Respiratory-deficient mutants of Candida glabrata, which present a reduced susceptibility to azole antifungals, are easily induced in vitro by exposure to azole drugs or ethidium bromide and may be selected in vivo in patients undergoing fluconazole prophylaxis or therapy. The present study was therefore conducted in order to define the pathogenicity of these petite mutants. To do this, two wild-type isolates of C. glabrata were compared with their respective fluconazole or ethidium bromide-induced petite mutants, regarding the carbohydrate and protein composition of the cell wall, as well as their surface physical properties and expression of CgEPA1 gene encoding an adhesin, but also their adherence capabilities and their virulence in mice. Thus, flow cytometric analysis of cell wall carbohydrates using several fluorescent lectins showed an increased binding of mutant cells to concanavalin A compared with their parent isolates, suggesting a greater availability or an increased amount of glucose-mannose residues at the cell surface in petite mutants. Likewise, quantitative differences were shown by SDS-PAGE in the surface proteins of blastoconidia between parent and mutant isolates. Regarding the surface physical properties, no significant differences were seen in the electrophoretic mobility determined by microelectrophoresis, but two-phase partitioning using hexadecane as the organic phase revealed a lower hydrophobicity for all petite mutants compared with parent isolates. Moreover, mutant cells exhibited an important overexpression of the gene CgEPA1 as revealed by real-time reverse transcription-PCR compared with their parent isolates, but the adherence capacities to Caco-2 cells, a human enterocyte cell line, were not significantly different. Finally, as suggested by their slower growth, petite mutants were less virulent than parent isolates in an experimental model of systemic infection using immunocompetent mice. Together these results suggest that one can disregard C. glabrata petite mutants clinically. However, due to their markedly decreased virulence, these mutants may constitute valuable tools for the characterization of virulence factors of this yeast, and proteomic studies of the changes associated with petite mutations are now in progress.
Full conference title:
- RICAI 24th (2004)