POSTERARO B, MORACE G, SANGLARD D, LA SORDA M, ROMANO L, SANGUINETTI M, FADDA G; Interscience Conference on Antimicrobial Agents and Chemotherapy.

Abstr Intersci Conf Antimicrob Agents Chemother Intersci Conf Antimicrob Agents Chemother. 2000 Sep 17-20; 40: 392.

Inst. Microbiol.-Catholic Univ. Sacred Heart, Rome, Italy

BACKGROUND: Molecular mechanisms operating in azole resistance of Cryptococcus neoformans have not still been elucidated. We hypothesized the involvement of drug efflux based on overexpression of multidrug transporter genes. Differential gene expression between two matched strains, a fluconazole-susceptible strain and its azole-resistant derivative, was investigated by using Suppression Subtractive Hybridization (SSH). Materials andMETHODS: A C. neoformans clinical isolate, CR22, fluconazole-susceptible (MIC 2 microg/ml) and its resistant derivative strain, CR22.17 (MIC 32 microg/ml), obtained by in vitro induction of resistance, were analysed by rhodamine 6G (R6G) accumulation assay. SSH procedure was performed on mRNAs extracted from the C. neoformans strains.RESULTS: The resistant strain CR22.17 accumulated less R6G than the susceptible one, suggesting a possible overexpression of multidrug transporter in CR22.17. To identify these genes, SSH was used to construct a library consisting of CR22.17 cDNAs subtracted with those of CR22. Several differentially expressed clones were obtained from this library and one of them (cH9) showed a significant nucleotide homology to PMR1 gene of Penicillium digitatum and other yeast multidrug transporter genes of the ATP Binding Cassette (ABC) family. The cDNA of cH9 showed in Northern blot analysis a 3-fold increase of expression in CR22.17. Conclusion: Our data demonstrated that 1) SSH could be used to detect and identify differentially expressed genes in C. neoformans; 2) as observed in other clinically important yeasts, the resistance of C. neoformans to fluconazole could be related to the overexpression of multidrug transporter genes of the ABC family.KEYWORDS: Azole resistance; Cryptococcus neoformans; SSH

Publication Types:

• Meeting Abstracts

Keywords:

• Antigens, Fungal
• Azoles
• Blotting, Northern
• Case-Control Studies
• Cryptococcus neoformans
• Fluconazole
• Gene Library
• Hybridization, Genetic
• In Vitro
• Membrane Transport Proteins
• Microbial Sensitivity Tests
• Nucleic Acid Hybridization
• Saccharomyces cerevisiae
• genetics
• immunology
• methods

Other ID:

• GWAIDS0010604

UI: 102248102

From Meeting Abstracts