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Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans
H Nakayama1, T Mio, S Nagahashi
1Department of Mycology, Nippon Roche K. K. Research Center, 200 Kajiwara, Kamakura, Kanagawa 247-8530, Japan. hironobu.nakayama@roche.com
Abstract:
Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.
Insights
Researchers developed a novel tetracycline-regulatable (TR) promoter system for Candida albicans. This system effectively controls gene expression, aiding in the study of fungal gene function both in vitro and in vivo.
Area of Science:
- Mycology
- Molecular Biology
- Biotechnology
Background:
- Candida albicans is a major human fungal pathogen.
- Limited tools exist for studying gene function in C. albicans.
- A need exists for controllable gene expression systems in this organism.
Purpose of the Study:
- To develop a tetracycline-regulatable (TR) gene expression system for Candida albicans.
- To validate the system's efficacy in controlling gene expression and impacting fungal growth and virulence.
Main Methods:
- Constructed a TR promoter system in C. albicans.
- Assessed gene expression using Renilla reniformis luciferase (RLUC1) reporter.
- Evaluated gene function by replacing native promoters with TR promoters.
- Tested the system's in vitro and in vivo efficacy in a mouse infection model.
Main Results:
- Doxycycline (DOX) treatment resulted in near-complete inhibition of RLUC1 expression.
- Gene expression was induced 400- to 1,000-fold in the absence of DOX.
- TR promoter-driven gene disruption caused DOX-dependent growth defects.
- In vivo, DOX administration protected infected mice and reduced fungal burden.
Conclusions:
- The developed TR promoter system offers robust, doxycycline-inducible control of gene expression in C. albicans.
- This system is effective for studying essential gene functions and virulence in both laboratory and host settings.
- The system provides a valuable tool for advancing research on this significant fungal pathogen.