Table 1.
Azole antifungal susceptibility and amino acid substitutions in CaErg11p for the clinical isolates of C. albicans.
Figure 1.
Expression of recombinant Pichia pastoris transformants.
(A): SDS-polyacrylamide gel electrophoresis analysis. P. pastoris CaErg11p transformants were induced for protein expression on BMMY medium for 72 h as described in Material and Methods. Soluble cytosolic proteins were loaded into a 10% SDS-PAGE gel and stained with Coomassie brilliant blue. (B): Western blot analysis of control and mutated CaErg11p proteins produced in P. pastoris. Cytosolic proteins were transferred to PVDF membranes and incubated with a 1∶100 dilution of a polyclonal rabbit anti-yeast Erg11p and a 1∶2000 dilution of goat-anti rabbit-HRP. Signals were visualized using supersignal west pico substrate detection reagent.
Figure 2.
In vitro antifungal activity of FLC against P. pastoris transformants.
(A): P. pastoris cells transformed with WT (□), K143R (▪), Y447H (○) and V456I (•) mutants of CaErg11p were tested according to the CLSI method with some modifications. MIC values were determined as the lowest antifungal concentration giving a 50% or less reduction in the optical density at 450 nm compared to the OD of the corresponding drug-free incubation medium. (B): Susceptibilities of CaErg11p P. pastoris transformants to azole fluconazole using spot assay. Serial dilutions of each control and mutant clones were spotted onto BMMY agar plates containing different concentrations of fluconazole and incubated for 72 h at 30°C. Untreated conditions (a), 2 µg/ml FLC (b), 4 µg/ml FLC (c) and 8 µg/ml FLC (d).
Figure 3.
CaErg11p activity of P. pastoris transformants in the presence of FLC. P. pastoris CaErg11p methanol-induced transformants were treated with FLC in BMMY medium for 24 h at 30°C.
Non-saponifiable lipids (sterols) were extracted as described in Material and Methods. Sterol identification was done in reference to the relative retention times and mass spectra previously reported [54], [55]. Activity results were expressed as the ratio of ergosterol biosynthesis compared to the lanosterol accumulation (E/L). (A): Untreated P. pastoris clones, (B): 4 µg/ml FLC and (C): 8 µg/ml FLC (n = 4).
Figure 4.
Localization of the major long insertion sequence of CaErg11p and azole-resistance substitutions mapping on CaErg11p structures obtained by homology modeling.
(A): ModWeb model with human CYP51 ketoconazole liganded (3ld6). (B): YASARA model with human CYP51 liganded with econazole (3jus) with protoporphyrin IX and econazole represented as a stick respectively in orange and yellow. (C): SwissModel with M. tuberculosis 4-phenylimidazole liganded structure as template (1e9x). (D): ModWeb model zebrafish prostacyclin synthase CYP450 8a1 free (3b98). All the models are aligned, based on their secondary structure with the N-terminal part of the beta-strand rich domain left and the alpha-helices rich domain right. The secondary structure of the models are color-coded according to their type, red: beta-stands, blue = alpha-helices, green = turn, cyan = coil, magenta = the long fungi specific amino acid sequence insertion. Amino acid positions that have been proven to be responsible for azole-resistance are indicated with the amino acid name of the WT CaCYP51 with yellow for substrate binding site hot-spots and magenta in the insertion fungi specific sequence. N- and C-terminal are labeled by single letters N and C.
Table 2.
Oligonucleotide primers used in this study.