Regulation of copper uptake by the SWI/SNF chromatin remodeling complex in Candida albicans affects susceptibility to antifungal and oxidative stresses under hypoxia

Abstract Candida albicans is a human colonizer and also an opportunistic yeast occupying different niches that are mostly hypoxic. While hypoxia is the prevalent condition within the host, the machinery that integrates oxygen status to tune the fitness of fungal pathogens remains poorly characterized. Here, we uncovered that Snf5, a subunit of the chromatin remodeling complex SWI/SNF, is required to tolerate antifungal stress particularly under hypoxia. RNA-seq profiling of snf5 mutant exposed to amphotericin B and fluconazole under hypoxic conditions uncovered a signature that is reminiscent of copper (Cu) starvation. We found that under hypoxic and Cu-starved environments, Snf5 is critical for preserving Cu homeostasis and the transcriptional modulation of the Cu regulon. Furthermore, snf5 exhibits elevated levels of reactive oxygen species and an increased sensitivity to oxidative stress principally under hypoxia. Supplementing growth medium with Cu or increasing gene dosage of the Cu transporter CTR1 alleviated snf5 growth defect and attenuated reactive oxygen species levels in response to antifungal challenge. Genetic interaction analysis suggests that Snf5 and the bona fide Cu homeostasis regulator Mac1 function in separate pathways. Together, our data underlined a unique role of SWI/SNF complex as a potent regulator of Cu metabolism and antifungal stress under hypoxia.


Introduction
Candida albicans is the most pr e v alent fungal colonizer of humans and it is also the first cause of inv asiv e fungal infections with a mortality r ate a ppr oac hing 40% despite the curr ent ther a peutic options (Sellam andWhiteway 2016 , Fisher et al. 2020 ).This yeast thriv es on m ucosal surfaces, occupying div erse nic hes c har acterized by varying and dynamic oxygen le v els.As a facultativ e anaerobe, C. albicans grow in both oxygen-rich environments such as skin and o xygen-de pleted settings such as internal or gans, v a gina, gastr ointestinal tr act, and e v en tumor al envir onments (Ernst and Tielker 2009, McKeown 2014, Gan and Ooi 2020 ).While hypoxia is the pr e v alent condition within the host, the mac hinery that integrates oxygen status to tune the growth and the fitness of C. albicans remains poorly characterized.
Hypoxia is known to impact different facets of the hostfungus interaction and determines the outcome of the infection.From the pathogen side, hypoxia positiv el y impacts fungal virulence, which can worsen the outcome of the infection.For instance , the in v asiv e filamentous gr owth of C. albicans is enhanced under micr oaer ophilic envir onments and regulators of this pr ocess ar e essential for host infection (Askew et al. 2009, Desai et al. 2015, Glazier 2022, Henry et al. 2022 ).As their bacterial analogs, fungal biofilms offer hypoxic en vironments , which dri ve the distincti ve antifungal-resistant phenotype of this sessile growth state and provide a favorable niche for the flourishing of human pathogenic bacteria (Sellam et al. 2009, Fox et al. 2014, Ko w alski et al. 2020 ).Furthermor e, hypoxia pr omotes the masking of C. albicans from the immune cells as a strategy to attenuate pha gocytic r ecognition and uptake ( Pr adhan et al. 2018 ).As many fungal metabolic and energetic routes (e.g.sterol biosynthesis and r espir ation) r equir e oxygen, fungal cells hav e to r epr ogr am these pr ocesses to accommodate the hypoxic lifestyle and sustain their fitness (Butler 2013, Sellam et al. 2014, Burgain et al. 2020 ).In this r egard, r ecent inv estigation has underscor ed the role of the chromatin remodeling complex SWI/SNF as a master regulator that governs the hypoxic metabolic adaptation and fungal fitness in vivo (Burgain et al. 2019 ).Furthermore, transcription factors such as Upc2 and Efg1, which govern the biosynthesis of the o xygen-de pendent metabolites, er goster ol and unsaturated fatty acids, respectively, were required for growth under o xygen-de pleted en vironments , emphasizing the importance of tuning metabolism for fungal adaptation to hypoxia (MacPher-son et al. 2005, Setiadi et al. 2006, Synnott et al. 2011, Puerner et al. 2023 ).
So far, only few investigations have assessed the impact of hypoxia on environmental stresses that C. albicans and other human fungal pathogens confront at the different colonized niches.These works focused mainly on antifungal stress and the effect of oxygen le v els on the sensitivity of C. albicans to different classes of standard antimycotics.Ov er all, hypoxia was found to potentiate the activity of amphotericin B and itraconazole on C. albicans (Warn et al. 2004 ).Our recent study also reported an increased sensitivity of C. albicans to other antifungals, including azoles (miconazole and fluconazole) and the echinocandin caspofungin in addition to cell wall and endoplasmic reticulum (ER) stressors under hypoxia (Burgain et al. 2020 ).The potentiation of azoles and amphotericin B activity by hypoxia could be explained by the fact that er goster ol metabolism, whic h is the tar get of these antifungal classes, becomes a rate-limiting process under oxygen shortage, whic h consequentl y exacerbates fungal growth inhibition (Burgain et al. 2020 ).So far, the mechanisms whereby oxygen status is integrated into the stress-responsive machinery in fungi remain elusive.
In the current study, we uncovered that Snf5, a subunit of the c hr omatin r emodeling complex SWI/SNF, pr ovides a nexus for integrating oxygen status and stress response.We found that Snf5 is r equir ed to modulate C. albicans gr owth in r esponse to azole and amphotericin B particularly under hypoxia.RNA-seq profiling of snf5 mutant strain exposed to amphotericin B and fluconazole under hypoxic conditions uncov er ed a signatur e that is reminiscent of copper (Cu) starv ation.Remarkabl y, the snf5 m utant exhibited ele v ated le v els of r eactiv e oxygen species (ROS) specifically under hypoxia, which might explain its increased antifungal sensitivity in this specific condition.Furthermore, our data sho w ed that under hypoxic and Cu-starved en vironments , Snf5 is critical for pr eserving Cu homeostasis.Accordingl y, supplementing gr owth medium with Cu or increasing gene dosage of the Cu transporter CTR1 alleviated snf5 growth defect and attenuated ROS le v els in response to antifungals under hypoxia.Genetic interaction analysis suggests that Snf5 and the bona fide Cu homeostasis regulator Mac1 function in parallel pathwa ys .Our in vestigation establishes an important role of the SWI/SNF complex as a potent hypoxic regulator of antifungal stress and Cu homeostasis.
Strains used in this study are listed in Supplementary Table S2 .Candida albicans strains were routinely grown at 30 • C under constant agitation (200 rpm) in YPD medium supplemented with uridine (1% yeast extract, 2% peptone, 2% dextrose, and 50 mg/ml uridine).The Ca CTR1 open reading frame (ORF) was amplified using the CTR1-OE-F/CTR1-OE-R primer pair and the r esulted pol ymer ase c hain r eaction (PCR) pr oducts wer e cloned in the CIp-Act-Cyc plasmid to generate CIp-Act-CTR1 -Cyc ov er expr essing construct (Blackwell et al. 2003 ).The plasmid was linearized with StuI restriction enzyme and integrated into the SN148 WT (Wild Type) strain or snf5 mutant strain (DHY3) (Finkel et al. 2012 ).snf5 mac1 double homozygous mutant was generated by deleting the two MAC1 alleles in snf5 (DHY3) strain using URA3 -and SAT1 -deletion cassettes (Schaub et al. 2006 ).All primers used in this study are listed in Supplementary Table S2 .
For growth assays in liquid medium, overnight cultures of C. albicans were resuspended in fresh YPD medium at an OD 600 of 0.1 and added to a flat-bottom 96-well plate in a total volume of 150 μl per well in addition of the tested compounds.For each experiment, a compound-free positive growth control and a cell-free negativ e contr ol wer e included.Gr owth assay curv es wer e performed in a BioTek ™ Cytation™ 5 plate reader at 30 • C under normoxic (21% O 2 ) or hypoxic (5% O 2 ) conditions with constant agitation.For spot dilution assa ys , o v ernight cultur es wer e diluted to an OD 600 of 0.1 and 5-fold serial dilutions wer e pr epar ed in distilled water.A total of 4 μl of each dilution was spotted on YPD plates containing drugs.Plates were incubated at 30 • C for 2 days under both normoxic (21% O 2 ) and hypoxic (5% O 2 ) conditions using the Heracell VIOS Tri-Gas incubator and imaged using the SP imager system.

Expression analysis by RNA-seq and RT-qPCR
Ov ernight cultur es of snf5 m utant and WT (day 185) str ains wer e diluted to an OD 600 of 0.1 in 40 ml of fresh YPD medium flushed with nitrogen to maintain a hypoxic environment (5% O 2 ) and grown at 30 • C under agitation (200 rpm) to an OD 600 of 0.4.Cultur es wer e then left untr eated or exposed to either fluconazole (1 μg/ml) or amphotericin B (0.5 μg/ml), and incubated at 30 • C for 30 min.For each condition, a total of two biological replicates wer e consider ed for RNA-seq anal ysis.Cells wer e then harv ested by centrifugation at 3000 × g for 5 min and the pellets were quickfrozen and stored at −80 • C.
RNA extr actions, libr ary pr epar ation, and RNA-seq pr ocedur es wer e performed as pr e viousl y described (Khemiri et al. 2020 ).The R pac ka ge limma was used to identify differ ences in gene expression levels between treated and nontreated samples (Ritchie et al. 2015 ).Nominal P -values were corrected for multiple testing using the Benjamini-Hochberg method.Differentiall y expr essed tr anscripts in Supplementary Table S1 were identified using a false discovery rate of 0.1 (fluconazole treatment) and 0.05 (amphotericin B treatment), and 1.5-fold enrichment cutoff.Gene ontology (GO) analysis was performed using the GO Term Finder of the Candida Genome Database (Skrzypek et al. 2017 ).
For r eal-time r e v erse tr anscription quantitativ e PCR (RT-qPCR), cell cultures and RN A extractions w ere performed as exactly as described for the RNA-seq experiment.cDNA was synthesized from 1 μg of total RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).The mixture was incubated at 25 • C for 10 min, 37 • C for 120 min, and 85 • C for 5 min.Two units per microliter of RNAse H (NEB) was added to the reactions and incubated at 37 • C for 20 min to r emov e RN A. qPCR w as performed using a LightCycler 480 Instrument (Roche Life Science) with the Po w erUp™ SYBR ® Green Master Mix (Applied Biosystems).The reactions were incubated at 50 • C for 2 min, 95 • C for 2 min, and cycled 40 times at 95 • C, 15 s; 54 • C, 30 s; and 72 • C, 1 min.Fold enrichment of each tested transcript was estimated using the comparative Ct method.To evaluate the gene expression level, the results were normalized using Ct values obtained from actin ( ACT1 , C1_13700W_A).Primer sequences used for qPCR are summarized in Supplementary Table S2 .

Cu quantification
A colorimetric Copper Assay Kit (Cat# MAK127; Sigma) was used to measur e intr acellular Cu concentr ations, using the manufactur er's pr otocol.Briefly, C. albicans cells wer e r esuspended at an OD 600 of 0.1 in fresh YPD medium or YPD supplemented with 400 μM BCS and gr own ov ernight at 30 • C. Cells pellets were collected and digested overnight at 100 • C using 20% nitric acid.

Measurement of intracellular ROS levels
Intr acellular ROS le v els wer e detected using the cell-permeable fluorescent dye 2 ,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA) (Cat# D6883; Sigma).Yeast cells grown to late exponential phase in YPD were pretreated with the indicated compounds for 4 h at 30 • C under either normoxia or hypoxia.After a 30-min incubation with 25 μM H 2 DCFDA in the dark, the cells were harvested, washed three times with PBS, and resuspended in the same buffer to 2 × 10 7 cells/ml.A total of 100 μl of each suspension was tr ansferr ed to a flat-bottom 96well microplate and fluorescence was measured using a BioTek Cytation 5 plate reader ( λ ex = 485 nm; λ em = 528 nm).

Differential role of hypoxia in sensitizing snf5 mutant to antifungals
We hav e pr e viousl y uncov er ed that the SWI/SNF subunit Snf5 is r equir ed for metabolic r epr ogr amming to sustain the growth of C. albicans under hypoxic environments (Burgain et al. 2019 ).Here, we tested whether this transcriptional regulator might have an o xygen-de pendent role in modulating antifungal sensitivity tow ar d the three-standar d class of antifungals r epr esented by fluconazole, amphotericin B, and caspofungin.Serial dilution assays on solid medium sho w ed that snf5 mutant was hypersensitive to both fluconazole and amphotericin B, specifically under hypoxia as compared to normoxia (Fig. 1 A).Nevertheless, snf5 growth was inhibited by caspofungin irr espectiv e of oxygen le v els.Quantitativ e gr owth assay on liquid medium confirmed the exacerbated hypoxic defect of snf5 to both fluconazole and amphotericin B, while only a modest inhibition was perceived under normoxic conditions (Fig. 1 B).Together, these observations suggest that the pr e viousl y c har acterized o xygen-de pendent role of SWI/SNF in metabolic flexibility is expanded to include the modulation of antifungal stress principally under hypoxia.

RNA-seq profiling of snf5 in response to both fluconazole and amphotericin B under hypoxic conditions uncovers a transcriptional signature reminiscent of Cu depriv a tion
To understand the contribution of Snf5 in mediating antifungal sensitivity specifically under hypoxia, we performed RNA-seq profiling of both WT and snf5 cells grown under hypoxic conditions and exposed to fluconazole or amphotericin B .W e compared the tr anscriptional r esponse of snf5 m utant exposed to eac h antifungal to that of WT cells treated similarly and found that, for either fluconazole or amphotericin B, Cu-r esponsiv e r egulon was significantl y upr egulated in snf5 (Fig. 2 A and B).This includes the Cu transporter CTR1 , the ferric reductases ( FRE7 , FRE30 , and orf19.7077)that reduce Cu to facilitate its uptake by Ctr1, and the master tr anscriptional activ ator Mac1 (Fig. 2 C).Tr anscripts associated with Cu efflux and detoxification, including the cytosolic small c ha per one ATX1 , the two metallothioneins CUP1 and CRD2 , and the Cu efflux pump CRP1 , wer e r epr essed.We also found that snf5 m utant upr egulated the Mn-dependent super oxide dism utase SOD3 and r epr essed the Cu-dependent SOD1.qPCR anal ysis confirmed the RNA-seq data and showed that the modulation of Cu metabolic genes was more marked under hypoxia as compared to normoxia (Fig. 2 D and E).This transcriptional signature, reminiscent of a Cu-de pri v ed envir onment, suggests that snf5 mutant is unable to maintain a homeostatic le v el of intracellular Cu upon challenge by antifungals under hypoxia.
Beyond the Cu regulon, genes upregulated in snf5 exposed to fluconazole were also enriched in aspartate and alpha-amino acid biosynthesis, while genes of lipid metabolism, including fatty acids and er goster ol, wer e upr egulated in r esponse to amphotericin B (Fig. 2 A and B).As compared to WT, snf5 mutant failed to activate genes related to cellular respiration and oxidationr eduction pr ocess in r esponse to fluconazole and amphotericin B, r espectiv el y.

Cu repletion revert snf5 susceptibility to antifungals under hypoxia
To assess whether the perceived antifungal sensitivity of snf5 under hypoxia is linked to an ina ppr opriate internalization of Cu in C. albicans , gro wth medium w as supplemented b y 2 mM CuSO 4 .Of note, at 2 mM of CuSO 4 , the growth of C. albicans is not altered and it is sufficient to r estor e gr owth defect of Cu-uptake mutants as pr e viousl y shown (Khemiri et al. 2020 ).Supplementing gr owth medium with Cu, was sufficient to correct snf5 sensitivity to fluconazole and amphotericin B under hypoxia at a le v el compar able to the untreated snf5 condition (Fig. 2 F).Furthermore, ov er expressing the Cu transporter Ctr1 reverted the snf5 sensitivity to antifungal stress under hypoxia to a le v el similar to that observed in the WT strain (Fig. 2 G).Together, these data suggest that the snf5 exacerbated sensitivity to antifungals under oxygen-depleted environment is a consequence of a perturbed Cu homeostasis.

Inactiv a tion of SNF5 results in Cu utilization defect under hypoxia
We and others hav e pr e viousl y shown that Cu homeostasis modulates antifungal tolerance in C. albicans (Hunsaker andFranz 2019 , Khemiri et al. 2020 ).Accor dingly, w e hypothesized that Snf5 might govern Cu homeostasis under O 2 -depleted environment, which in turn impacts antifungal sensitivity in this condition.To test this hypothesis, we tested the ability of snf5 mutant to grow in a medium starved or containing sublethal concentration of Cu.We found that snf5 mutant exhibited high sensitivity to Cu chelation (BCS) and a better tolerance to w ar d excess of Cu as compared to the WT particularly under hypoxia (Fig. 3 A  and B).Measurement of Cu content sho w ed that under Cu sufficiency (YPD), snf5 exhibited more than two order magnitude reduction of Cu as compared to the WT, regardless of oxygen status (Fig. 3 C).This suggests that Snf5 is essential for preserving Cu homeostasis in C. albicans .When Cu was depleted from the medium using BCS, snf5 exhibited a similar Cu amount as compared to WT under normoxia.Ho w ever, under hypoxia, intracellular Cu was almost undetectable in snf5 as compared to the WT par ental str ain (Fig. 3 C).This underlines that Snf5 has a hypoxicdependent contribution to Cu homeostasis exclusiv el y under Culimiting en vironments .T his is corr obor ated by the fact that, under Cu scar city, snf5 w as not able to activate the transcription of the high affinity Cu transporter Ctr1, specifically under hypoxia (Fig. 3 D).Ov er all, these data underscore that Snf5 might integrate both oxygen and Cu availability to tune fungal growth in hypoxic niches.

Loss of SNF5 impairs resistance to oxidati v e stress and increases ROS levels under hypoxia
In addition to their primary targets, antifungals such as amphotericin B and azoles are known to mediate cell death by inducing o xidati v e dama ges (K oba y ashi et al. 2002, Belenk y et al. 2013, Mesa-Arango et al. 2014 ).We hypothesized that snf5 might have high basal levels of ROS under hypoxia as compared to normoxia, which might explain the differential sensitivity to the ROSgenerating antifungals, azoles and amphotericin B. Notably, Cu is a k e y cofactor for supero xide dism utases, whic h ar e enzymes that pr omote pr otection a gainst R OS (Hw ang et al. 2002(Hw ang et al. , F rohner et al. 2009 ) ).In this regard, as snf5 has low Cu contents, this might impede Cu-SOD activity, which might lead to the accumulation of high le v els of ROS and consequentl y explain the exacerbated antifungal sensitivity of snf5 under hypoxia.We found that snf5 exhibited higher le v els of ROS as compar ed to the WT in either unstressed or H 2 O 2 -treated cells particularly when oxygen was depleted (Fig. 4 A).We also tested the impact of SNF5 inactivation on the sensitivity to H 2 O 2 and uncov er ed that snf5 mutant was hypersensitive to 4 mM H 2 O 2 specially under hypoxia (Fig. 4 B).Supplementation of growth media with Cu mitigated snf5 sensitivity to o xidati v e str ess, though to a lesser degr ee compar ed to the observ ed alle viation in the WT str ain (Fig. 4 C).Ov er all, deletion of SNF5 leads to higher le v els of intrinsic o xidati v e str ess specifically under hypoxia, which might explain snf5 hypersensitivity to H 2 O 2 .When treated with fluconazole and amphotericin B under hypoxia, snf5 exhibited higher ROS le v els as compared to the WT and the untreated conditions (Fig. 4 D).This phenotype was attenuated with Cu supplementation as ROS le v els dr opped significantly in snf5 mutant, the WT, and the complemented strains (Fig. 4 D).These findings suggest that Snf5 govern antifungal sensitivity under hypoxia by preserving Cu homeostasis to promote Cu-de pendent antio xidant response in C. albicans cells.

Functional relationship between Snf5 and the master regulator of Cu homeostasis Mac1
We have previously reported that mac1 mutant was hypersensitive to azoles and amphotericin B under normoxic conditions (Khemiri et al. 2020 ).As transcription factors require chromatin remodeling complexes to facilitate their accessibility to their target DNA (Burns andPeterson 1997 , Neely et al. 2002 ), we hypothesized that Mac1 and SWI/SNF might operate synergistically to control the transcription of the Cu regulon in C. albicans .To test potential epistatic relationship between the two regulators, we deleted MAC1 in snf5 mutant and tested the sensitivity of the resulting double mutant to fluconazole and amphotericin B under both nor-  mo xia and hypo xia.Ov er all, under either str essed or unstr essed conditions and regardless of oxygen status, snf5 mac1 double mutant exhibited an ad diti v e gr owth defect as compar ed to mac1 or snf5 single mutants (Fig. 5 ).This synthetic sickness of mac1 snf5 indicates that both regulators function in parallel pathways controlling the same biological pr ocess.Intriguingl y, while mac1 exhibited hypersensitivity to w ar d the tested antifungals under normoxia as pr e viousl y r eported, this defect was completely buffered by hypoxia.

Discussion
We have previously found that Snf5 was critical for reprogramming different metabolic routes to accommodate C. albicans growth under hypoxic environments (Burgain et al. 2019 ).In this study, we ha ve unco vered a no vel role of Snf5 in mediating stress response and Cu metabolism under hypoxia, which underscores the global role of this regulator in tuning fungal fitness in oxygende pri v ed nic hes.In metazoans, hypoxia is known to cause ROS accumulation as a consequence of inefficient electron transfer across the mitoc hondrial electr on tr ansport c hain (Hamanaka andChandel 2009 , Kung-Chun Chiu et al. 2019 ).We found that this phenomenon was conserved in C. albicans as ROS levels were increased by 3-fold under hypoxia as compared to normoxia (Fig. 4 A).Under unstressed conditions, snf5 accumulated more ROS in hypoxia, which indicates that ROS production exceeds elimination in this mutant.This suggests that Snf5 is r equir ed to pr eserv e r edox homeostasis and/or to detoxify ROS as an ada ptativ e mec hanism to surviv e in o xygen-de pleted niches.Our prior metabolomics analysis of snf5 cells experiencing hypoxia uncov er ed r educed le v els of metabolic intermediates of the pentose phosphate pathway (PPP), an essential metabolic route for the production of the NADPH that is critical for cellular redox balance (Burgain et al. 2019 ).Given the importance of PPP for C. albicans fitness under hypoxia (Burgain et al. 2020 ), SWI/SNF might act as a transcriptional regulator of this pathway to promote NADPH supply and accommodate the hypoxic growth.This hypothesis is also supported by our genomewide occupancy anal ysis wher e pr omoters of the majority of the PPP genes were bound by the Snf2 subunit, suggesting a direct tr anscriptional contr ol of this metabolic r oute by SWI/SNF complex (Tebbji et al. 2020 ).In addition to the potential contribution to the biosynthesis of NADPH, SWI/SNF might modulate o xidati ve str ess indir ectl y by contr olling the uptake of Cu, which is a prerequisite for Cu-dependent SODs to detoxify superoxide radicals.T hus , SWI/SNF might modulate redox status of the cells by means of multiple mechanisms in C. albicans .
When challenged by either azole or amphotericin B, snf5 exhibited a str ong gr owth defect under hypoxia and displayed a transcriptional signature of Cu scarcity.snf5 mutant has a reduced intracellular amount of Cu and was not able to properly modulate the Cu transporter Ctr1, especially under Cu limitation.Together, these data suggest that SWI/SNF is an important regulator of Cu uptake in C. albicans .Cu homeostasis is modulated by the transcription factor Mac1, which is activated by Cu limitation to promote Cu uptake in C. albicans (Woodacre et al. 2008, Tebbji et al. 2020 ).As a c hr omatin r emodeler, SWI/SNF might thus facilitate Mac1 access to their target promoter through nucleosome rearrangement of Mac1-occupied promoters.Ho w ever, this hypothesis is not supported by the synthetic sick phenotype of mac1 -snf5 double m utant, whic h suggests that Mac1 and Snf5 operate in parallel pathways to pr eserv e Cu homeostasis.Thus, SWI/SNF might cooperate with another transcription factor yet to be discovered or act dir ectl y thr ough a differ ent mec hanism to modulate the Cu regulon in C. albicans .
Intriguingly, C. albicans hypoxic cells internalized more Cu than cells growing under normoxic conditions, underscoring the im-portance of this metal for fungal adaptation in hypoxia.A similar phenomenon was observed in murine macrophages where an increased Cu uptake and redistribution into the secretory pathway in response to hypoxia was perceived (Sarkar et al. 2003, White et al. 2009 ).This study uncov er ed that hypoxic macr opha ges prioritize Cu delivery to post-Golgi vesicles to assist the function of the m ulticopper ferr oxidase in ir on (F e) uptake , underlining that Cu and Fe homeostasis are intimately intertwined.As Cu is also r equir ed for r eductiv e Fe uptake in C. albicans (Knight et al. 2002, Weissman et al. 2002, Fourie et al. 2018 ), it is tempting to speculate that a similar mechanism is taking place under hypoxia where Cu demand is increased to sustain the function of multicopper ferroxidase and Fe assimilation.

Figure 2 .
Figure 2. snf5 sensitivity to antifungals under hypoxia is associated with deregulation of Cu homeostasis.(A and B) Gene functions and biological pr ocesses enric hed in the tr anscriptional pr ofiles of snf5 m utant exposed to either fluconazole (A) or amphotericin B (B). (C) Modulation of Cu r egulon by antifungal treatment under hypoxia.Heat map visualization of the transcript levels of the Cu homeostasis pathway in C. albicans as defined pr e viousl y (Khemiri et al. 2020 ).(D and E) qPCR validation of RNA-seq data.Transcript levels of Cu-utilization genes ( MAC1 , CTR1 , FRE7 , and SOD3 ) were assessed under both hypoxia (D) and normoxia (E) in response to fluconazole (Fcz) and amphotericin B (AmB).Fold-changes were calculated using the compar ativ e Ct method.Data were normalized using Ct values obtained from the actin gene in each condition.Significance was determined using the two-tailed Student's t -test ( * * P < .01,* * * P < .001,and * * * * P < .0001;ns: nonsignificant).(F) Cu supplementation by 2 mM of CuSO 4 r e v ert snf5 susceptibility to antifungals under hypoxia.(G) Increasing CTR1 dosage in snf5 alleviates its hypersensitivity to antifungals under hypoxia.Spot assay was used to assess the effect of fluconazole (Fcz) and amphotericin B (AmB) on the growth of WT (ASJC1) and snf5 (DHY3-CipAct), and their equivalent str ains ov er expr essing the Cu tr ansporter CTR1 (WT p CTR1 and snf5 p CTR1 ).

Figure 3 .
Figure 3. Snf5 is r equir ed for Cu homeostasis under hypoxia.Effect of Cu chelation using BCS on the growth of WT (day185), snf5 , and snf5 complemented strains ( snf5 -p SNF5 ) in YPD liquid (A) and YPD-agar (B) media.For Cu excess in YPD-agar, cells were treated with 8 mM of CuSO 4 .(C) Impact of snf5 mutation and Cu chelation (400 μM BCS) on intracellular Cu contents under both normoxia and hypoxia.(D) Transcript levels of CTR1 assessed by qPCR in WT and snf5 strains under hypoxia.WT and snf5 cells were grown in either YPD or YPD supplemented with 400 μM BCS.Tr anscript le v els of CTR1 wer e normalized to the untr eated condition (YPD).Results r epr esent mean fold c hanges of at least thr ee r eplicates.Significance was determined using the two-tailed Student's t -test ( * P < .05,* * P < .01,* * * P < .001,and * * * * P < .0001;ns: nonsignificant).

Figure 4 .
Figure 4. Snf5 modulates o xidati v e str ess under hypoxia.(A) R OS quantification b y H 2 DCFDA probe .WT (da y 185), snf5 , and snf5 -complemented ( snf5-p SNF5 ) strains were grown under either hypoxia or normoxia in unstressed and stressed (3 mM H 2 O 2 ) conditions.Data represent the percentage of ROS le v els in the different strains relative to the WT under normoxia.Results are the mean of at least three replicates.(B) Impact of H 2 O 2 on snf5 growth under both normoxia and hypoxia.Results r epr esent mean growth inhibition of at least three replicates.(C) Cu supplementation by 2 mM of CuSO 4 mitigates snf5 susceptibility to H 2 O 2 under hypoxia.Strains were grown in liquid YPD medium with or without CuSO 4 and H 2 O 2 as indicated.Results are the mean of three replicates.(D) Impact of Cu supplementation (2 mM CuSO 4 ) on ROS levels generated by fluconazole (Fcz) and amphotericin B (AmB) treatment in snf5 mutant.The level of ROS production was measured 4 h after treatment with either 10 μg/ml of fluconazole or 10 μg/ml of amphotericin B under hypoxic conditions.Data r epr esent the mean of three replicates.Statistical significance (WT versus snf5 ) was determined using the two-tailed Student's t -test ( * P < .05,* * P < .01,* * * P < .001,and * * * * P < .0001;ns: nonsignificant).

Figure 5 .
Figure 5. Genetic interaction between Snf5 and Mac1.Ad diti ve effect of snf5 and mac1 mutations on growth and sensitivity to antifungals.Cultures wer e gr own ov ernight and spotted on YPD plates with or without antifungals (fluconazole: Fcz; amphotericin B: AmB) as indicated.