Systematic Identification of Essential Transcription Factors and Their Roles in Cryptococcus neoformans

Systematic Identification of Essential Transcription Factors and Their Roles in Cryptococcus neoformans

Seung-Heon Lee1, Yu-Byeong Jang1, Jin-Tae Choi1, Kyung-Tae Lee1, Alexander Idnurm2, Doyeon Won1, Kyung-Tae Lee3*, and Yong-Sun Bahn1*

1Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea

2School of BioSciences, The University of Melbourne, Parkville Campus, Parkville, VIC, 3010, Australia

3Korea Zoonosis Research Institute, Jeonbuk National University, Jeollabuk-do, Korea

Cryptococcus neoformans causes cryptococcosis, which is one of the leading causes of death among HIV patients. Current antifungal treatments are limited, therefore, developing antifungal drugs is crucial. In this regard, essential proteins can be notable as they are required for growth and therefore considered as targets. In this study, we aim to identify essential TFs of C. neoformans and characterize their roles. Our previous research revealed that 23 genes could be essential TFs in the fungus because they cannot be deleted. We constructed conditional expression strains for the 17 TFs by replacing their promoters with the copper-regulated CTR4 promoter. Under repressive condition, conditional expression strains of 10 TFs showed defects, implying that these TFs are required for growth. To verify their essentiality for the viability of the fungus, we constructed heterozygous mutants with diploid strain using the drug resistance marker and performed spore analysis. After harvesting spores from the heterozygous mutant, we analyze the genotypes of progenies and identified 16 TFs essential TFs of C. neoformans. To investigate the role of the essential TFs, we constructed constitutive overexpression strains and observed their phenotypic traits under various stress-containing conditions. Particularly, three TFs exhibited high divergence from other eukaryotes, and one of them showed significant involvement in antifungal drug susceptibility, stress responses, and the pathogenicity of C. neoformans. Our findings reveal the complicated signaling networks of the TF and indicate multiple essential TFs, giving the targets for cryptococcosis therapy.

*Correspondence to Yong-Sun Bahn (ysbahn@yonsei.ac.kr) and Kyung-Tae Lee (lee.kt@jbnu.ac.kr)