A Chimeric ORF Fusion Phenotypic Reporter for Cryptococcus neoformans

TL;DR

This paper introduces a new genetic reporter system for studying gene function in the fungus Cryptococcus neoformans.

Contribution

A novel chimeric ORF fusion reporter system is developed, combining fluorophores and counter-selectable markers for Cryptococcus neoformans.

Findings

Multiple fusion reporter variants were created and evaluated for expression differences.

Promoters from TEF1 and GAL7 genes revealed novel expression patterns in C. neoformans.

The fusion ORF reporter is smaller and more versatile than traditional systems like lacZ.

Abstract

The plethora of genome sequences produced in the postgenomic age has not resolved many of our most pressing biological questions. Correlating gene expression with an interrogatable and easily observable characteristic such as the surrogate phenotype conferred by a reporter gene is a valuable approach to gaining insight into gene function. Many reporters including lacZ, amdS, and the fluorescent proteins mRuby3 and mNeonGreen have been used across all manners of organisms. Described here is an investigation into the creation of a robust, synthetic, fusion reporter system for Cryptococcus neoformans that combines some of the most useful fluorophores available in this system with the versatility of the counter-selectable nature of amdS. The reporters generated include multiple composition and orientation variants, all of which were investigated for differences in expression. Evaluation of known promoters from the TEF1 and GAL7 genes was undertaken, elucidating novel expression tendencies of these biologically relevant C. neoformans regulators of transcription. Smaller than lacZ but providing multiple useful surrogate phenotypes for interrogation, the fusion ORF serves as a superior whole-cell assay compared to traditional systems. Ultimately, the work described here bolsters the array of relevant genetic tools that may be employed in furthering manipulation and understanding of the WHO fungal priority group pathogen C. neoformans.

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