Upc2-mediated mechanisms of azole resistance in Candida auris

ABSTRACT Candida auris is an emerging yeast pathogen of major concern because of its ability to cause hospital outbreaks of invasive candidiasis and to develop resistance to antifungal drugs. A majority of C. auris isolates are resistant to fluconazole, an azole drug used for the treatment of invasive candidiasis. Mechanisms of azole resistance are multiple, including mutations in the target gene ERG11 and activation of the transcription factors Tac1b and Mrr1, which control the drug transporters Cdr1 and Mdr1, respectively. We investigated the role of the transcription factor Upc2, which is known to regulate the ergosterol biosynthesis pathway and azole resistance in other Candida spp. Genetic deletion and hyperactivation of Upc2 by epitope tagging in C. auris resulted in drastic increases and decreases in susceptibility to azoles, respectively. This effect was conserved in strains with genetic hyperactivation of Tac1b or Mrr1. Reverse transcription PCR analyses showed that Upc2 regulates ERG11 expression and also activates the Mrr1/Mdr1 pathway. We showed that upregulation of MDR1 by Upc2 could occur independently from Mrr1. The impact of UPC2 deletion on MDR1 expression and azole susceptibility in a hyperactive Mrr1 background was stronger than that of MRR1 deletion in a hyperactive Upc2 background. While Upc2 hyperactivation resulted in a significant increase in the expression of TAC1b, CDR1 expression remained unchanged. Taken together, our results showed that Upc2 is crucial for azole resistance in C. auris, via regulation of the ergosterol biosynthesis pathway and activation of the Mrr1/Mdr1 pathway. Notably, Upc2 is a very potent and direct activator of Mdr1. IMPORTANCE Candida auris is a yeast of major medical importance causing nosocomial outbreaks of invasive candidiasis. Its ability to develop resistance to antifungal drugs, in particular to azoles (e.g., fluconazole), is concerning. Understanding the mechanisms of azole resistance in C. auris is important and may help in identifying novel antifungal targets. This study shows the key role of the transcription factor Upc2 in azole resistance of C. auris and shows that this effect is mediated via different pathways, including the regulation of ergosterol biosynthesis and also the direct upregulation of the drug transporter Mdr1.

1st Editorial Decision Re: Spectrum03526-23 (Upc2-mediated mechanisms of azole resistance in Candida auris) Dear Prof. Frederic Lamoth: Thank you for the privilege of reviewing your work.Below you will find my comments, instructions from the Spectrum editorial office, and the reviewer comments.
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Reviewer #1 (Comments for the Author): This is a strong manuscript that characterizes the role of Upc2 in C. auris azole susceptibility.The data strongly support the conclusions and add interesting observations that make important extensions to what has been observed for the homologous TF in other Candida spp.I have only a few suggestions that could improve the manuscript.1.It would be nice to use the HA tag of the hyperactive Upc2 to confirm that protein levels are also increased.2. The statistical tests need to be corrected for multiple comparisons.I do not think this will affect the results but it needs to be corrected to maintain rigor.3. A figure to summarize the interactions of Upc2 might make the discussion a little more straightforward for the reader, particularly those not familiar with the azole field.4. Line 42. Azoles are not a first line therapy for any candida spp.causing invasive disease according to IDSA and European guidelines.This is because of the likelihood of resistance and the fact that echinocandins are more effecctive clinically.
Reviewer #2 (Comments for the Author): The manuscript provides interesting and meaningful results about the role of transcription factor Upc2 on azole resistance mechanisms in Candida auris, that 90-100 clinical isolates are fluconazole resistant.The manuscript is well written and easy to read and interpret the results.Minor comments: insert the statistical test used in the captions.In the figures, try changing the arrows by other symbols that indicate the differences between the compared strains.It's confusing.