{delta}-Aminolaevulinic acid modulates the resistance to fluconazole in a hem1 mutant of Saccharomyces cerevisiae

Dimitrios P. Kontoyiannisa,* and Gregory S. Mayb

a Department of Internal Medical Specialties and b Department of Research Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, TX, USA

Sir,

Fluconazole, a widely used triazole, selectively inhibits the cytochrome P450-dependent C-14 lanosterol demethylase (P450 14-DM), a key enzyme in ergosterol biosynthesis in fungi.1 Saccharomyces cerevisiae, a genetically tractable fungus closely related to the human pathogen Candida glabrata, provides an attractive experimental system to study the mechanisms of toxicity of azoles.1 We have previously shown that fluconazole toxicity requires intact mitochondria, because petite strains of S. cerevisiae (rho-) that lack mitochondrial DNA are resistant to the drug.2 Furthermore, a relationship between mitochondrial DNA deficiency and azole resistance has been demonstrated recently in azole-resistant isolates of C. glabrata.3 However, the exact contribution of mitochondria to fluconazole toxicity is not known. We have previously suggested that protection from fluconazole toxicity in the setting of mitochondrial inhibition could be the result of preservation of ergosterol.2 This preservation of ergosterol could be the combined result of overexpression of cytochrome P450 demethylase (Erg11p) and to derepression of another enzyme later in the ergosterol pathway.2 In the present work, we additionally asked whether the toxic effects of fluconazole could be dependent on the cytochrome activity. We used standard procedures to prepare the media and to manipulate S. cerevisiae. Growth was examined by plating c. 105 cells on synthetic complete (SC) agar plates containing 500 µg of fluconazole on a paper disc (100 µL of 5 mg/mL stock solution). The cells were grown aerobically for 2 days at 30°C. The zone of inhibition was then measured. The {delta}-aminolaevulinate (ALA) synthase (hem1) mutant in S. cerevisiae has an early lesion in the pathway of prophyrin biosythesis.4 This results in the loss of all haem-containing enzymes, including the mitochondrial cytochromes, and prevents the synthesis of components whose formation requires haem-containing enzymes, including unsaturated fatty acids and ergosterol.4,5 It has been shown previously that the sterol, unsaturated acid and cytochrome content of cells of a hem1 mutant could be manipulated by growing the organism in media containing defined supplements of {delta}-ALA.5 More specifically, if unsaturated fatty acids are added to a growth medium such as Tween-80, then in the presence of low (0–2 mg/L) supplements of {delta}-ALA, only unsaturated fatty acid biosynthesis is restored; however, much higher concentrations (>20 mg/L) are needed to restore cytochrome biogenesis, and even higher levels (500 mg/L) are necessary for normal sterol synthesis.5 Finally, the addition of {delta}-ALA to the wild-type cells is capable of boosting the cytochrome content, respiratory activity and lipid content of the cells, presumably by bypassing the normal control of the porphyrin pathway, which is principally exerted at the level of {delta}-ALA synthetase.4,5

We constructed a hem1 mutant by transforming the strain 10480-6D (MATa ura3-52 leu2::hisG his::hisG::hisG rho+)2 with plasmid 249 containing the HEM1 gene disrupted by the LEU2 gene (R. Zitomer, unpublished) and plating it in SC-leucine + {delta}-ALA medium (50 mg/L). The plasmid 249 was linearized by digestion with the enzymes HindIII and PstI. The putative hem1 mutants, in contrast to the isogenic wild-type strain, were unable to grow in the absence of {delta}-ALA, were unable to grow in a nonfermentable carbon source (YPGE medium), were hypersensitive to oxidation in SC-leucine + {delta}-ALA 50 mg/L + peroxide 0.4 mM plates and were resistant to nystatin as described previously.4 We then plated both the wild type and the isogenic hem1 mutants in SC-leucine + 1% Tween 80 plates containing different concentrations of {delta}-ALA (0, 0.5, 2, 5, 50 and 500 mg/L). As shown in panel a of the FigureGo, low doses (0.5–2 mg/L) of {delta}-ALA restored growth in the hem1 {triangleup}LEU2 mutant on SC-leucine + 1% Tween-80 plates and attenuated toxicity to fluconazole on the same plates. In contrast, higher concentrations of {delta}-ALA (5–100 mg/L) dose dependently restored both growth and toxicity to fluconazole in the hem1 mutant (Figure, aGo). No effect of {delta}-ALA was seen in the isogenic wild-type strain (Figure, bGo). Finally, when placed on a disc, {delta}-ALA did not affect the zone of inhibition around the fluconazole disc (500 µg) in the isogenic petite (rho-) wild-type strain as well as in erg3 rho+ and erg3 rho- strains2 in SC plates (data not shown). Because of the pleiotropic consequences of haem deficiency,4,5 the level of such haem-dependent enzymic reaction is not known. For example, the lack of effect of {delta}-ALA in both the wild-type (ERG3) rho+ and rho- and erg3 rho+ and rho- mutants could be consistent, however, with the alternative hypothesis, that a cytochrome-dependent but mitochondrially independent enzyme reaction (e.g. Erg11p) protects from azole toxicity. Turi & Loper6 have shown that the ERG11 transcript is induced by haem in haem-deficient cells and that {delta}-ALA increases the ERG11 message in the hem1 mutant. Further biochemical studies to measure the lanosterol contents of the hem1 mutant exposed to different concentrations of {delta}-ALA and fluconazole are needed to test this hypothesis. This preliminary work suggests that a hem1/{delta}-ALA selection strategy could also serve as a titratable system to explore further the mechanisms of azole toxicity in S. cerevisiae.




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Figure. Growth of (a) hem1 mutant and (b) isogenic wild-type strain on SC-leucine + 1% Tween-80 + {delta}-ALA plate with fluconazole 500 µg disc. The numbers in (a) indicate {delta}-ALA concentrations in mg/L.

 

Acknowledgments

We thank Dr R. Zitomer from the State University of New York at Albany, NY, for providing the plasmid 249.

Notes

J Antimicrob Chemother 2000; 46: 1044–1046

* Correspondence address. Department of Internal Medicine Specialties, Section of Infectious Diseases, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 47, Houston, TX 77030, USA. Tel: +1-713-792-6237; Fax: +1-713-794-4351; E-mail: dkontoyi{at}notes.mdacc.tmc.edu Back

References

1 . Kelly, S. L., Arnoldi, A. & Kelly, D. E. (1993). Molecular genetic analysis of azole antifungal mode of action. Biochemistry Society Transactions 21, 1034–8.

2 . Kontoyiannis, D. P. (2000). Modulation of fluconazole sensitivity by the interaction of mitochondria and Erg3p in Saccharomyces cerevisiae. Journal of Antimicrobial Chemotherapy 46, 191–7.[Abstract/Free Full Text]

3 . Defontaine, A., Bouchara, J.-P., Declerk, P., Planchenault, C., Chabasse, D. & Hallett, J.-N. (1999). In-vitro resistance to azoles associated with mitochondrial DNA deficiency in Candida glabrata. Journal of Medical Microbiology 48, 663–70.[Abstract]

4 . Henri, S. A. (1982). Membrane lipids of yeast: biochemical and genetic studies, In The Molecular and Cellular Biology of the Yeast Saccharomyces. (Strathern, J., Jones, E. W. & Broach, J., Eds), pp. 101–58. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

5 . Haslam, J. M. & Astin, A. M. (1979). The use of heme-deficient mutants to investigate mitochondrial function and biogenesis in yeast. Methods in Enzymology 56, 558–68.[Medline]

6 . Turi, T. G. & Loper, J. C. (1992). Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14{alpha}-demethylase (ERG11). Journal of Biological Chemistry 267, 2046–56.[Abstract/Free Full Text]





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