Adaptation of the tetracycline-repressible system for modulating the expression of essential genes in Cryptococcus neoformans

Abstract

The opportunistic human fungal pathogen Cryptococcus neoformans has an enormous impact on human health as the causative agent of cryptococcal meningitis, an AIDS-defining illness that results in significant human mortality. To combat C. neoformans infections, there is a dire need to expand our current antifungal arsenal. Essential gene products often serve as ideal targets for antimicrobials, and thus identifying and characterizing essential genes in a pathogen of interest is critical for drug development. Unfortunately, characterization of essential genes in C. neoformans is limited due to its haploid nature and lack of genetic tools for generating effective conditional-expression mutants. To date, the copper-repressible promoter pCTR4 is the mostly widely used system to regulate essential gene expression, however, its expression is leaky and copper has pleiotropic effects. In diverse fungal species, including Saccharomyces cerevisiae, Candida albicans, and Candida auris, the tetracycline-repressible promoter system is a powerful tool to regulate gene expression, however, it has yet to be adapted for C. neoformans. In this study, we successfully implemented the tetracycline-repressible system in C. neoformans to regulate the expression of the essential gene HSP90. Supplementation of cultures with the tetracycline analog doxycycline efficiently depleted HSP90 at both transcript and protein levels and inhibited C. neoformans growth and viability. Thus, this work unveils a novel approach to generate conditional-expression mutants in C. neoformans, providing unprecedented potential to systematically study essential gene function in this important human fungal pathogen.