A heterologous expression system allows rapid testing and discovery of resistance-conferring mutations from multiple pathogenic fungi
- 1Université Laval
Abstract
To face the fungal infections that affect millions of people every year, a better understanding of resistance-conferring mutations is necessary. One obstacle is the considerable time and effort necessary to construct point mutations in pathogenic species, which is required to validate their role in resistance. To overcome this problem, we have constructed a series of heterologous expression plasmids that allow expression of the ERG3, ERG6 and ERG11 orthologs from Candida albicans, Candidozyma auris, Nakaseomyces glabratus, Candida parapsilosis, Candida tropicalis, and the CYP51A and CYP51B genes of Aspergillus fumigatus. All orthologs are codon-optimized and synthesized for expression in the budding yeast Saccharomyces cerevisiae. We also constructed host strains in which the native ERG genes are either deleted or under the control of a repressible promoter. We showed that all orthologs complement the function of the native genes when expressed from the S. cerevisiae native promoters. We recreated previously reported resistance-conferring mutations, and showed that expression in S. cerevisiae replicates known resistance phenotypes. We then used the system to test corresponding mutations across orthologs, and showed that most resistance phenotypes are conserved. The heterologous expression system allows rapid construction of mutants, by taking advantage of the abundant genetic tools available for S. cerevisiae. Screening of mutants can be done within a few experimental steps, greatly increasing the throughput compared to the introduction of mutations in pathogenic fungi. As a proof-of-concept, we used the system to discover new resistance-conferring mutations, using random mutagenesis. All plasmids are made publicly available.
Related
- Map-based cloning and functional validation of Xa50, a wall-associated kinase gene conferring broad-spectrum bacterial blight resistance in rice
- Heterologous Expression of Saussurea involucrata SiPIP1;5b Enhances Freezing Tolerance in Arabidopsis and Chilling Tolerance in Tomato
- Heterologous expression of the membrane protein OmpA from bovine Pasteurella multocida and establishment of an indirect ELISA for antibody detection
- Relative inhibitory activities of taniborbactam and xeruborbactam against KPC variants conferring reduced susceptibility or resistance to ceftazidime/avibactam, cefiderocol, and cefepime/taniborbactam
- Cloning and heterologous expression of recombinant microcin S in Escherichia coli
BibTeX
@misc{jordan2026heterologous,
title = {{A heterologous expression system allows rapid testing and discovery of resistance-conferring mutations from multiple pathogenic fungi}},
author = {D. F. Jordan and M. Roland-Caveriviere and C. Bedard and A. K. Dube and I. Gagnon-Arsenault and C. R. Landry},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.18.751866},
url = {https://www.biorxiv.org/content/10.64898/2026.09.18.751866v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A heterologous expression system allows rapid testing and discovery of resistance-conferring mutations from multiple pathogenic fungi
AU - D. F. Jordan
AU - M. Roland-Caveriviere
AU - C. Bedard
AU - A. K. Dube
AU - I. Gagnon-Arsenault
AU - C. R. Landry
PY - 2026
DA - 2026/09/18
PB - bioRxiv
DO - 10.64898/2026.09.18.751866
UR - https://www.biorxiv.org/content/10.64898/2026.09.18.751866v1
AB - To face the fungal infections that affect millions of people every year, a better understanding of resistance-conferring mutations is necessary. One obstacle is the considerable time and effort necessary to construct point mutations in pathogenic species, which is required to validate their role in resistance. To overcome this problem, we have constructed a series of heterologous expression plasmids that allow expression of the ERG3, ERG6 and ERG11 orthologs from Candida albicans, Candidozyma auris, Nakaseomyces glabratus, Candida parapsilosis, Candida tropicalis, and the CYP51A and CYP51B genes of Aspergillus fumigatus. All orthologs are codon-optimized and synthesized for expression in the budding yeast Saccharomyces cerevisiae. We also constructed host strains in which the native ERG genes are either deleted or under the control of a repressible promoter. We showed that all orthologs complement the function of the native genes when expressed from the S. cerevisiae native promoters. We recreated previously reported resistance-conferring mutations, and showed that expression in S. cerevisiae replicates known resistance phenotypes. We then used the system to test corresponding mutations across orthologs, and showed that most resistance phenotypes are conserved. The heterologous expression system allows rapid construction of mutants, by taking advantage of the abundant genetic tools available for S. cerevisiae. Screening of mutants can be done within a few experimental steps, greatly increasing the throughput compared to the introduction of mutations in pathogenic fungi. As a proof-of-concept, we used the system to discover new resistance-conferring mutations, using random mutagenesis. All plasmids are made publicly available.
ER -