1887

Abstract

The cytochrome P450 14α-demethylase, encoded by the ) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine strains were used in this study. The base sequence of seven isolates, of which only two were azole-sensitive, were determined. The base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G465S). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the C-terminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.

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1999-10-01
2019-08-22
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References

  1. Aoyama, Y., Horiuchi, T., Gotoh, O., Noshiro, M. & Yoshida, Y. ( 1998; ). CYP51-like gene of Mycobacterium tuberculosis actually encodes a P450 similar to eukaryotic CYP51. J Biochem 124, 694-696.[CrossRef]
    [Google Scholar]
  2. Bard, M., Lees, N. D., Turi, T., Craft, D., Cofrin, L., Barbuch, R., Koegel, C. & Loper, J. C. ( 1993; ). Sterol synthesis and viability of ERG11 (cytochrome P450 lanosterol demethylase) mutations in Saccharomyces cerevisiae and Candida albicans. Lipids 28, 963-967.[CrossRef]
    [Google Scholar]
  3. Bellamine, A., Mangla, A. T., Nes, D. & Waterman, M. R. (1998). Is the Mycobacterium tuberculosis CYP51-like P450 a 14α-demethylase? In Program and Abstracts of the Fourth International Symposium on P450 Biodiversity and Biotechnology, Strasbourg, France. IC-11.
  4. Bodey, G. P. ( 1997; ). Resistance to antimicrobial agents revisited. Curr Opin Infect Dis 10, 419-421.[CrossRef]
    [Google Scholar]
  5. Boscott, P. E. & Grant, G. H. ( 1994; ). Modeling cytochrome P450 14α-demethylase (Candida albicans) from P450cam. J Mol Graph 12, 185-192.[CrossRef]
    [Google Scholar]
  6. Bradford, M. M. ( 1976; ). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248-254.[CrossRef]
    [Google Scholar]
  7. Cartledge, J. D., Midgley, J. & Gazzard, B. G. ( 1997; ). Clinically significant azole cross-resistance in Candida isolates from HIV-positive patients with oral candidosis. AIDS 11, 1839-1844.[CrossRef]
    [Google Scholar]
  8. De Muri, G. P. & Hostetter, M. K. ( 1995; ). Resistance to antifungal agents. Pediatr Clin N Am 42, 665-685.
    [Google Scholar]
  9. Denning, D. W., Baily, G. G. & Hood, S. V. ( 1997; ). Azole resistance in Candida. Eur J Clin Microbiol Infect Dis 16, 261-280.[CrossRef]
    [Google Scholar]
  10. Doignon, F., Aigle, M. & Ribereau-Gayon, P. ( 1993; ). Resistance to imidazoles and triazoles in Saccharomyces cerevisiae as a new dominant marker. Plasmid 30, 224-233.[CrossRef]
    [Google Scholar]
  11. Dupont, B. ( 1995; ). Azole antifungal agents: emerging and inherent resistance. Curr Opin Infect Dis 8, 424-427.[CrossRef]
    [Google Scholar]
  12. Frosco, M. & Barrett, J. F. ( 1998; ). Importance of antifungal drug-resistance: clinical significance and need for novel therapy. Exp Opin Invest Drugs 7, 175-198.[CrossRef]
    [Google Scholar]
  13. Hartman, P. G. & Sanglard, D. ( 1997; ). Inhibitors of ergosterol as antifungal agents. Curr Pharm Des 3, 177-208.
    [Google Scholar]
  14. Hitchcock, C. A., Russell, N. J. & Barrett-Bee, K. J. ( 1987; ). Sterols in Candida albicans mutants resistant to polyene or azole antifungals, and of a double mutant C. albicans 6.4. Crit Rev Microbiol 15, 111-115.[CrossRef]
    [Google Scholar]
  15. Ishida, N. Y., Aoyama, Y., Hatanaka, R. & 10 other authors ( 1988; ). A single amino substitution converts cytochrome P45014dm to an inactive form, cytochrome P450SG1: complete primary structure deduced from cloned DNAs. Biochem Biophys Res Commun 155, 317–323.[CrossRef]
    [Google Scholar]
  16. Johnson, E. M. & Warnock, D. W. ( 1995; ). Azole drug resistance in yeast. J Antimicrob Chemother 36, 751-755.[CrossRef]
    [Google Scholar]
  17. Joseph-Horn, T. & Hollomon, D. W. ( 1997; ). Molecular mechanisms of azole resistance in fungi. FEMS Microbiol Lett 149, 141-149.[CrossRef]
    [Google Scholar]
  18. Kallakuri, S., Manavathu, E. K., Arganoza, M. T., Vazquez, J. A. & Sobel, J. D. (1996). Molecular characterization of the gene encoding cytochrome P450-dependent lanosterol 14α-demethylase from a fluconazole-resistant clinical isolate of Candida albicans. In Program and Abstracts of the 96th American Society for Microbiology General Meeting, New Orleans, Louisiana, 1996, Abstract F-63, p. 84. Washington, DC: American Society for Microbiology.
  19. Kelly, S. L., Lamb, D. C., Kelly, D. E., Manning, N. J., Loeffler, J., Schumacher, H. U. & Einsele, H. ( 1997; ). Resistance to fluconazole and cross-resistance to amphotericin B in Candida albicans from AIDS patients caused by defective sterol delta 5,6-desaturation. FEBS Lett 400, 80-82.[CrossRef]
    [Google Scholar]
  20. Kolaczkowski, M. & Goffeau, A. ( 1997; ). Active efflux by multidrug transporters as one of the strategies to evade chemotherapy and novel practical implications of yeast pleiotropic drug resistance. Pharmacol Ther 76, 219-242.[CrossRef]
    [Google Scholar]
  21. Lai, M. H. & Kirsch, D. R. ( 1989; ). Nucleotide sequence of cytochrome P450 L1A1 (lanosterol 14α demethylase) from Candida albicans. Nucleic Acids Res 17, 804.[CrossRef]
    [Google Scholar]
  22. Lamb, D. C., Kelly, D. E., Schunck, W.-H., Shyadehi, A. Z., Akhtar, M., Lowe, D., Baldwin, B. C. & Kelly, S. L. ( 1997; ). The mutation T315A in Candida albicans sterol 14α-demethylase causes reduced enzyme activity and fluconazole resistance through reduced affinity. J Biol Chem 272, 5682-5688.[CrossRef]
    [Google Scholar]
  23. Löffler, J., Kelly, S. L., Hebart, H., Schumacher, U., Lass-Flörl, C. & Einsele, H. ( 1997; ). Molecular analysis of cyp51 from fluconazole-resistant Candida albicans strains. FEMS Microbiol Lett 151, 263-268.[CrossRef]
    [Google Scholar]
  24. Manavathu, E. K., Kallakuri, S., Arganoza, M. T., Pierson, C. & Vazquez, J. A. (1996). Amino acid variations of cytochrome P450-dependent lanosterol 14α-demethylase (P450LDM) from a fluconazole-resistant clinical isolate of Candida albicans. In Program and Abstracts of the 36th Interscience Conference on Antimicrobial Agents and Chemotherapy, New Orleans, Louisiana, 1996, Abstract C68, p. 46. Washington, DC: American Society for Microbiology.
  25. Marichal, P. & Vanden Bossche, H. ( 1995; ). Mechanisms of resistance to azole antifungals. Acta Biochim Pol 42, 509-516.
    [Google Scholar]
  26. Marichal, P., Gorrens, J., Coene, M.-C., Le Jeune, L. & Vanden Bossche, H. ( 1995; ). Origin of differences in susceptibility of Candida krusei to azole antifungal agents. Mycoses 38, 111-117.[CrossRef]
    [Google Scholar]
  27. Marichal, P., Vanden Bossche, H., Odds, F. C., Nobels, G., Warnock, D. W., Timmerman, V., Van Broeckhoven, C., Fay, S. & Mose-Larsen, P. ( 1997; ). Molecular biological characterization of an azole-resistant Candida glabrata isolate. Antimicrob Agents Chemother 41, 2229-2237.
    [Google Scholar]
  28. Marr, K. A., Lyons, C. N., Rustad, T., Bowden, R. A. & White, T. C. ( 1998; ). Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR. Antimicrob Agents Chemother 42, 2584-2589.
    [Google Scholar]
  29. National Committee for Clinical Laboratory Standards (1995). Reference method for broth dilution susceptibility testing of yeasts. Tentative standard M27-A. Villanova, PA: National Committee for Clinical Laboratory Standards.
  30. Odds, F. C. ( 1992; ). Antifungal susceptibility testing of Candida spp. by relative growth measurement at single concentrations of antifungal agents. Antimicrob Agents Chemother 36, 1727-1737.[CrossRef]
    [Google Scholar]
  31. Odds, F. C. ( 1998; ). Should resistance to azole antifungals in vitro be interpreted as predicating clinical non-response? Drug Res Updates 1, 11-15.[CrossRef]
    [Google Scholar]
  32. Odds, F. C., Vranckx, L. & Woestenborghs, F. ( 1995; ). Antifungal susceptibility testing of yeasts: Evaluation of technical variables for test automation. Antimicrob Agents Chemother 39, 2051-2060.[CrossRef]
    [Google Scholar]
  33. Okonogi, K., Asia, K., Tsuchimori, N., Perfect, J. R., Gotoh, O. & Yoshida, Y. (1998). A novel mechanism for emerging azole-resistant Candida albicans in AIDS patients. In Program and Abstracts of the Fourth International Symposium on P450 Biodiversity and Biotechnology, Strasbourg, France.
  34. Orozco, A. S., Higginbotham, L. M., Hitchcock, C. A., Parkinson, T., Falconer, D., Ibrahim, A. S., Ghannoum, M. A. & Filler, S. G. ( 1998; ). Mechanism of fluconazole resistance in Candida krusei. Antimicrob Agents Chemother 42, 2645-2649.
    [Google Scholar]
  35. Pearce, M. A. & Howell, S. A. ( 1991; ). Restriction fragment length polymorphism analysis of azole-resistant and azole-susceptible Candida albicans strains. J Clin Microbiol 29, 1364-1367.
    [Google Scholar]
  36. Rex, J. H., Rinaldi, M. G. & Pfaller, M. A. ( 1995; ). Resistance of Candida species to fluconazole. Antimicrob Agents Chemother 39, 1-8.[CrossRef]
    [Google Scholar]
  37. Rodriguez, R., Low, C., Bottema, C. & Parks, L. ( 1985; ). Multiple functions for sterols in Saccharomyces cerevisiae. Biochim Biophys Acta 837, 336-343.[CrossRef]
    [Google Scholar]
  38. Ryder, N. S. & Favre, B. (1997). Resistance to azoles in Candida albicans caused by mutations in the lanosterol 14α-demethylase gene. In Program and Abstracts of the 37th Interscience Conference on Antimicrobial Agents and Chemotherapy, Toronto, Canada, 1997, Abstract C-13, p. 48. Washington, DC: American Society for Microbiology.
  39. Sanglard, D., Kuchler, K., Ischer, F., Pagani, J. L., Monod, M. & Bille, J. ( 1995; ). Mechanisms of resistance to azole antifungal agents in Candida albicans isolates from AIDS patients involve specific multidrug transporters. Antimicrob Agents Chemother 39, 2378-2386.[CrossRef]
    [Google Scholar]
  40. Sanglard, D., Ischer, F., Calabrese, D., de Micheli, M. & Bille, J. ( 1998a; ). Multiple resistance mechanisms to azole antifungals in yeast clinical isolates. Drug Res Updates 1, 255-265.[CrossRef]
    [Google Scholar]
  41. Sanglard, D., Ischer, F., Koymans, L. & Bille, J. ( 1998b; ). Amino acid substitutions in the cytochrome P450 lanosterol 14alpha-demethylase (CYP51) from azole-resistant Candida albicans clinical isolates contributing to the resistance to azole antifungal agents. Antimicrob Agents Chemother 42, 241-253.[CrossRef]
    [Google Scholar]
  42. Smith, K. J., Warnock, D. W., Kennedy, C. T. C., Johnson, E. M., Hopwood, V., Van Cutsem, J. & Vanden Bossche, H. ( 1986; ). Azole resistance in Candida albicans. J Med Vet Mycol 24, 133-144.[CrossRef]
    [Google Scholar]
  43. Supply, P., Wach, A., Thines-Sempoux, D. & Goffeau, A. ( 1993; ). Proliferation of intracellular structures upon overexpression of the PMA2 ATPase in Saccharomyces cerevisiae. J Biol Chem 268, 19744-19752.
    [Google Scholar]
  44. Vanden Bossche, H. ( 1997; ). Mechanisms of antifungal resistance. Rev Iberoam Micol 14, 44-49.
    [Google Scholar]
  45. Vanden Bossche, H. & Koymans, L. ( 1997; ). Cytochromes P450 in fungi. Mycoses 41, 32-38.
    [Google Scholar]
  46. Vanden Bossche, H., Willemsens, G., Cools, W., Cornelissen, F., Lauwers, W. F. & Van Cutsem, J. M. ( 1980; ). In vitro and in vivo effects of the antimycotic drug ketoconazole on sterol synthesis. Antimicrob Agents Chemother 17, 922-928.[CrossRef]
    [Google Scholar]
  47. Vanden Bossche, H., Bellens, D., Cools, W. & 10 other authors ( 1986; ). Cytochrome P-450: target for itraconazole. Drug Dev Res 8, 287–298.[CrossRef]
    [Google Scholar]
  48. Vanden Bossche, H., Marichal, P., Gorrens, J., Bellens, D., Verhoeven, H., Coene, M.-C., Lauwers, W. & Janssen, P. W. J. ( 1987; ). Interaction of azole derivatives with cytochrome P-450 isozymes in yeast, fungi, plants and mammalian cells. Pestic Sci 21, 1–13.[CrossRef]
    [Google Scholar]
  49. Vanden Bossche, H., Marichal, P., Gorrens, J., Bellens, D., Moereels, H. & Janssen, P. A. J. ( 1990; ). Mutation in cytochrome P450-dependent 14α-demethylase results in decreased affinity for azole antifungals. Biochem Soc Trans 18, 56-59.
    [Google Scholar]
  50. Vanden Bossche, H., Marichal, P., Odds, F. C., Le Jeune, L. & Coene, M.-C. ( 1992; ). Characterization of an azole-resistant Candida glabrata isolate. Antimicrob Agents Chemother 36, 2602-2610.[CrossRef]
    [Google Scholar]
  51. Vanden Bossche, H., Marichal, P. & Odds, F. C. ( 1994; ). Molecular mechanisms of drug resistance in fungi. Trends Microbiol 2, 393-400.[CrossRef]
    [Google Scholar]
  52. Vanden Bossche, H., Dromer, F., Improvisi, L., Lozano-Chiu, M., Rex, J. H. & Sanglard, D. ( 1998; ). Antifungal drug resistance in pathogenic fungi. Med Mycol 36, (suppl. 1) 119–128.[CrossRef]
    [Google Scholar]
  53. Vergeres, G., Yen, T. S., Aggeler, J., Lausier, J. & Waskell, L. ( 1993; ). A model system for studying membrane biogenesis. Overexpression of cytochrome b5 in yeast results in marked proliferation of the intracellular membrane. J Cell Sci 106, 249-259.
    [Google Scholar]
  54. Watson, P. F., Rose, M. E., Ellis, S. W., England, H. & Kelly, S. L. ( 1989; ). Defective sterol C5–6 desaturation and azole resistance: a new hypothesis for the mode of action of azole antifungals. Biochem Biophys Res Commun 164, 1170-1175.[CrossRef]
    [Google Scholar]
  55. White, T. C. ( 1997; ). The presence of an R467K amino acid substitution and loss of allelic variation correlate with an azole-resistant lanosterol 14α-demethylase in Candida albicans. Antimicrob Agents Chemother 41, 1488-1494.
    [Google Scholar]
  56. White, T. C., Bowden, R. A. & Marr, K. A. ( 1998; ). Clinical, cellular and molecular factors that contribute to antifungal drug resistance. Clin Microbiol Rev 11, 382-402.
    [Google Scholar]
  57. Yoshida, Y. ( 1988; ). Cytochrome P450 of fungi, primary target for azole antifungal agents. Curr Top Med Mycol 2, 388-418.
    [Google Scholar]
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