1887

Abstract

In this study the role of the gene in fungal drug susceptibility was investigated by disrupting and overexpressing the gene in . MIC determination and a spot assay showed that a Δ/Δ null mutant (strain T2bc) was more resistant to the antifungals tested than the wild-type (strain CAI4). Real-time RT-PCR and rhodamine 6G efflux examination showed that 2 did not influence the activity of drug efflux pumps. Sterol analysis with GC/high-resolution MS indicated that the intracellular ergosterol composition of the Δ/Δ mutant was significantly increased. Subsequently, fluorescence polarization measurements also revealed that Top2-deprived cells displayed a decrease in membrane fluidity, resulting in enhanced passive diffusion of the drugs. Quantitative real-time RT-PCR analysis further confirmed that the gene, an essential gene in ergosterol biosynthesis, was upregulated. These results demonstrate a close relationship between the gene and drug susceptibility in .

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2010-07-01
2019-10-24
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References

  1. Akins, R. A. ( 2005; ). An update on antifungal targets and mechanisms of resistance in Candida albicans. Med Mycol 43, 285–318.[CrossRef]
    [Google Scholar]
  2. Albertson, G. D., Niimi, M., Cannon, R. D. & Jenkinson, H. F. ( 1996; ). Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance. Antimicrob Agents Chemother 40, 2835–2841.
    [Google Scholar]
  3. Anderson, J. B. ( 2005; ). Evolution of antifungal-drug resistance: mechanisms and pathogen fitness. Nat Rev Microbiol 3, 547–556.[CrossRef]
    [Google Scholar]
  4. Care, R. S., Trevethick, J., Binley, K. M. & Sudbery, P. E. ( 1999; ). The MET3 promoter: a new tool for Candida albicans molecular genetics. Mol Microbiol 34, 792–798.[CrossRef]
    [Google Scholar]
  5. Cline, S. D. & Hanawalt, P. C. ( 2006; ). Topoisomerase deficiencies subtly enhance global genomic repair of ultraviolet-induced DNA damage in Saccharomyces cerevisiae. DNA Repair (Amst) 5, 611–617.[CrossRef]
    [Google Scholar]
  6. Dassanayake, R. S., Ellepola, A. N., Samaranayake, Y. H. & Samaranayak, L. P. ( 2002; ). Molecular heterogeneity of fluconazole-resistant and -susceptible oral Candida albicans isolates within a single geographic locale. APMIS 110, 315–324.[CrossRef]
    [Google Scholar]
  7. Espinel-Ingroff, A., Rodríguez-Tudela, J. L. & Martínez-Suárez, J. V. ( 1995; ). Comparison of two alternative microdilution procedures with the National Committee for Clinical Laboratory Standards reference macrodilution method M27-P for in vitro testing of fluconazole-resistant and -susceptible isolates of Candida albicans. J Clin Microbiol 33, 3154
    [Google Scholar]
  8. Fonzi, W. A. & Irwin, M. Y. ( 1993; ). Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717–728.
    [Google Scholar]
  9. Ghannoum, M. A. & Rice, L. B. ( 1999; ). Antifungal agents: mode of action, mechanism of resistance and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 12, 501–517.
    [Google Scholar]
  10. Howell, S. A., Mallet, A. I. & Noble, W. C. ( 1990; ). A comparison of the sterol content of multiple isolates of the Candida albicans Darlington strain with other clinically azole sensitive and resistant strains. J Appl Bacteriol 69, 692–696.[CrossRef]
    [Google Scholar]
  11. Keller, B. A., Patel, S. & Fisher, L. M. ( 1997; ). Molecular cloning and expression of the Candida albicans TOP2 gene allows study of fungal DNA topoisomerase II inhibitors in yeast. Biochem J 324, 329–339.
    [Google Scholar]
  12. Kim, R. A. & Wang, J. C. ( 1989; ). Function of DNA topoisomerases as replication swivels in Saccharomyces cerevisiae. J Mol Biol 208, 257–267.[CrossRef]
    [Google Scholar]
  13. Kohli, A., Smriti, Mukhopadhyay, K., Rattan, A. & Prasad, R. ( 2002; ). In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry. Antimicrob Agents Chemother 46, 1046–1052.[CrossRef]
    [Google Scholar]
  14. Loffler, J., Einsele, H., Hebart, H., Schumacher, U., Hrastnik, C. & Daum, G. ( 2000; ). Phospholipid and sterol analysis of plasma membranes of azole-resistant Candida albicans strains. FEMS Microbiol Lett 185, 59–63.[CrossRef]
    [Google Scholar]
  15. Morschhauser, J. ( 2002; ). The genetic basis of fluconazole resistance development in Candida albicans. Biochim Biophys Acta 1587, 240–248.[CrossRef]
    [Google Scholar]
  16. Mukhopadhyay, K., Kohli, A & Prasad, R. ( 2002; ). Drug susceptibilities of yeast cells are affected by membrane lipid composition. Antimicrob Agents Chemother 46, 3695–3705.[CrossRef]
    [Google Scholar]
  17. Nitiss, J. L. ( 1998; ). Investigating the biological functions of DNA topoisomerases in eukaryotic cells. Biochim Biophys Acta 1400, 63–81.[CrossRef]
    [Google Scholar]
  18. Pappas, P. G., Rex, J. H., Lee, J., Hamill, R. J., Larsen, R. A., Powderly, W., Kauffman, C. A., Hyslop, N., Mangino, J. E. & other authors ( 2003; ). A prospective observational study of candidemia: epidemiology, therapy, and influences on mortality in hospitalized adult and pediatric patients. Clin Infect Dis 37, 634–643.[CrossRef]
    [Google Scholar]
  19. Perea, S. & Patterson, T. F. ( 2002; ). Antifungal resistance in pathogenic fungi. Clin Infect Dis 35, 1073–1080.[CrossRef]
    [Google Scholar]
  20. Perea, S., López-Ribot, J. L., Kirkpatrick, W. R., McAtee, R. K., Santillán, R. A., Martínez, M., Calabrese, D., Sanglard, D. & Patterson, T. F. ( 2001; ). Prevalence of molecular mechanisms of resistance to azole antifungal agents in Candida albicans strains displaying high-level fluconazole resistance isolated from human immunodeficiency virus-infected patients. Antimicrob Agents Chemother 45, 2676–2684.[CrossRef]
    [Google Scholar]
  21. Prasad, T., Saini, P., Gaur, N. A., Vishwakarma, R. A., Khan, L. A., Haq, Q. M. & Prasad, R. ( 2005; ). Functional analysis of CaIPT1, a sphingolipid biosynthetic gene involved in multidrug resistance and morphogenesis of Candida albicans. Antimicrob Agents Chemother 49, 3442–3452.[CrossRef]
    [Google Scholar]
  22. Sanglard, D., Ischer, F., Monod, M. & Bille, J. ( 1996; ). Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob Agents Chemother 40, 2300–2305.
    [Google Scholar]
  23. Schvartzman, J. B. & Stasiak, A. ( 2004; ). A topological view of the replicon. EMBO Rep 5, 256–261.[CrossRef]
    [Google Scholar]
  24. Shao, P. L., Huang, L. M. & Hsueh, P. R. ( 2007; ). Recent advances and challenges in the treatment of invasive fungal infections. Int J Antimicrob Agents 30, 487–495.[CrossRef]
    [Google Scholar]
  25. Silver, P. M., Oliver, B. G. & White, T. C. ( 2004; ). Role of Candida albicans transcription factor Upc2p in drug resistance and sterol metabolism. Eukaryot Cell 3, 1391–1397.[CrossRef]
    [Google Scholar]
  26. Umeyama, T., Nagai, Y., Niimi, M. & Uehara, Y. ( 2002; ). Construction of FLAG tagging vectors for Candida albicans. Yeast 19, 611–618.[CrossRef]
    [Google Scholar]
  27. Wang, J. C. ( 2002; ). Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol 3, 430–440.[CrossRef]
    [Google Scholar]
  28. White, T. C., Holleman, S., Dy, F., Mirels, L. F. & Stevens, D. A. ( 2002; ). Resistance mechanisms in clinical isolates of Candida albicans. Antimicrob Agents Chemother 46, 1704–1713.[CrossRef]
    [Google Scholar]
  29. Wisplinghoff, H., Bischoff, T., Tallent, S. M., Seifert, H., Wenzel, R. P. & Edmond, M. B. ( 2004; ). Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis 39, 309–317.[CrossRef]
    [Google Scholar]
  30. Zini, N., Martelli, A. M., Sabatelli, P., Santi, S., Negri, C., Astaldi Ricotti, G. C. & Maraldi, N. M. ( 1992; ). The 180-kDa isoform of topoisomerase II is localized in the nucleolus and belongs to the structural elements of the nucleolar remnant. Exp Cell Res 200, 460–466.[CrossRef]
    [Google Scholar]
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