Yeast sphingolipid bypass mutants as indicators of antifungal agents selectively targeting sphingolipid synthesis

doi:10.1016/S0006-291X(03)01164-1

Biochemical and Biophysical Research Communications

Volume 307, Issue 2, 25 July 2003, Pages 369-374

https://doi.org/10.1016/S0006-291X(03)01164-1 Get rights and content

Abstract

Standard methods for evaluating the target specificity of antimicrobial agents often involve the use of microorganisms with altered expression of selected targets and thus either more resistant or more susceptible to target specific inhibitors. In this study we present an alternative approach that utilizes physiological bypass mutants. The Saccharomyces cerevisiae sphingolipid bypass mutant strain AGD is able to grow without making sphingolipids and importantly, tolerates loss-of-function mutations in the otherwise essential genes for both serine palmitoyltransferase (SPT) and inositol phosphorylceramide (IPC) synthase. We found that strain AGD was >1000-fold more resistant than the wild-type strain to selective inhibitors of SPT and IPC synthase. In contrast, strain AGD, which due to abnormal composition of the plasma membrane is sensitive to a variety of environmental stresses, was more susceptible than the wild-type to amphotericin B, voriconazole, and to cycloheximide. We show that in a simple growth assay the AGD strain is an appropriate and useful indicator for inhibitors of IPC synthase, a selective antifungal target.

Section snippets

Materials and methods

Strains and growth conditions. Yeast strains SJ21R (MATa ura3-52 leu2-3 ade 1), AGD27-61 (MATa/MATα ura3-52 leu2-3,112 lys2-80amber ade1 lcb1::URA3 SLC1-1 ipc1-1), 4R3 (MATa ura3-52 leu2-3,112 ade1 lcb::URA3 SLC1-1), 7R6 (MATa ura3-52 leu2-3,112 ade1 lcb::URA3 SLC1-1), and 7R4 (MATa ura3-52 leu2-3,112 ade1 lcb::URA3 SLC2-1) were obtained from Dr. Bob Dickson (Univ. Kentucky) and were typically maintained in PYED medium (1% yeast extract, 2% Bacto-peptone, 2% glucose, 50 mM sodium succinate, pH

Results and discussion

Yeast sphingolipid bypass mutants are able to grow in the absence of sphingolipid biosynthesis due to their unusual ability to synthesize novel compensatory phospholipids (Fig. 1). The sphingolipid bypass mutants (SLC strains) carry two mutations. One consists of a deletion of the LCB1 gene, which encodes a subunit of serine palmitoyltransferase (SPT), and results in a lack of activity of this normally essential enzyme. The second mutation is a gain of function mutation, SLC1-1, that affects

Acknowledgements

We thank Bob Dickson (Univ. Kentucky) for strains, Hans Achenbach for providing galbonolide A, and Vince Marshall, Joyce Cialdella, Ming-Shang Kuo, and Barclay Shilliday for purifying samples of australifungin and khafrefungin.

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Again, NBD-ceramide-labeled samples were imaged using the FLA3000 laser fluorescence imaging system (Fuji). Agar Diffusion Assay—Wild type YPH499 yeast and the transgenic strain YPH499-HIS-GAL-AUR1 complemented with ScAUR1 or LmIPCS were assayed for susceptibility to aureobasidin A (Takara Bio Inc.), myriocin (Sigma) and cycloheximide (Sigma) as previously described (30). Briefly, 2.4 × 107 logarithmically dividing cells were embedded in 15 ml of YPD-agarose (1% yeast extract, 2% peptone, 2% dextrose, 0.8% agarose) on 100-mm2 square Petri dishes (Sarstedt).
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¹: Present address: Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA.

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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Results and discussion

Acknowledgements

References (19)

Sphingolipid synthesis as a target for antifungal drugs. Complementation of the inositol phosphorylceramide synthase defect in a mutant strain of Saccharomyces cerevisiae by the AUR1 gene

J. Biol. Chem.

Inositol phosphoryl transferases from human pathogenic fungi

Biochim. Biophys. Acta

Mutant strains of Saccharomyces cerevisiae lacking sphingolipids synthesize novel inositol glycerophospholipids that mimic sphingolipid structures

J. Biol. Chem.

A suppressor gene that enables Saccharomyces cerevisiae to grow without making sphingolipids encodes a protein that resembles an Escherichia coli fatty acyltransferase

J. Biol. Chem.

Khafrefungin, a novel inhibitor of sphingolipid synthesis

J. Biol. Chem.

Specificity of inhibitors of serine palmitoyltransferase (SPT), a key enzyme in sphingolipid biosynthesis, in intact cells. A novel evaluation system using an SPT-defective mammalian cell mutant

Biochem. Pharmacol.

Rustmicin, a potent antifungal agent, inhibits sphingolipid synthesis at inositol phosphoceramide synthase

J. Biol. Chem.

Isolation and characterization of novel inhibitors of sphingolipid synthesis: australifungin, viridiofungins, rustmicin, and khafrefungin

Methods Enzymol.

A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme

Proc. Natl. Acad. Sci. USA

Cited by (12)

The pore-forming action of polyenes: From model membranes to living organisms

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Sphingolipid biosynthesis in pathogenic fungi: Identification and characterization of the 3-ketosphinganine reductase activity of Candida albicans and Aspergillus fumigatus

Oceanapiside, a Marine Natural Product, Targets the Sphingolipid Pathway of Fluconazole-Resistant Candida glabrata

Antileishmanial Chemotherapy through Clemastine Fumarate Mediated Inhibition of the Leishmania Inositol Phosphorylceramide Synthase

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