ABSTRACT Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans . Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Dysfunction of ERG251 resulted in transcriptional upregulation of the alternate sterol biosynthesis pathway and ZRT2 , a Zinc transporter. Notably, we determined that overexpression of ZRT2 is sufficient to increase azole tolerance in C. albicans . Our combined transcriptional and phenotypic analyses revealed the pleiotropic effects of ERG251 on stress responses including cell wall, osmotic and oxidative stress. Interestingly, while loss of either allele of ERG251 resulted in similar antifungal drug responses, we observed functional divergence in filamentation regulation between the two alleles of ERG251 ( ERG251-A and ERG251-B ) with ERG251-A exhibiting a dominant role in the SC5314 genetic background. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study provides extensive genetic, transcriptional and phenotypic analysis for the effects of ERG251 on drug susceptibility, fitness, filamentation and stress responses. AUTHOR SUMMARY Invasive infections caused by the fungal pathogen Candida albicans have high mortality rates (20-60%), even with antifungal drug treatment. Numerous mechanisms contributing to drug resistance have been characterized, but treatment failure remains a problem indicating that there are many facets that are not yet understood. The azole class of antifungals targets production of ergosterol, an essential component of fungal cell membranes. Here, we provide insights into the contributions of ERG251, a component of the ergosterol biosynthesis pathway, to increased growth in azoles as well as broad scale effects on stress responses filamentation and pathogenicity. One of the most striking results from our study is that even a single nucleotide change in one allele of ERG251 in diploid C. albicans can lead to azole tolerance. Tolerance, a distinct phenotype from resistance, is the ability of fungal cells to grow above the minimum inhibitory concentration in a drug concentration-independent manner. Tolerance frequently goes undetected in the clinic because it is not observable in standard assays. Strikingly, azole tolerance strains lacking one allele of ERG251 remained virulent in a mouse model of infection highlighting the potential for mutations in ERG251 to arise and contribute to treatment failure in patients.

Erg251 has complex and pleiotropic effects on azole susceptibility, filamentation, and stress response phenotypes