oxidative phosphorylation and antioxidant system, this study also provides novel mechanistic insights associated with S. culicis H2O2 resistance.

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Osh2 mediates Candida resistance to miltefosine by regulating zymosterol transport

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Osh2 mediates <i>Candida</i> species resistance to miltefosine by regulating zymosterol accumulation.

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Osh2 mediates Candida resistance to miltefosine by regulating zymosterol transport

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Osh2 mediates &lt;i&gt;Candida&lt;/i&gt; species resistance to miltefosine by regulating zymosterol accumulation.

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OmicsDI is part of the ELIXIR infrastructure

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Osh2 mediates <i>Candida</i> species resistance to miltefosine by regulating zymosterol accumulation.