Dataset Information

Cdn-Cna1-GFP-IP from Cryptococcus neoformans, Yadav and Heitman

ABSTRACT: Differential protein interaction analysis following IP, LC-MS/MS, and label-free quantitation on an Orbitrap Astral

INSTRUMENT(S):

ORGANISM(S): Cryptococcus Neoformans Bt1

SUBMITTER: Erik Soderblom

LAB HEAD: Joseph Heitman

PROVIDER: PXD060167 | Pride | 2025-08-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	20250121_162909_10723_Vikas_012125.sne	Other
	ID124816_01_SPD30_OA10034_10723_011025.raw	Raw
	ID124817_01_SPD30_OA10034_10723_011025.raw	Raw
	ID124818_01_SPD30_OA10034_10723_011025.raw	Raw
	ID124819_01_SPD30_OA10034_10723_011025.raw	Raw

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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.

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Cdn-Cna1-GFP-IP from Cryptococcus neoformans, Yadav and Heitman

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