Enhanced extracellular production of aspartyl proteinase, a virulence factor, by Candida albicans isolates following growth in subinhibitory concentrations of fluconazole

We examined the production of secreted aspartyl proteinase (Sap), a putativ e virulence factor of Candida albicans, by a series of 17 isolates represen ting a single strain obtained from the oral cavity of an AIDS patient befor e and after the development of clinical and in vitro resistance to fluconaz ole. Isolates were grown in Sap-inducing yeast carbon base-bovine serum alb umin medium containing 0, 0.25, 0.5, or 1 MIC of fluconazole, and cultures were sampled daily for 14 days to determine extracellular Sap activity by e nzymatic degradation of bovine serum albumin. Extracellular Sap activity wa s significantly decreased in a dose-dependent manner for the most fluconazo le-susceptible isolate (MIC, 1.0 mu g/ml) and significantly increased in a dose-dependent manner for the most fluconazole-resistant isolate (MIC, >64 mu g/ml). Enhanced extracellular Sap production could not be attributed to cell death or nonspecific release of Sap, because there was no reduction in the number of CFU and no significant release of enolase, a constitutive en zyme of the glycolytic pathway. Conversely, intracellular Sap concentration s were significantly increased in a dose-dependent manner in the most fluco nazole-susceptible isolate and decreased in the most fluconazole-resistant isolate. Enhanced Sap production correlated with the overexpression of a ge ne encoding a multidrug resistance (MDR1) efflux pump occurring in these is olates. These data indicate that exposure to subinhibitory concentrations o f fluconazole can result in enhanced extracellular production of Sap by iso lates with the capacity to overexpress MDR1 and imply that patients infecte d with these isolates and subsequently treated with suboptimal doses of flu conazole may experience enhanced C. albicans virulence in vivo.