Azole antifungals induce up-regulation of SAP4, SAP5 and SAP6 secreted proteinase genes in filamentous Candida albicans cells in vitro and in vivo

Objectives: Expression of fungal virulence factors can be influenced by exposure to antifungal agents. To test this hypothesis, we determined the effects of subinhibitory concentrations of three antifungal agents on expression of three secreted proteinase genes associated with virulence in filamentous forms of Candida albicans. Methods: GFP-SAP promoter constructs and fluorescence measurement, transcript profiling and RT-PCR in vitro and in an animal model of disseminated Candida infection. Results: Exposure of C. albicans to subinhibitory concentrations of fluconazole in RPMI 1640 in the absence of serum led to up-regulation of the virulence-associated genes SAP4, SAP5 and SAP6 in hyphae and long pseudohyphae. Measurements with green fluorescent protein (GFP)-tagged promoters showed that the fluorescence of SAP4 and SAP6 under these conditions was strongest in the apical tip compartments of these filamentous cells and declined in compartments more proximal to the parent yeast cell. By contrast, SAP5-GFP fluorescence was expressed at similar levels in all cell compartments. Exposure to fluconazole led to significant increases in GFP-SAP4 and -SAP6 fluorescence in the filaments; itraconazole exposure also significantly increased GFP-SAP4 fluorescence, whereas flucytosine had no effect on any of the constructs. In experimentally infected animals, fluorescence of the GFP-SAP promoter fungal cells in kidney tissues was greater than that was seen in vitro for all four SAP constructs: treatment of animals with fluconazole did not significantly increase SAP promoter expression as measured by GFP fluorescence. Conclusions: Azole antifungal agents stimulated up-regulation of SAP4 and SAP6 genes in filamentous C. albicans cells in vitro and may therefore influence virulence as well as growth of the fungus. However, such effects appear to be transient in vivo.