Adaptation of the tetracycline-repressible system for modulating the expression of essential genes in <i>Cryptococcus neoformans</i>

The opportunistic human fungal pathogen <i>Cryptococcus neoformans</i> has an enormous impact on human health as the causative agent of cryptococcal meningitis, and there is a dire need to expand our current antifungal arsenal. Essential gene products often serve as ideal targets for antimicrobials, and identifying and characterizing essential genes in a pathogen of interest is critical for drug development. Unfortunately, characterization of essential genes in <i>C. neoformans</i> is limited due to its haploid nature and lack of genetic tools for generating effective conditional-expression mutants. To date, the copper-repressible promoter <i>pCTR4</i> is the most widely used system to regulate essential gene expression; however, its expression is leaky and copper has pleiotropic effects. In diverse fungal species, including <i>Saccharomyces cerevisiae</i>, <i>Candida albicans</i>, and <i>Candida auris</i>, the tetracycline-repressible promoter system is a powerful tool to regulate gene expression; however, it has yet to be adapted for <i>C. neoformans</i>. In this study, we successfully implemented the tetracycline-repressible system in <i>C. neoformans</i> to regulate the expression of the essential genes <i>HSP90</i> and <i>FKS1</i>. Supplementation of cultures with the tetracycline analog doxycycline efficiently depleted <i>HSP90</i> at both transcript and protein levels and inhibited <i>C. neoformans</i> growth and viability. Similarly, the depletion of <i>FKS1</i> with doxycycline enhanced sensitivity of the strain to the echinocandin caspofungin, an antifungal that targets the glucan synthase but is generally ineffective against <i>C. neoformans</i>. Thus, this work unveils a novel approach to generate conditional-expression mutants in <i>C. neoformans,</i> providing unprecedented potential to systematically study essential gene function in this important human fungal pathogen.IMPORTANCEInvasive fungal infections cause millions of deaths annually, while the number of antifungals available to combat these pathogens is limited to only three classes: polyenes, azoles, and echinocandins. The largest source of novel antifungal drug targets are essential gene products, which are required for cellular viability. However, tools to identify and characterize essential genes in <i>C. neoformans</i> are extremely limited. Here, we adapted the tetracycline-repressible promoter system, that has been widely used in other organisms, to study essential gene function in <i>C. neoformans</i>. By placing this regulatable promoter upstream of the essential genes <i>HSP90</i> and <i>FKS1</i>, we confirmed that the growth of the strains in the presence of the tetracycline analog doxycycline results in the depletion of essential gene expression. This approach provides a significant advance for the systematic study of essential genes in <i>C. neoformans</i>.