Contribution of mutations in the cytochrome P450 14α-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans

Abstract

The cytochrome P450 14α-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals. Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism. Nine Candida albicans strains were used in this study. The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined. The ERG11 base sequences of the other two strains have been published previously. In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G465S). In addition, 16 silent mutations were found. Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole. Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates. A three-dimensional model of C. albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences. These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions. Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance. In the Cterminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis.

Genetic and phenotypic characterization of Candida albicans strains isolated from infectious disease patients in Shanghai

“…Four ERG11 sequences containing novel V332L mutations (KM052652, KM052653, KM052654 and KM052655) were submitted to GenBank. In agreement with previous studies, the majority of missense mutations were located in three hot spot regions: amino acids 105-165, 266-287 and 405-488 (Marichal et al, 1999). The combination of A114S and Y257H mutations in the N-terminal regions was detected in a total of seven strains, two of which were itraconazole resistant, four were itraconazole S-DD, one was pan-azole-resistant and none was present in any of the azolesensitive strains.…”

“…Their contribution to fluconazole resistance is still uncertain and requires further study. Single mutations of D116E, K128T, E266D, V437I or V488I may not contribute to azole resistance because they were found in both azole-resistant and azole-susceptible strains (Marichal et al, 1999;Morio et al, 2010;Perea et al, 2001;Sanglard et al, 1998;Ying et al, 2013). The novel V332L mutation was detected in nine strains, three of which were azole-resistant (33.33 %), two were S-DD (22.22 %) and four were azole-susceptible (44.44 %); therefore we suggest that V332L mutation probably does not contribute to azole resistance.…”

“…Y132H, the most well-characterized mutation associated with azole resistance, is situated in the B-B9 helix cluster, a region believed to play a role in the entry of the substrate into the pocket (Kelly et al, 1999;Marichal et al, 1999;Park et al, 2011). The A114S substitution is near the substrate channel and Y257H, which is located in the G helix, and may be primarily related to azole resistance (Xiang et al, 2013).…”

Endosomal Trafficking Defects Can Induce Calcium-Dependent Azole Tolerance in Candida albicans

“…Several specific mechanisms that contribute to the development of azole resistance in the prevalent human fungal pathogen Candida albicans have been described. These mechanisms include increased expression of the target enzyme, point mutations that alter the target enzyme's affinity for the azoles (4,5), and increased expression of efflux pumps, such as Cdr1p and Mdr1p, that export the azoles out of the fungal cell (6)(7)(8). However, the azole resistance of many fungal isolates is not fully accounted for by these well-characterized mechanisms.…”

X-ray Structure of 4,4′-Dihydroxybenzophenone Mimicking Sterol Substrate in the Active Site of Sterol 14α-Demethylase (CYP51)

“…These highly dynamic regions are enclosed in mutational hot spots in azole-resistant clinical isolates of C. albicans (13,36,37), indicating their either direct or indirect (via protein dynamics) involvement in the substrate or inhibitor binding. A critical mutation hot spot, the BC-loop (36), could not be fully defined in previously reported structures due to insufficient electron density in this region.…”