Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)

A candidate CYP51 gene encoding sterol 14 alpha-demethylase from the fish oomycete pathogen Saprolegnia parasitica (SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in Escherichia coli, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (K-s, 3 to 5 mu M). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (K-d, <= 3 nM) and prothioconazole- desthio the lowest (K-d, similar to 51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC(50)s) of 0.17 to 2.27 mu M using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting S. parasitica, Saprolegnia diclina, and Saprolegnia ferax growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit Saprolegnia growth (MIC100, >256 mu g ml(-1)). The remaining azoles inhibited Saprolegnia growth only at elevated concentrations (MIC100 [the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20 degrees C], 16 to 64 mu g ml(-1)) with the exception of clotrimazole, which was as potent as malachite green (MIC100, similar to 1 mu g ml(-1)). Sterol profiles of azole-treated Saprolegnia species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC50, similar to 1 mu M) and the concentration inhibiting the growth of Saprolegnia species in vitro (MIC100, similar to 1 to 2 mu g ml(-1)) suggest that clotrimazole could be used against Saprolegnia infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.