Summary auto-generated
Researchers developed yeast-enhanced GFP (yEGFP3), a fluorescent protein reporter for studying gene expression in Candida albicans and Saccharomyces cerevisiae. The wild-type green fluorescent protein from jellyfish failed to function in C. albicans, likely due to codon usage incompatibilities. Although changing a problematic CTG codon to TTG did not resolve the issue, creating a completely codon-optimized GFP gene using codons preferred by C. albicans succeeded. The yEGFP3 construct also incorporated two mutations (S65G and S72A) that dramatically increase fluorescence brightness. Cells expressing yEGFP3 displayed strong fluorescence detectable by microscopy and flow cytometry analysis. The reporter functioned under both constitutive and inducible promoters, showing 20-fold induction under maltose control. Notably, C. albicans expressing yEGFP3 were readily visible in infected mouse kidney tissue, demonstrating utility for in vivo imaging. This codon-optimized GFP represents a valuable new tool for fungal molecular biology and should have applications across multiple species.
Key findings
- Wild-type GFP from Aequorea victoria failed to express in C. albicans despite mRNA production, and changing the CTG codon to TTG did not rescue expression, indicating multiple codon usage problems
- A synthetic GFP gene with all 239 amino acids encoded by codons optimal for C. albicans successfully produced functional fluorescent protein with 75-fold brightness enhancement from chromophore mutations
- yEGFP3 showed strong fluorescence in both C. albicans and S. cerevisiae, with 2000-fold greater fluorescence than wild-type GFP in S. cerevisiae and 20-fold induction under maltose promoter control
- C. albicans cells expressing yEGFP3 were readily detected in infected mouse kidney tissue 24 hours post-infection, demonstrating utility as an in vivo reporter
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Abstract
The green fluorescent protein (GFP) of Aequorea victoria has been developed here as a reporter for gene expression and protein localization in Candida albicans. When wild-type (wt) GFP was expressed in C. albicans, it was not possible to detect fluorescence or a translation product for the wt protein. Since this was probably due in part to the presence of the non-canonical CTG serine codon in the Aequorea sequence, this codon was changed to the leucine codon TTG. C. albicans cells expressing this construct contained GFP mRNA but were non-fluorescent and contained no detectable translation product. Hence a codon-optimized GFP gene was constructed in which all of the 239 amino acids are encoded by optimal codons for C. albicans. In this gene were also incorporated two previously identified mutations in the chromophore that increase GFP fluorescence. C. albicans cells expressing this yeast-enhanced GFP gene (yEGFP3) are fluorescent and contain GFP protein. yEGFP3 can be used as a versatile reporter of gene expression in C. albicans and Saccharomyces cerevisiae and the optimized GFP described here should have broad applications in these and other fungal species.