PXD052472-1

PXD052472 is an original dataset announced via ProteomeXchange.

Title	Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1
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.
HostingRepository	PRIDE
AnnounceDate	2024-06-21
AnnouncementXML	Submission_2024-06-21_01:57:59.674.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Victoriano Corpas-Lopez
SpeciesList	scientific name: Leishmania donovani BPK282A1; NCBI TaxID: 981087;
ModificationList	No PTMs are included in the dataset
Instrument	Orbitrap Astral

Revision	Datetime	Status	ChangeLog Entry
0	2024-05-22 02:23:32	ID requested
⏵ 1	2024-06-21 01:58:00	announced

Dataset with its publication pending

submitter keyword: visceral leishmaniasis, mechanism of action, P450 reductase and sterol 14-alpha demethylase (CYP51),Drug resistance, Leishmania, sterol metabolism

Susan Wyllie
contact affiliation	WCAIR, School of Life Sciences, BCDD, University of Dundee
contact email	swyllie@dundee.ac.uk
lab head
Victoriano Corpas-Lopez
contact affiliation	University of Dundee
contact email	vcorpaslopez@dundee.ac.uk
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD052472
     1. Label: PRIDE project
     2. Name: Sterol 14-alpha demethylase (CYP51) activity in Leishmania donovani is likely dependent upon cytochrome P450 reductase 1