Characterization and functional analysis of sterol 14 a-demethylase genes (McCyp51) in Mucor circinelloides

ObjectiveSeveral members of Mucoromycotina are considered as opportunistic human pathogens, which can cause frequently fatal systemic infections in immunocompromised patients. In the recent years, the number of patients with mucormycosis has significantly increased worldwide. In clinics, triazoles are frequently used to treat invasive mycoses. These compounds inhibit the activity of the enzyme, sterol 14 u03b1-demethylase, which participated in the ergosterol biosynthesis. However, Mucoral fungi are generally resistant to the majority of the presently available azoles. The aim of the present study has been the functional characterization of Cyp51 genes encoding sterol 14 u03b1-demethylases in the model organism Mucor circinelloides. MethodsRelative transcript levels of McCyp51 genes were measured under different growth conditions (i.e. different cultivation temperatures and azole treatments) using the qRT-PCR method. A CRISPR-Cas9 system was used to disrupt the two McCyp51 genes of M. circinelloides. The isolated mutants were characterized and their ergosterol content and sensitivity to various azoles were measured.ResultsThe genes McCyp51a and McCyp51b showed a constitutively high and a moderate transcription level, respectively, under the tested conditions. Fluconazole had no effect on the transcription level of McCyp51 genes while itraconazole and ketoconazole treatment increased the relative transcript level of both genes. Growth rate of the disrupted mutants was similar to the wild type, but their ergosterol content significantly decreased. ConclusionOur result suggested that both McCyp51 genes play role in the ergosterol biosynthesis of M. circinelloides. Azoles have different effect on the transcription of these genes because ketoconazole and itraconazole treatment resulted higher relative transcript level in McCyp51b gene than in McCyp51a gene.The study was supported by the grants LP2016-8/2016 and GINOP-2.3.2-15-2016-00035.