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Fungal azole resistance and the role of cytochrome P450. / Nadja Rodrigues de Melo

Swansea University Author: Nadja Rodrigues de Melo

Abstract

The incidence of fungal infections and particularly infections caused by non- albicans Candida species has increased considerably in the past two decades. The azole antifungal drugs are central to the management of fungal infections and like the use of other antibiotics drug resistance has emerged....

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Published: 2007
Institution: Swansea University
Degree level: Doctoral
Degree name: Ph.D
URI: https://cronfa.swan.ac.uk/Record/cronfa42593
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Abstract: The incidence of fungal infections and particularly infections caused by non- albicans Candida species has increased considerably in the past two decades. The azole antifungal drugs are central to the management of fungal infections and like the use of other antibiotics drug resistance has emerged. Factors associated with antifungal resistance were investigated including azole susceptibility, drug accumulation and membrane sterol composition. Intracellular drug accumulation in Candida strains and species was examined and a clear correlation to microbial sensitivity was not observed. Also the membrane sterol profiles of some resistant strains were also altered with an increase in intermediates sterols, but no clear evidence of new mutants of sterol biosynthetic enzymes was uncovered. Some clinical strains were found to be resistant to specific azoles where other strains were resistant to a broad spectrum of azoles. The fungal cell wall and its biosynthesis are essential for cell viability and as such are potential targets for novel antifungal agents. CYP56 in S. cerevisiae encodes a cytochrome P450 enzyme that catalyses the biosynthesis of dityrosine during spore wall biogenesis. The role of the C. albicans CYP56 gene in growth and drug susceptibility was investigated. The full-length CYP56 DNA sequence was determined and the gene disrupted using the SAT 1-flipper technique. The mutant exhibited increased susceptibility to caspofungin and nikkomycin compared to the wild-type parental strain, whereas susceptibility to azoles and other metabolic inhibitors tested was unaffected. Phenotypically, the wild-type and mutant strains were morphologically similar on rich media, however in minimal media the cyp56tDelta mutant exhibits hyphal growth in contrast to the yeast only form of the parental wild-type strain. Also the mutant failed to form chlamydospores. Over- expression of CYP56 protein in E. coli produced a membrane-associated cytochrome P450 enzyme that catalysed the conversion of N-formyl tyrosine to N-formyl dityrosine when reconstituted with a fungal cytochrome P450 reductase in liposomes. The present study aimed to further the understanding of the biological, genetic and physiological aspects azole effect on clinical strains, on fungal CYPs (including CYP56) and for sterol biology associated with fungi, particularly in Candida species.
Keywords: Medicine.;Pharmacology.
College: Swansea University Medical School