UPC2 Is Universally Essential for Azole Antifungal Resistance in Candida albicans

Abstract

In Candida albicans, the transcription factor Upc2 is central to the regulation of ergosterol biosynthesis. UPC2-activating mutations contribute to azole resistance, whereas disruption increases azole susceptibility. In the present study, we investigated the relationship of UPC2 to fluconazole susceptibility, particularly in azole-resistant strains. In addition to the reduced fluconazole MIC previously observed with UPC2 disruption, we observed a lower minimum fungicidal concentration (MFC) for a upc2⌬/⌬ mutant than for its azole-susceptible parent, SC5314. Moreover, the upc2⌬/⌬ mutant was unable to grow on a solid medium containing 10 g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed higher fungistatic activity against the upc2⌬/⌬ mutant than against SC5314. UPC2 disruption in strains carrying specific resistance mutations also resulted in reduced MICs and MFCs. UPC2 disruption in a highly azole resistant clinical isolate containing multiple resistance mechanisms likewise resulted in a reduced MIC and MFC. This mutant was unable to grow on a solid medium containing 10 g/ml fluconazole and exhibited increased susceptibility and a clear zone of inhibition by Etest. Time-kill analysis showed increased fungistatic activity against the upc2⌬/⌬ mutant in the resistant background. Microarray analysis showed attenuated induction by fluconazole of genes involved in sterol biosynthesis, iron transport, or iron homeostasis in the absence of UPC2. Taken together, these data demonstrate that the UPC2 transcriptional network is universally essential for azole resistance in C. albicans and represents an attractive target for enhancing azole antifungal activity.

Disruption of the Transcriptional Regulator Cas5 Results in Enhanced Killing of Candida albicans by Fluconazole

“…We identified four TF mutants that met the criteria: CAS5, RPN4, UPC2, and CZF1 ( Table 1). The observation that disruption of UPC2 increased susceptibility to fluconazole was not surprising, as this was reported previously (20,32,33), validating the results of our screen. The remaining three TF deletion mutants that exhibited reductions in MFCs in both RPMI and YPD media were prioritized as being of the greatest interest, and independent mutants were generated in the SC5314 background (Table 2).…”

“…The disruption of RPN4 did not display as strong of an effect, suggesting that RPN4 influences fungistatic azole activity to a lesser extent, while CZF1 disruption had no impact on the killing activity of fluconazole. As previously observed for disruption of UPC2, CAS5 disruption enhanced the killing activity of fluconazole (20). Upon disruption of CAS5 in fluconazole-resistant clinical isolates 12-99, G5, Gu5, and S2, which carry combinations of the four most common mechanisms of resistance, there was no marked change in fluconazole killing activity, although there was a slight change in susceptibility in the G5 background.…”

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses

“…7B). These results confirmed the existence of a close relationship between plant sterol metabolism and Fe homeostasis, a connection that is well established in other organisms such as fungi (Craven et al, 2007;Blatzer et al, 2011;Hosogaya et al, 2013;Thomas et al, 2013;Chung et al, 2014;Vasicek et al, 2014) but is much less known in plants (Urbany et al, 2013). In general, depletion of sterol biosynthesis has been correlated with both higher membrane permeability and increased uptake of metal ions in roots, although the underlying molecular mechanism remains unknown (Diener et al, 2000;Khan et al, 2009;Urbany et al, 2013;Wagatsuma et al, 2015).…”

The Celecoxib Derivative AR-12 Has Broad-Spectrum Antifungal Activity In Vitro and Improves the Activity of Fluconazole in a Murine Model of Cryptococcosis

“…This indicates that AR-12 is active against C. albicans strains that are resistant to FLU by common molecular mechanisms. To further confirm this observation, we tested AR-12 against C. albicans clinical isolates with gain-of-function mutations in two transcription factors that regulate efflux pump expression in C. albicans MRR1 and TAC1 mutants (23). As shown in Table 2 (entries 6 and 7), the AR-12 MIC was unchanged from that of the reference strain (4 g/ml).…”