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Updated: Jun 14, 2025
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Published on: July 12, 2013
An efficient gene targeting system using Δku80 and functional analysis of Cyp51A in Trichophyton rubrum
Masaki Ishii1, Tsuyoshi Yamada2,3, Shinya Ohata4
1Research Institute of Pharmaceutical Sciences, Faculty of Pharmacy, Musashino University, Tokyo, 202-8585, Japan. m_ishii@musashino-u.ac.jp.
Abstract:
Trichophyton rubrum is one of the most frequently isolated fungi in patients with dermatophytosis. Despite its clinical significance, the molecular mechanisms of drug resistance and pathogenicity of T. rubrum remain to be elucidated because of the lack of genetic tools, such as efficient gene targeting systems. In this study, we generated a T. rubrum strain that lacks the nonhomologous end-joining-related gene ku80 (Δku80) and then developed a highly efficient genetic recombination system with gene targeting efficiency that was 46 times higher than that using the wild-type strain. Cyp51A and Cyp51B are 14-α-lanosterol demethylase isozymes in T. rubrum that promote ergosterol biosynthesis and are the targets of azole antifungal drugs. The expression of cyp51A mRNA was induced by the addition of the azole antifungal drug efinaconazole, whereas no such induction was detected for cyp51B, suggesting that Cyp51A functions as an azole-responsive Cyp51 isozyme. To explore the contribution of Cyp51A to susceptibility to azole drugs, the neomycin phosphotransferase (nptII) gene cassette was inserted into the cyp51A 3'-untranslated region of Δku80 to destabilize the mRNA of cyp51A. In this mutant, the induction of cyp51A mRNA expression by efinaconazole was diminished. The minimum inhibitory concentration for several azole drugs of this strain was reduced, suggesting that dermatophyte Cyp51A contributes to the tolerance for azole drugs. These findings suggest that an efficient gene targeting system using Δku80 in T. rubrum is applicable for analyzing genes encoding drug targets.
Insights
Researchers developed a new gene targeting system for Trichophyton rubrum, a common cause of fungal infections. This system enhances the study of drug resistance, particularly azole antifungal drug tolerance, by targeting the Cyp51A gene.
Area of Science:
- Medical Mycology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Trichophyton rubrum is a prevalent cause of dermatophytosis.
- Understanding T. rubrum's drug resistance and pathogenicity is hindered by a lack of efficient genetic tools.
Purpose of the Study:
- To develop an efficient gene targeting system for T. rubrum.
- To investigate the role of Cyp51A in azole antifungal drug resistance.
Main Methods:
- Generated a ku80-deficient (Δku80) T. rubrum strain for enhanced genetic recombination.
- Developed a gene targeting system with significantly improved efficiency.
- Created a mutant by inserting the nptII gene into the cyp51A 3'-untranslated region to destabilize cyp51A mRNA.
- Assessed the impact of this mutation on efinaconazole-induced cyp51A expression and azole drug susceptibility.
Main Results:
- The Δku80 strain exhibited a 46-fold increase in gene targeting efficiency compared to the wild-type.
- Expression of cyp51A mRNA was induced by efinaconazole, unlike cyp51B.
- The cyp51A mutant showed diminished cyp51A mRNA induction and reduced minimum inhibitory concentrations for azole drugs.
- These findings indicate Cyp51A contributes to azole drug tolerance in T. rubrum.
Conclusions:
- An efficient gene targeting system using Δku80 in T. rubrum is established.
- This system is valuable for analyzing genes involved in drug resistance, such as Cyp51A.
- Cyp51A plays a significant role in dermatophyte tolerance to azole antifungal drugs.
