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

Abstract

ABSTRACTErgosterol 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 geneERG251is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds ofCandida albicans. Heterozygous point mutations led to single allele dysfunction ofERG251and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance inC. albicans.Importantly, single allele dysfunction ofERG251in combination with recurrent chromosome aneuploidies resulted inbona fideazole resistance. Homozygous deletions ofERG251caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Dysfunction ofERG251resulted in transcriptional upregulation of the alternate sterol biosynthesis pathway andZRT2, a Zinc transporter. Notably, we determined that overexpression ofZRT2is sufficient to increase azole tolerance inC. albicans. Our combined transcriptional and phenotypic analyses revealed the pleiotropic effects ofERG251on stress responses including cell wall, osmotic and oxidative stress. Interestingly, while loss of either allele ofERG251resulted in similar antifungal drug responses, we observed functional divergence in filamentation regulation between the two alleles ofERG251(ERG251-AandERG251-B) withERG251-Aexhibiting a dominant role in the SC5314 genetic background. Finally, in a murine model of systemic infection, homozygous deletion ofERG251resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study provides extensive genetic, transcriptional and phenotypic analysis for the effects ofERG251on drug susceptibility, fitness, filamentation and stress responses.AUTHOR SUMMARYInvasive infections caused by the fungal pathogenCandida albicanshave 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 ofERG251,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 ofERG251in diploidC. albicanscan 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 ofERG251remained virulent in a mouse model of infection highlighting the potential for mutations inERG251to arise and contribute to treatment failure in patients.