Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, IndiaLaboratoire de génomique des microorganismes, CNRS FRE3214, Université Pierre et Marie Curie, 15 rue de l’école de médecine, 75006 Paris, France
r t i c l e i n f o
rticle history:eceived 24 July 2010eceived in revised form0 November 2010ccepted 18 November 2010
a b s t r a c t
Steroids are known to induce pleiotropic drug resistance states in hemiascomycetes, with tremendouspotential consequences on human fungal infections. The proteins capable of binding to steroids such asprogesterone binding protein (PBP), estradiol binding proteins (ESP) are found in yeasts, however, thewell known receptor mediated signaling present in higher eukaryotic cells is absent in yeasts and fungi.Steroids are perceived as stress by yeast cells which triggers general stress response leading to activationof heat shock proteins, cell cycle regulators, MDR transporters, etc. In this article, we review the response
of yeast to human steroid hormones which affects its cell growth, morphology and virulence. We discussthat a fairly conserved response to steroids at the level of transcription and translation exists betweenpathogenic and non-pathogenic yeasts.
Human steroid hormones affect growth, morphogenesis andrug susceptibilities of pathogenic and non-pathogenic yeast cells.owever, the molecular basis of steroids action and signaling
emains unresolved [1–5]. Relevance of human steroids in yeasthysiology has emerged from several observations, which showedhat yeast cells such as pathogenic Candida albicans undergoeshanges in its morphology and growth rate upon supplementa-ion of the growth medium with steroids [3,4,6–9]. Another factorhat relates steroids with the patho-physiology of C. albicans is therevalence of vulvovaginal candidiasis (VVC), which most oftenccurs in women during the late luteal phase of the menstrualycle, when estrogen and progesterone levels are elevated [10].he presence of human steroid hormones in Candida’s host would
lso imply that these could act as environmental cues in control-ing its virulence [6,9]. Taken together, though steroids are knowno influence a variety of processes in yeasts, yet, very little is
understood in the context of steroid signaling in these organisms.This acquires further significance when one considers the fact thatthe well-characterized steroid receptor pathways found in highereukaryotes are non-existent not only in Candida but also in otheryeasts and fungi as well [2]. Interestingly, steroid binding proteinssuch as estradiol binding protein (EBP) [11] corticosteroid bind-ing protein (CBP) [12] and progesterone binding protein (PBP) [13]do exist in Candida. However, they appear to be non-DNA bindingproteins [11], and their role in steroid signaling in yeast is not veryclear.
There are very limited studies where steroid response inyeasts has been examined. Therefore, transcriptome and proteomeresponses to steroids are particularly discussed from our own anda few other studies [14–17]. Nonetheless, these limited studiesalready demonstrate that yeast cells are extremely responsive tosteroids in affecting its morphology, growth and expression profileof several genes and proteins.
1.1. Steroids as substrates of MDR pump proteins
C. albicans and other pathogenic species of Candida derivetheir importance not only from the severity of their infectionsbut also for their ability to develop resistance against antifungals,such as azoles, in patients undergoing long-term or prophylac-
tic treatment. Although several mechanisms which contribute tothe azole-resistance in clinical isolates have been identified, up-regulation of drug extrusion pump encoding genes belonging to thesuperfamily of either ABC (ATP-binding cassette, e.g. CaCDR1 and
62 R. Prasad et al. / Journal of Steroid Biochemistry & Molecular Biology 129 (2012) 61– 69
spons
CmaCwtwChwpcICcM
tcdsgmt[��d�ec
egitQ
Fig. 1. Physiological re
aCDR2) or MF (major facilitator, e.g. CaMDR1) represents one of theost prevalent mechanisms of drug resistance [18–20]. Steroids act
s substrates of ABC proteins. For example, multidrug transporteraCdr1p can specifically transport �-estradiol and corticosterone,hich could be blocked by molar excesses of �-estradiol, cor-
icosterone, ergosterol or dexamethasone. Notably, progesteronehich generates good response in yeasts, is not a substrate ofaCdr1p [21]. Interestingly, some of the drugs such as cyclo-eximide, chloramphenicol, fluconazole and o-phenanthroline, tohich an overexpression of CaCDR1 confers resistance, could alsorevent efflux and enhance accumulation of �-estradiol implyingommon binding site sharing between steroids and drugs [22].n conclusion, human steroid hormones are the substrates foraCdr1p and the energy dependent transport mediated by it is spe-ific for �-estradiol and corticosterone (Fig. 1). Unlike CaCdr1p, theFS multidrug transporter CaMdr1p does not transport steroids.Kolaczkowski et al. [23], earlier demonstrated that ABC
ransporter ScPdr5p (homologue of CaCdr1p) of baker’s yeast Sac-haromyces cerevisiae mediates resistance to progesterone andeoxycorticosterone by showing hyper susceptibility of �Scpdr5train to both of these steroids. They also observed that both pro-esterone and deoxycorticosterone competitively inhibit Pdr5pediated rhodamine 6G (R6G) transport, which strongly suggested
hat they are indeed transport substrates of ScPdr5p. Mahe et al.24], have monitored (3H)-estradiol accumulation in �Scpdr5 and
Scsnq2 null strains and observed that it was increased by 3-fold inScpdr5 as compared to wild type strain, however, �Scsnq2 nulls
id not show much difference. The subsequent deletion of bothScpdr5 and �Scsnq2 led to even higher accumulation of (3H)-
stradiol implying that ScSnq2p works in concert with ScPdr5p toontribute for �-estradiol export in S. cerevisiae cells.
Steroids are well known substrates of MDR proteins of higherukaryotes as well. For example, human ABC transporter P-
lycoprotein (Pgp) which is present in various tumor cells andmparts drug resistance to them can export various steroids. Usinghree-dimensional quantitative structure activity relationship (3D-SAR), cortisol, aldosterone, dexamethasone, 11-deoxycortisol and
e of steroids in yeasts.
corticosterone were identified as substrates while pregnanedioneand progesterone were strong inhibitors of human Pgp [25].Recently, a steroid-binding element has been identified in themembrane domain of ABCG2 an ATP binding cassette transporterthat confers drug resistance to cancer cells. Steroid binding to thiselement results in modulation of ABCG2 activity [26].
1.2. Steroid as growth and morphogenesis regulator
Morphological switching from yeast to hyphal form has beenregarded as one of the important virulence traits [27]. This switchdepends on various environmental cues inside the host. Steroidpresents one such environmental cue that affect hyphal devel-opment of C. albicans. White’s group demonstrated the effect ofmammalian hormones viz. estradiol, testosterone, cholesterol onclinical isolates of C. albicans and showed that there was a noteddelay in the ability of steroid stripped serum to facilitate the germi-nation, which was partially restored upon the addition of estradiol[7]. Cheng et al. [15] focused on the effect of 17-� estradiol, ethynylestradiol and estriol on several C. albicans strains and observedan increase in germ tube length in a dose and strain dependentmanner. This study has further revealed a potential relationshipbetween 17-�-estradiol and the upregulation of CaPDR16 andCaPLD1, which promote hyphae formation in C. albicans. Recently,in a transcriptome based study, Banerjee et al. [16] observed dif-ferential regulation of genes associated with hyphal developmentand showed that progesterone is a morphogenetic regulator influ-encing expression of many morphological genes such as CaEFG1,CaCPH1, CaNRG1, CaALS1 etc. [16].
1.3. Steroids and drug resistance
The micro-dilution tests (MIC80) on the C. albicans SC5314 cells
and in a matched pair of azole sensitive (AS) and azole resis-tant (AR) clinical isolates, in the presence and absence of thehuman steroid hormones progesterone and �-estradiol revealeda consistent increase in MIC80 for the drugs like fluconazole and
mistry
kapeapr
1
rt[riadatestioucttDrHDDtas
1r
hc(h[pg
gtgphffA(Sto[ssi
R. Prasad et al. / Journal of Steroid Bioche
etoconazole [16]. Recently, azole susceptible clinical isolates of C.lbicans have been shown to acquire transient drug resistance inresence of steroids �-estradiol and progesterone. The raised lev-ls of R6G efflux in progesterone and �-estradiol induced AS strainsnd their ability to withstand higher doses of azole drugs as com-ared with basal levels, confirmed that steroid induction impartsesistance to otherwise susceptible clinical isolates [28].
.4. Steroids transcriptionally activate MDR genes of yeasts
Recent efforts have revealed that the CaCDR1 promoter not onlyesponds to multitude of drugs but also to other environmen-al stimuli such as heat shock, heavy metals and human steroids29–31]. Karnani et al. [32] have identified presence of steroidesponsive region (SRR) that confers �-estradiol and progesteronenducibility to CaCDR1 promoter. SRR spans a region between −696nd −521 bp upstream of transcription start site. SRR was furtherivided into two progesterone responsive sequences (−628 to −594nd −683 to −648) and one �-estradiol responsive sequence (−628o −577). Even more deletions in the SRR delimited it to two distinctlements that were called SRE1 and SRE2. Both SREs are specific forteroids, SRE1 respond only to progesterone whereas SRE2 respondo both progesterone and �-oestradiol. de Micheli et al. [33], alsodentified the presence of cis regulatory elements in the promoterf CaCDR1 and CaCDR2 responsive to estradiol. To identify the reg-latory elements, the promoter deletions of both these genes wereloned in frame with Renilla luciferase reporter gene and exposedo estradiol for transient induction. This led to the identification ofwo regulatory elements i.e. BEE (basal expression element) andREI (drug responsive element) in the CaCDR1 promoter; BEE is
equired for basal expression and DRE for estradiol responsiveness.owever, only one regulatory element was found in CaCDR2 calledREII that was responsive for estradiol responsiveness. DREI andREII share a 21 base pair consensus sequence that is not common
o any known eukaryotic element. Unlike SRE1 and SRE2, whichre exclusively responsive to steroids, DREs are responsive to bothteroids and drugs.
.5. The transcriptome response of steroids mimics stressesponses
Whole genome cDNA microarray analyses in response to theuman steroid hormone progesterone were performed in C. albi-ans and in S. cerevisiae, using a supra-physiological concentration1 mM) of progesterone and a 30 min time-point, which gave theighest expression response of CaCDR1 and CaCDR2 in C. albicans16]. Of note, it was reported that using a physiological or near-hysiological concentration of oestrogen had very few effects onene expression in C. albicans cells [15].
In S. cerevisiae, the overall scenario of differentially regulatedenes suggested that the short-term progesterone treatment leadso a stress response in yeast. For example, transport facilitationenes, which are involved in drug and other xenobiotic efflux, com-rise a large percentage of up-regulated genes (12%). These genesave been previously shown to be up-regulated by transcription
actors ScPDR1 and ScPDR3, heat shock, H2O2, and other stress-ul conditions [34–36], (http://www.transcriptome.ens.fr/ymgv/).dditionally, several genes belonging to the cell cycle control
ScAPC11, ScCIS1, ScCDC34, ScPCL6, ScMID2, ScCTF13, ScNUF2, andcTRF4) and to the biogenesis of cell wall were induced by proges-erone exposure. Interestingly these genes also respond to variousther cellular stresses (http://www.transcriptome.ens.fr/ymgv/)
37]. Notably, ScMID2, which encodes a potential cell wall stressensor and upstream activator of cell integrity pathway [38], wasignificantly up-regulated by progesterone exposure. The genesnvolved in carbohydrate utilization, regulation and transport
& Molecular Biology 129 (2012) 61– 69 63
(ScYAL061W, ScTDH1, ScFBP1, ScGAL80, ScSHR5, ScYOL007C, ScHXT6,ScHXT7), which are induced by various stresses [37], also exhib-ited up-regulation by progesterone treatment. The STREs (havingcore consensus AG4 or C4T) have been demonstrated to occur inthe upstream promoter region of a number of genes responsiveto stress signals [39–42]. The analysis of steroid affected genesrevealed that several affected gene promoters had STRE sequencespresent at least two-times or more. The high doses of progesteronewhich are perceived as a stress response by the S. cerevisiae cellswas further evident from among the down-regulated genes. Dur-ing stressful conditions, when the cell needs to devote more ofits resources to diffuse the stress, ribosomal protein synthesis isshut down [43] Notably, a number of genes encoding ribosomalproteins (ScRPS3, ScRPS5, ScRPS4B, ScRPS6B, ScRPL4B, and ScRPL17B)were significantly down-regulated by steroids.
Similarly, in C. albicans, many of the genes induced by proges-terone belong to drug resistance and its associated gene categories.Some hyphae-associated genes like CaGPX1 (CaEfg1p, CaRfg1p,CaCyr1p, CaTup1p and CaNrg1p regulated), CaDUR1,2 (CaNrg1pregulated), CaMIG1 (CaTUP1 dependent and independent), CaGDH3,CaHSP90 (hyphal surface localization), CaGLG2 (hyphal induced),CaCDC19 (mutation affects filamentation), CaPDC11 (found onlyin hyphal cells) were also induced. The hyphae-associated genesaffected by progesterone reflect the similarity between steroidresponse and dimorphic transition regulation [16]. The up-regulated categories of genes included stress-associated geneslike CaDDR48 (gene consistently up-regulated under conditionsfavoring filamentation), CaPRB1 (heat regulated, GlcNAc induced),CaSSA4 (heat shock protein), CaKAR2 (similar to chaperone of Hsp70family; expression greater in high iron), CaHSP90, CaHSP12 andCaHSP60 (heat shock chaperone proteins) and CaIPF6629 (oxidativestress response). Notably, C. albicans CaHsp90p can provide steroiddependent activation of a mammalian steroid receptor when bothproteins are expressed in S. cerevisiae [34]. Interestingly, the SRRconsensus sequences are present in many responsive genes. It alsoincludes an inverted CCAAT box which in combination with otherconserved sequences is attributed towards human CaMDR1 respon-siveness to cellular stresses [44–46].
Thus, yeast cells perceive high doses of steroids as a cellu-lar stress. Several similarities can be observed between yeastcells exposed to human steroid hormones and those exposed toother stress conditions such as antifungal drugs of various classes,hypoxia, heat shock and oxidative stress [29,47–49]. For instance,steroids induce hyphae specific genes and hypoxia is also known tobe a very good inducer of filamentation in C. albicans. Several geneswere commonly up-regulated in the two conditions [16].
1.6. Steroids induce a pleiotropic drug response
In a recent study [17], a kinetic microarray analysis of both S.cerevisiae and C. albicans cells to low doses (ranging from 0.1 mMdown to 1 nM) of the human steroid hormone progesterone wasconducted. Some interesting insights into the patho-physiologicalsimilarities and differences between the two yeast species emergedfrom that study. In S. cerevisiae, two pathways are mainly respon-sible for multidrug-resistance phenotypes: the PDR pathway,controlled by the ScPdr1p and ScPdr3p transcription factors, andthe ergosterol biosynthesis pathway [50–52]. Gene ontology andDNA regulatory motif mining of microarray data indicated thatprogesterone specifically and extensively activated these two path-ways. The DNA consensus motif bound by ScPdr1p and ScPdr3p(named PDRE) was found to be significantly correlated with pro-
gesterone induction. These genes represent about 80% of theScPdr1p/ScPdr3p targets defined previously. Such a complete PDRresponse had been only observed in the case of constitutivelymultidrug-resistant strains harboring ScPDR1 or ScPDR3 gain of
unction alleles [50,53–56]. This makes progesterone the most effi-ient inducer of PDR pathway of S. cerevisiae.
The early PDR response of progesterone exposure was followedy the induction of genes involved in the ergosterol biosynthe-is. As a consequence, the ergosterol metabolism pathway washe main GO (gene ontology) category to be significantly enrichedf cells were exposed to progesterone for longer time (∼90 min).hese genes encode enzymes from the ERG pathway (ScERG25,cERG11, ScERG5, ScERG4, ScERG3 and ScERG28), the ScPdr16p pro-ein, etc. Interestingly, the transcriptional activator of the ERGenes, ScUpc2p, was itself similarly induced. Moreover, the globalxpression of ScUpc2p DNA binding motif-containing genes wasignificantly correlated with progesterone induction [17]. ScUpc2ps reported to confer resistance to antifungal drugs [57] and thus
ay play a role in drug export and resistance. Noteworthy, the PDRnd ERG response to progesterone was very stable, being observedp to 7 h after the beginning of the treatment.
.7. The PDR response of steroids is partially conserved in C.lbicans
In C. albicans, our knowledge of MDR regulation is rather limited.ultidrug resistance can be due to the up-regulation of multidrug
ransporters belonging to the ABC (ATP-binding cassette) trans-orter family (e.g. CaCDR1 and CaCDR2), which is the equivalent ofhe PDR pathway of budding yeast, or of the major facilitator familye.g. CaMDR1), which is the equivalent of the YAP1/FLR1 pathway in. cerevisiae. In one transcriptome-based study, CaCDR1 and CaCDR2enes were not induced by MIC50 concentrations of drugs likemphotericin B, caspofungin and fluorocytosine and only ketocona-ole could induce both CaCDR1/2 [58]. Of note, only fluphenazinead been shown to induce a large CaCDR1/CaCDR2 response in C.lbicans. Remarkably, progesterone induced many genes known toe co-regulated with CaCDR1/CaCDR2 in C. albicans [59,60]. This
ncluded CaCDR1, CaCDR2, CaIFU5, CaRTA3 and CaHSP12. The lev-ls of transcription factor CaMMR1 which exclusively regulatesaMDR1, did not change in response to progesterone treatment.his would support the fact that the regulation of CaMDR1 is dif-erent from those of ABC transporters. The CaCDR1 gene expressionrofile was similar to the ScPDR5 expression profile, indicating thathe mechanisms involved in the progesterone induction of theseenes may be conserved from S. cerevisiae to C. albicans. Remark-bly, this is in spite of the fact that the promoters of the two genesarbor different regulatory elements.
To achieve a global qualitative and quantitative estimation ofhe conservation of the PDR and ERG responses to progesteronen a non-pathogenic and a pathogenic yeast species, two indepen-ent hierarchical clustering of the PDR and ERG genes induced byrogesterone in S. cerevisiae and of their closest homologues in C.lbicans were made and directly compared the gene expressionrofiles of the homologous gene pairs using Pearson correlationistances. No conservation of the ERG response identified in S.erevisiae and in C. albicans was evident. In contrast, a full conser-ation of the induction of the oxidoreductase (Gre2p-like) and ofhe putative flippase (Rsb1p-like) encoding genes involved in theDR response, two homologous genes being similarly induced inoth species, was observed. However, in the case of the ABC trans-orter family, only a partial conservation was seen, with just twoandida genes (CaCDR1 and CaCDR2) being up-regulated by proges-erone while four of their homologues (ScPDR5, ScPDR15, ScPDR10nd ScSNQ2) were induced in S. cerevisiae [17] (Fig. 2).
Notably, about 20 ScPdr1/ScPdr3 target genes get induced in S.
erevisiae, when only 9 homologues of these genes were similarlyegulated in C. albicans. One simple explanation would be that theDR pathways in C. albicans involve genes which are not clear
omologues of the S. cerevisiae PDR genes but which play a similar
& Molecular Biology 129 (2012) 61– 69
role. Actually, this is the case of the C. albicans MDR transcriptionalregulator, CaTac1p, which is not strictly homologous to the S. cere-visiae ScPdr1p and ScPdr3p but which belongs to the same family ofGal4p-like transcription factors, and exhibited a similar expressionprofile in response to progesterone. Many known putative CaTac1ptargets were induced by progesterone (discussed in Section 1.9)[17].
1.8. Several transcription factors are induced in response toprogesterone
As mentioned above, yeast and fungi are responsive to steroidexposure but are not known to have their receptors as in highereukaryotes. The Candida transcription factors induced by pro-gesterone in the microarray experiments and thus tentativelyinvolved in steroid response are: CaTAC1 (transcriptional activatorof drug-responsive genes including CaCDR1 and CaCDR2), CaGAT2(mutation affects filamentous growth), CaCZF1 (hyphal growthregulator), CaZNC1 (Zn(2)–Cys(6) binuclear cluster, regulated byCaGcn2p and CaGcn4p), CaZCF39 (filament induced), CaIPF2822(Zn-finger TF) and CaCAS5 (cell wall damage response; down-regulated in core stress response). The down-regulated TFs wereCaCBF1 (sulfur amino acid biosynthesis; mutant defective in mor-phology), CaFCR1 (Zn-cluster TF; negative regulator of fluconazole,ketoconazole, brefeldin-A resistance; transposon mutation affectsfilamentous growth), CaCRZ2 (homozygous Cacrz1 null mutationsuppresses fluconazole resistance of homozygous Cacka2 null(defective in CK2 kinase), CaSTP4 (zinc finger DNA-binding motif;induced in core caspofungin response) and CaUPC2 (Zn(2)–Cys(6)binuclear cluster TF, involved in ergosterol biosynthesis and steroluptake; binds CaERG2 promoter). Interestingly, ScUPC2 and its tar-get genes were clearly induced in S. cerevisiae, revealing a strongdivergence of the regulation of these genes through evolution [61].
1.9. Steroids and evolution of the PDR networks
In S. cerevisiae, ScPdr1p and ScPdr3p share a very similar setof target genes and recognize the same DNA consensus sequence(PDRE) in their promoters. The ScPDR3 gene itself has been shownto be regulated by ScPdr3p and ScPdr1p and was sensitive to pro-gesterone. Most of the PDR genes were insensitive to progesteronein a �Scpdr1/�Scpdr3 strain. Remarkably, ScPdr1p was dispens-able for the regulation of ScPDR3 in these conditions, although itconstitutively binds to its promoter, suggesting that the proges-terone induction of ScPDR3 occurs through autoregulation [53].Progesterone was used to investigate the respective physiologicalroles of ScPdr1p and ScPdr3p in the PDR response to drug and con-firmed that ScPDR3 has a dispensable role in drug response. Basedon their ScPDR3 dependency in a �Scpdr1 background, one coulddistinguish between three different groups of PDR targets. ScPdr1pand ScPdr3p have identical activity on the genes encoding most ofthe main transporters involved in pleiotropic drug export (ScPDR5,ScPDR15, ScSNQ2, and ScTPO1), they have an overlapping effectwith a predominance of ScPdr1p on lipid metabolism genes, andsome co-regulated proteins of unknown function (ScRSB1, ScPDR16,ScYGR035c, and ScYLR346c) and ScPdr1p specifically regulates geneswhich (for most of them), are sensitive to other stress responsepathways (ScGRE2, YPL088w, YLL056c, ScICT1, etc.) (Fig. 3). Theauthors found a correlation between these groups and the natureand number of PDRE present in the corresponding promoters, sug-gesting that the DNA binding affinity of ScPdr1p and ScPdr3p mayexplain these different gene behaviors [17].
In C. albicans, CaTac1p is the only clear regulator of the PDRresponse identified till date [60]. CaTac1p belongs to the same fam-ily of Gal4p-like transcription factors as ScPdr1p and ScPdr3p andthe progesterone induction profile of CaTAC1 was very similar to
R. Prasad et al. / Journal of Steroid Biochemistry & Molecular Biology 129 (2012) 61– 69 65
Fig. 2. Comparison of C. albicans homolouges or orthologues genes in S. cerevisiae (mainly PDR and ERG gene family) according to their expression levels following progesteronetreatment in microarray experiment. Color coding indicates the main PDR gene families in both species: blue, ABC transporters; red, oxidoreductases from the Gre2 family;b ol dehc anerjr
tiCt
FSG
rown, putative flippases from the RTA family; gray, ICT1; green, putative aryl alcohorrelation distance between their expression profiles is significantly low (d < 0.4) (Beader is referred to the web version of the article.)
hat of ScPDR3. Noteworthy, CaTAC1 may also be subjected to pos-tive autoregulation, as suggested by the binding of CaTac1p to theaTAC1 promoter [62]. A deletion of CaTAC1 is enough to decreasehe basal level of expression of CaCDR1 and CaCDR2. This is differ-
-0,5
0
0,5
1
1,5
2
2,5
3
3,5
1BSR1ROY2ERG c530RGYw880LPY c650LLY1TCI PDR1 0
UORGGROUP I
Log2(fold induct ion)
-0,5
0
0,5
1
1,5
2
2,5
3
3,5
1BSR1ROY2ERG c530RGYw880LPY c650LLY1TCI PDR1 0
UORGGROUP I
Log2(fold induct ion)
ig. 3. Progesterone induction of PDR genes depending on expression levels in microarra. cerevisiae. Group I represents genes exclusively dependent on ScPdr1p, Group II represroup III represents genes in which absence of ScPdr1p is fully complemented by ScPdr3
ydrogenases; black, others. An arrow connects two homologues when the Pearsonee et al. [17]). (For interpretation of the references to color in this figure legend, the
ent from the situation in S. cerevisiae, where the double deletionof ScPDR1 and ScPDR3 was needed to observe the same effect.CaTac1p has been shown to recognize the DRE DNA consensusmotif present in their promoters, which is different from the S. cere-
51RDP5RDP 3RDP2QNS61RDPc643RLY YAL061 wTPO1
WT
pdr1Δpdr1Δpdr 3Δpdr3Δ
III PUORGII P
51RDP5RDP 3RDP2QNS61RDPc643RLY YAL061 wTPO1
WT
pdr1Δpdr1Δpdr 3Δpdr3Δ
III PUORGII P
y study in ScPDR1 deletant, ScPDR3 deletant and ScPDR1/ScPDR3 deletant strains inents genes in which absence of ScPdr1p is partially complemented by ScPdr3p andp (taken from Banerjee et al. [17]).
66 R. Prasad et al. / Journal of Steroid Biochemistry & Molecular Biology 129 (2012) 61– 69
PROGESTERONEPdr1p
Pdr3p Tac1p
??
snacibla .Ceaisiverec .S
??
IFU5
CDR1CDR2RTA 3
??
GRE2 , YOR1, YPL088 w, ICT1, YL L056 c, PD R10
RSB1, YLR34 6c, YGR035c, PDR16
PDR5, PDR15, SNQ 2,TPO1, YAL061w
PROGESTERONEPdr1p
Pdr3p Tac1p
??
snacibla .Ceaisiverec .S
??
IFU5
CDR1CDR2RTA 3
??
GRE2 , YOR1, YPL088 w, ICT1, YL L056 c, PD R10
RSB1, YLR34 6c, YGR035c, PDR16
PDR5, PDR15, SNQ 2,TPO1, YAL061w
F albicand
vibiCmCagtodtilistnCtodT
ttttPsbttcslaEtdttitoaasa
ig. 4. Comparison of effect of Progesterone on PDR network in S. cerevisiae and C.
ispensable for its progesterone induction (taken from Banerjee et al. [17]).
isiae PDRE [33,60]. However, the definition of the CaTAC1 regulonn C. albicans is not clear-cut. A genome-wide study has identifiedinding of CaTac1p at 37 promoters, but only 17 of the correspond-
ng genes have their expression modulated in strains harboringaTac1p gain of function mutants while, 25 CaTac1p bound pro-oters do not contain DRE [62,63]. To date, 5 genes (CaCDR1,
aCDR2, CaIFU5, CaRTA3, CaPDR16) are unambiguously considereds direct CaTAC1 targets. Remarkably, they were all induced by pro-esterone, which made steroid response a good model to explorehe CaTac1p-related transcriptional pathway (Fig. 4). Surprisingly,nly CaCDR1, CaCDR2, CaRTA3, CaMET15 and CaHSP70 were depen-ent on CaTac1p for their steroid induction. These results suggesthat other, yet unknown, transcription factors may second CaTac1pn steroid response, like ScPdr3p seconds ScPdr1p in S. cerevisiae. Ateast three ScPDR1/ScPDR3 homologues, named CaFCR1-3 have beendentified in C. albicans, with apparently no role in the basal expres-ion of CaCDR1 and CaCDR2 [64], but which are obvious candidateso be tested for their role in steroid response. It is interesting toote as to how much the CaCDR1/ScPDR5, CaCDR2/ScPDR15 and theaRTA3/ScRSB1 expression patterns have been conserved, when theranscription factors, the DNA regulatory motifs and the structuref the corresponding transcriptional networks have significantlyiverged between the two species. The role of the other putativeFs also needs to be explored.
The fact that PDR was the most sensitive pathway to proges-erone suggests that progesterone acts directly on the signal(s) thatrigger the PDR response. These signals are unknown to date andheir discovery is a key challenge in the combat against fungal infec-ions. Some inferences can be drawn from the existing literature.rogesterone, as an ergosterol analogue, may alter the lipid compo-ition and the properties of the plasma membrane. This is suggestedy the induction of ERG genes in S. cerevisiae, which occurs laterhan the PDR response. However, specific inhibitors of the ergos-erol biosynthesis pathway like azoles, although able to induce alear ERG response, are poor inducers of the PDR genes [22]. Thisuggests that the PDR and ERG response would not be necessarilyinked. Of note, in Candida, the response of ERG genes to steroid ismbiguous, CaERG1 and CaERG4 being induced while most of theRG genes are repressed, even when a clear CaCDR1/CaCDR2 induc-ion is measured. Alternatively, progesterone could act on PDR byirectly modifying the activity and properties of some PDR ABCransporters. A feedback may exist between the activity of the PDRransporters and the transcriptional regulation of the correspond-ng genes, since a knock-out of ScPDR5 has been shown to increasehe expression of ScSNQ2 and ScYOR1, this effect being dependentn ScPdr1p. The inhibition by progesterone of ScPdr5p, ScPdr15p
nd other transporter activity may thus cause ScPdr1p/ScPdr3pctivation. Although it was shown that progesterone is a good sub-trate of ScPdr5p and CaCdr1p, it is unlikely that progesteronects just by competing “natural” ScPdr5p or CaCdr1p substrates
s. Plain arrows show regulatory interaction, dashed arrows show the regulation is
for transport since: (1) other good substrates of ScPdr5p/CaCdr1p,like fluconazole, are poor inducers of the PDR response [21], (2)fluphenazine, which is not a substrate of ScPdr5p, is a good inducerof the PDR response [65] and (3) very low (1 �M) doses of proges-terone are enough to trigger an efficient ScPDR5/CaCDR1 induction[17]. Recently, estradiol-derivatives have been shown to efficientlyinhibit the drug transport and ATPase activities of ScPdr5p andCaCdr1p [66]. This suggests that human steroids act directly on theactivity of the PDR transporters through a mechanism which is yetto be determined, but which has important and long-term implica-tions on the activity of the PDR transcriptional networks and, thus,the MDR status of yeast strains.
At last but not least, it was recently shown that, in S. cere-visiae and in Candida glabrata, ScPdr1p is able to directly binddrugs like ketoconazole, which stimulates its activity at the pro-moter of MDR genes. This sensor-like property of ScPdr1p, whichis also harbored by ScPdr3p in S. cerevisiae, is reminiscent of themammalian nuclear receptors PXR, which control the expressionof human MDR genes [67]. Although the protein domains involvedin this interaction need to be more precisely identified and thefunctional consequences of xenobiotic transcription factor bindinginvestigated in more details, this work opens the interesting pos-sibility that steroids could be directly sensed by ScPdr1p, ScPdr3por CaTac1p with a high affinity. This would explain the impressivesensitivity and the very fast response of PDR to progesterone in S.cerevisiae, although S. cerevisiae strains rarely meet steroids in theirenvironment. Therefore, it is tempting to speculate that ScPdr1p,ScPdr3p and CaTac1p would act as bona fide steroid nuclear recep-tors in yeasts.
1.10. Proteomic response of C. albicans to steroid treatment
Banerjee et al., [61] used nucleo-cytoplasmic extracts andidentified changes in the proteome of C. albicans in responseto progesterone treatment. An attempt was made to findout if the transcriptional response to progesterone that wasevident within 30 min gets converted into the translationalresponse. A comparison of the gels led to the identifica-tion of a limited number of spots on the 2D-gels that wereover expressed in the progesterone treated protein samples.These protein spots were then analyzed by MALDI-TOF–TOFand the differentially over expressed proteins were identi-fied.
The proteomic analysis of progesterone response showed anover expression of proteins belonging to ribosomal protein syn-thesis machinery (CaRPL12, CaRPL28.3), transcription (CaIPF670),
R. Prasad et al. / Journal of Steroid Biochemistry & Molecular Biology 129 (2012) 61– 69 67
Table 1Proteins over-expressed in response to progesterone.
Gene name Common name Description
CA1691 CaPGK1 Phosphoglycerate kinase; enzyme of glycolysis; localizes to cell wall and to cytoplasm; antigenic during murine orhuman systemic infection; biofilm, Hog1p, GCN-induced; down-regulated upon phagocytosis; induction bysymbiont of host defense response, interaction with host.
CA5714 CaIPF2431 Protein of TSA/alkyl hydroperoxide peroxidase C (AhPC) family; similar to thiol-dependent peroxidases, roles inoxidative stress signaling; immunogenic; on hyphal surface, nucleus; yeast-form nucleus, cytoplasm; neutrophil,peroxide induced.
CA5339 CaIPF885 Protein described as similar to glucan 1,3-beta-glucosidase; regulated by Nrg1p, Tup1p; possibly regulated byTac1p; induced upon biofilm formation; induced by nitric oxide; induced during cell wall regeneration.
CA5180 CaFBA1 Putative fructose-bisphosphate aldolase; enzyme of glycolysis; antigenic in murine or human infection; regulatedon yeast-hyphal switch; induced by Efg1p, Gcn4p, Hog1p, biofilm growth, or fluconazole; phagocytosis-repressed;fungal-specific.
CA4765 CaADH1 Alcohol dehydrogenase; at surface of yeast-form cells but not hyphae; soluble in hyphae; immunogenic in humanor mouse; complements S. cerevisiae adh1 adh2 adh3 mutation; regulated by growth phase, carbon source;fluconazole-induced.
and tRNA synthetases are down-regulated upon phagocytosis by murine macrophage; translation, cytosolic largeribosomal subunit.
CA5998 CaIPF670 Nucleosome disassembly, Ada2/Gcn5/Ada3 transcription activator complex.CA4817 CaPOM152 mRNA-binding (hnRNP) protein import into nucleus, nuclear pore organization and biogenesis, protein export
from nucleus, rRNA export from nucleus, ribosomal protein import into nucleus.CA4362 CaATP2 Protein described as a similar to F1 beta subunit of F1F0 ATPase complex; antigenic in human; transcription
up-regulated in response to ciclopirox olamine; flucytosine induced; caspofungin repressed;macrophage/pseudohyphal-induced.
CA1489 CaATP3 Predicted ORF from Assemblies 19 and 20; flucytosine induced; caspofungin repressed;macrophage/pseudohyphal-induced.
CA4001 CaRPL12 Predicted ribosomal protein; down-regulated in the presence of human whole blood or polymorphonuclear(PMN) cells; genes encoding cytoplasmic ribosomal subunits are down-regulated upon phagocytosis by murinemacrophage.
CA3773 CaIPF11153 Predicted ORF in Assemblies 19 and 20; regulation correlates with clinical development of fluconazole resistance.CA2582 CaTAL1 Predicted ORF from Assembly 19; oxidative stress-induced via Cap1p; induced by nitric oxide in yhb1 mutant;
transaldolase activity, pentose-phosphate shunt, cytoplasm.CA2162 CaPDB1 Protein described as similar to pyruvate dehydrogenase; fluconazole-induced.CA0433 CaSHM1 Mitochondrial serine hydroxymethyltransferase; complements the glycine auxotrophy of an S. cerevisiae shm1
null shm2 null gly1-1 triple mutant; mitochondrial glycine hydroxy-methyl-transferase activity.CA5822 CaYRB1 Functional homolog of S. cerevisiae Yrb1p, which regulates Gsp1p GTPase activity and thereby affects
nucleocytoplasmic transport and cytoskeletal dynamics; transcription is not regulated by white-opaque switching
lies 19tory p
nrltsott
wssttanticprofbipph
or by dimorphic transition.CA5528 CaRPN7 Predicted ORF from Assemb
process, proteasome regula
ucleo-cytoplasmic transport proteins (CaYRB1, CaPOM152), stresselated proteins like CaTAL1 (oxidative stress), proteosome regu-atory protein (CaRPN4), etc. Interestingly, many of the proteinshat were found up-regulated in the 2D-gels were either theame as the genes up-regulated in the microarray experimentsr belonged to similar pathways. Table 1 lists the identified pro-eins, which were over expressed in response to progesteronereatment.
Although, this is a single study by Banerjee et al.,here proteomic approach has been employed to examine
teroid/progesterone response, it amply demonstrates that uponteroid exposure of pathogenic yeast, the up-regulation of geneshat is seen at the transcriptional level, is also relevant at theranslational level and proteins can be seen up-regulated event short progesterone exposure time-point. Notably, since onlyucleo-cytoplasmic extracts was used to analyze differential pro-ein expression in response to progesterone treatment, the mostnduced membrane bound MDR genes such as CaCDR1/CaCDR2ould not be detected in that proteomic study. Indeed, a detailedroteomic analyses is required to examine global translationesponse to progetesrone in Candida cells. The implementationf proteomics in the post-genomic era of this important humanungal pathogen can provide important information about itsiological complexity and pathogenic traits. Proteomics is an
mportant tool in C. albicans research, particularly to addressroblems that cannot be solved by genomic studies such as proteinost-translational modifications that can affect pathogenicity andost-response. It is expected that in the near future, the results
and 20; regulated by Gcn2p and Gcn4p; ubiquitin-dependent protein catabolicarticle.
from proteomic experiments will lead to novel approaches for themanagement of candidiasis.
2. Conclusions
Studies so far amply demonstrate that yeast cells respondto human steroid hormones and affect their cell growth, mor-phology and virulence. The response can be detected at bothtranscriptional and translation levels. Notably, these responsespresented by yeast upon exposure to steroids lack the involve-ment of any receptor mediated signaling cascade, which commonlyoccurs in higher eukaryotes. Steroid exposure to baker’s yeastS. cerevisiae and pathogenic yeast C. albicans show reasonablyconserved transcriptome and proteome response. The up regula-tion of MDR genes upon progesterone exposure in yeasts is mostnoteworthy as it induces both a wider spectrum of PDR genesand induces them to higher levels of expression, as comparedto well known inducer drugs. Steroids also act as substrates ofMDR transporters and transiently induce levels of MDR trans-porter encoding genes making resistant the otherwise susceptibleCandida strains to azoles. Progesterone response in S. cerevisiaehelped uncovering PDR targets based on their PDR3 dependencyin a pdr1� background one could detect otherwise undistinguish-able three different groups of PDR targets. A correlation between
PDR target groups and the nature and number of PDRE presentin the promoters of the corresponding genes, suggested that theDNA binding affinities of ScPdr1p and ScPdr3p may be different.The response towards PDR and ERG gene in S. cerevisiae strains
6 mistry
ac
R
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
[
8 R. Prasad et al. / Journal of Steroid Bioche
ppeared to be only partially conserved in pathogenic C. albi-ans.
eferences
[1] S. Tanaka, S. Hasegawa, F. Hishinuma, S. Kurata, Estrogen can regulate the cellcycle in the early phase of yeast by increasing the amount of adenylate cyclasemRNA, Cell 57 (1989) 675–681.
[2] M. Zakelj-Mavrik, A. Kastelic-Suhadolk, A. Plementas, T.L. Rizner, I. Belic, Steroidhormone signaling system and fungi, Comp. Biochem. Physiol. 112B (1995)637–642.
[3] P.R. Gujjar, M. Finucane, B. Larsen, The effect of estradiol on Candida albicansgrowth, Ann. Clin. Lab. Sci. 27 (1997) 151–156.
[4] X. Zhang, M. Essman, E.T. Burt, B. Larsen, Estrogen effects on Candida albicans: apotential virulence-regulating mechanism, J. Infect. Dis. 181 (2000) 1441–1446.
[5] L. Romani, F. Bistoni, P. Puccetti, Adaptation of Candida albicans to the host envi-ronment: the role of morphogenesis in virulence and survival in mammalianhosts, Curr. Opin. Microbiol. 6 (2003) 338–343.
[6] O.S. Kinsman, A.E. Collard, Hormonal factors in vaginal candidiasis in rats, Infect.Immun. (1986) 498–504.
[7] S. White, B. Larsen, Candida albicans morphogenesis is influenced by estrogen,Cell Mol. Life Sci. 53 (1997) 744–749.
[8] X. Zhang, M. deMicheli, S.T. Coleman, D. Sanglard, W.S. Moye-Rowley, Analysisof the oxidative stress regulation of the Candida albicans transcription factor,Cap1p, Mol. Microbiol. 36 (2000) 618–629.
[9] O.S. Kinsman, K. Pitblado, C.J. Coulson, Effect of mammalian steroid hormonesand luteinizing hormone on the germination of Candida albicans and implica-tions for vaginal candidosis, Mycoses 31 (2002) 617–626.
10] Jr.P.L. Fidel, J. Cutright, C. Steele, Effects of reproductive hormones on experi-mental vaginal candidiasis, in: Anonymous, 2001, pp. 651–657.
11] N.D. Madani, P.J. Malloy, P. Rodrigues-Pombo, A.V. Krishnan, D. Feldman, Can-dida albicans estrogen-binding protein gene encodes an oxidoreductase that isinhibited by estradiol, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 922–926.
12] P.J. Malloy, X. Zhao, N.D. Madani, D. Feldman, Cloning and expression of thegene from Candida albicans that encodes a high-affinity corticosteroid-bindingprotein, Proc. Natl. Acad. Sci. U.S.A. 90 (1993) 1902–1906.
13] M. Das, A. Datta, Steroid binding protein(s) in yeasts, Biochem. Int. 11 (1985)171–176.
14] D. Banerjee, B. Pillai, N. Karnani, G. Mukhopadhyay, R. Prasad, Genome-wideexpression profile of steroid response in Saccharomyces cerevisiae, Biochem.Biophys. Res. Commun. 317 (2004) 406–413.
15] G. Cheng, K.M. Yeater, L.L. Hoyer, Cellular and molecular biology of Candidaalbicans estrogen response, Eukaryot. Cell 5 (2006) 180–191.
16] D. Banerjee, N. Martin, S. Nandi, S. Shukla, A. Dominguez, G. Mukhopadhyay,R. Prasad, A genome-wide steroid response study of the major human fungalpathogen Candida albicans, Mycopathologia 164 (2007) 1–17.
17] D. Banerjee, G. Lelandais, S. Shukla, G. Mukhopadhyay, C. Jacq, F. Devaux, R.Prasad, Responses of pathogenic and nonpathogenic yeast species to steroidsreveal the functioning and evolution of multidrug resistance transcriptionalnetworks, Eukaryot. Cell 7 (2008) 68–77.
18] M.E. Fling, J. Kopf, A. Tamarkin, J.A. Gorman, H.A. Smith, Y. Koltin, Analysisof a Candida albicans gene that encodes a novel mechanism for resistance tobenomyl and methotrexate, Mol. Gen. Genet. 227 (1991) 318–329.
19] R. Prasad, P.D. Worgifosse, A. Goffeau, E. Balzi, Molecular cloning and charac-terisation of a novel gene of C. albicans, CDR1, conferring multiple resistance todrugs and antifungals, Curr. Genet. 27 (1995) 320–329.
20] D. Sanglard, F. Ischer, M. Monod, J. Bille, Cloning of Candida albicans genes con-ferring resistance to azole antifungal agents: characterisation of CDR2, a newmultidrug ABC transporter gene, Microbiology 143 (1997) 405–416.
21] S. Krishnamurthy, V. Gupta, P. Snehlata, R. Prasad, Characterisation of humansteroid hormone transport mediated by Cdr1p, multidrug transporter of Can-dida albicans, belonging to the ATP binding cassette super family, FEMSMicrobiol. Lett. 158 (1998) 69–74.
22] S. Krishnamurthy, U. Chatterjee, V. Gupta, R. Prasad, P. Das, P. Snehlata, S.E.Hasnain, R. Prasad, Deletion of transmembrane domain 12 of CDR1, a multidrugtransporter from Candida albicans, leads to altered drug specificity: expressionof a yeast multidrug transporter in Baculovirus expression system, Yeast 14(1998) 535–550.
23] M. Kolaczkowski, M.E. van der Rest, A. Cybularz-Kolaczkowska, J.-P. Soumillion,W.N. Konings, A. Goffeau, Anticancer drugs, ionophoric peptides, and steroidsas substrates of the yeast multidrug transporter Pdr5p, J. Biol. Chem. 271 (1996)31543–31548.
24] Y. Mahe, A. Parle-McDermott, A. Nourani, A. Delahodde, A. Lamprecht, K. Kuch-ler, The ATP-binding cassette multidrug transporter Snq2 of Saccharomycescerevisiae: a novel target for the transcription factors Pdr1 and Pdr3, Mol.Microbiol. 20 (1996) 109–117.
25] Y. Li, Y.H. Wang, L. Yang, S.W. Zhang, C.H. Liu, S.L. Yang, Comparison of steroidsubstrates and inhibitors of P-glycoprotein by 3D-QSAR analysis, J. Mol. Struct.733 (2005) 111–118.
26] S. Velamakanni, T. Janvilisri, S. Shahi, H.W. van Veen, A functional steroid-
binding element in an ATP-binding cassette multidrug transporter, Mol.Pharmacol. 73 (2008) 12–17.
27] E. Leberer, D. Harcus, I.D. Broadbent, K.L. Clark, D. Dignard, A.Y. Schmidt,N.A. Gow, A.J. Brown, D. Thomas, Signal transduction through homologsof the Ste20p and Ste7p protein kinases can trigger hyphal formation in
28] R. Manoharlal, M. Sharma, R. Prasad, Molecular determinants of transient andreversible induced up-regulation of CaCDR1 in azole susceptible clinical iso-lates of Candida albicans, Biosci. Rep. (2010).
29] S. Krishnamurthy, V. Gupta, R. Prasad, S.L. Panwar, R. Prasad, Expression ofCDR1, a multidrug resistance gene of Candida albicans: In vitro transcriptionalactivation by heat shock, drugs and human steroid hormones, FEMS Microbiol.Lett. 160 (1998) 191–197.
30] M.L. Hernaez, C. Gil, J. Pla, C. Nombela, Induced expression of the Candida albi-cans multidrug resistance gene CDR1 in response to fluconazole and otherantifungals, Yeast 14 (1998) 517–526.
31] N. Puri, S. Krishnamurthy, S. Habib, S.E. Hasnain, S.K. Goswami, R. Prasad, CDR1,a multidrug resistance gene from Candida albicans, contains multiple regulatorydomains in its promoter and the distal AP-1 element mediates its induction bymiconazole, FEMS Microbiol. Lett. 180 (1999) 213–219.
32] N. Karnani, N.A. Gaur, S. Jha, N. Puri, S. Krishnamurthy, S.K. Goswami, G.Mukhopadhyay, R. Prasad, SRE1, SRE2 are two specific steroid-responsive mod-ules of Candida drug resistance gene 1 (CDR1) promoter, Yeast 21 (2004)219–239.
33] M. de Micheli, J. Bille, C. Schueller, D. Sanglard, A common drug-responsive ele-ment mediates the upregulation of the Candida albicans ABC transporters CDR1and CDR2, two genes involved in antifungal drug resistance, Mol Microbiol 43(2002) 1197–1214.
34] B. Panaretou, P.W. Piper, Plasma-membrane ATPase action affects severalsstress tolerances of Saccharomyces cerevisiae and Schizosaccharomyces pombeas well as the extent and duration of the heat shock response, J. Gen. Microbiol.136 (1990) 1763–1770.
35] K. Miyahara, D. Hirata, T. Miyakawa, yAP-1- and yAP-2-mediated, heat shock-induced transcriptional activation of the multidrug resistance ABC transportergenes in Saccharomyces cerevisiae, Curr. Genet. 29 (1996) 103–105.
36] H. Wolfger, Y.M. Mamnun, K. Kuchler, Fungal ABC proteins: pleiotropic drugresistance, stress response and cellular detoxification, Res. Microbiol. 152(2001) 375–389.
37] A.P. Gasch, A.P. Spellman, M.C. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D.Botstein, P.O. Brown, Genomic expression programs in the response of yeastcells to environmental changes, Mol. Biol. Cell 11 (2000) 4241–4257.
38] T. Ketala, R. Green, H. Bussey, Saccharomyces cerevisiae Mid2p is a potentialcell wall stress sensor and upstream activator of the PKC1-MPK1 cell integritypathway, J. Bacteriol. 181 (1999) 3330–3340.
39] G. Marchler, C. Schuller, G. Adam, H. Ruis, A Saccharomyces cerevisiae UAS ele-ment controlled by protein kinase A activates transcription in response to avariety of stress conditions, EMBO J. (1993) 1997–2003.
40] N. Kobayashi, K. McEntee, Identification of cis and trans components of a novelheat shock stress regulatory pathway in Saccharomyces cerevisiae, Mol. Cell.Biol. 13 (1993) 248–256.
41] J.L. DeRisi, V.R. Iyver, P.O. Brown, Exploring the metabolic and genetic controlof gene expression on a genome scale, Science 278 (1997) 680–686.
42] E. Moskvina, C. Schuller, C.T.C. Maurer, W.H. Mager, H. Ruis, Yeast functionalanalysis reports, Yeast 14 (1998) 1041–1050.
43] J.L. Parrou, M.A. Teste, J. Francois, Effects of various types of stress on themetabolism of reserve carbohydrates in Saccharomyces cerevisiae: genetic evi-dence for a stress-induced recycling of glycogen and trehalose, in: Anonymous,1997, pp. 1891–1900.
44] K Barnes, B. Dickstein, G.B. Cutler, T. Fojo, S.E. Bates, Steroid accumulation,and antagonism of P-glycoprtein in multidrug resistant cells, Biochemistry 35(1996) 4820–4827.
45] M. Sukhai, M. Piquette-Miller, Regulation of the multidrug resistance genes bystress signals, in: Anonymous, 2000, pp. 268–280.
46] S. Labialle, L. Gayet, E. Marthinet, D. Rigal, L.G. Baggetto, Transcriptional reg-ulators of the human multidrug resistance 1 gene: recent reviews, Biochem.Pharmacol. 64 (2003) 943–948.
47] P.D. Rogers, K.S. Barker, Evaluation of differential gene expression influconazole-susceptible and -resistant isolates of Candida albicans by cDNAanalysis, Antimicrob. Agents Chemother. 46 (2002) 3412–3417.
48] R. Ernst, R. Klemm, L. Schmitt, K. Kuchler, Yeast ATP-binding cassette trans-porters: cellular cleaning pumps Methods, Enzymology 400 (2005) 460–484.
49] Y. Wang, Y.Y. Cao, X.M. Jia, Y.B. Cao, P.H. Gao, X.P. Fu, K. Ying, W.S. Chen, Y.Y.Jiang, Cap1p is involved in multiple pathways of oxidative stress response inCandida albicans, Free Radic. Biol. Med. 40 (2006) 1201–1209.
50] J. DeRisi, B.v.d. Hazel, P. Marc, E. Balzi, P. Brown, C. Jacq, A. Goffeau, Genomemicroarray analysis of transcriptional activation in multidrug resistance yeastmutants, FEBS Lett. 470 (2000) 156–160.
51] M. Sidorova, E. Drobna, V. Dzugasova, I. Hikkel, J. Subik, Loss-of-function pdr3mutations convert the Pdr3p transcription activator to a protein suppressingmultidrug resistance in Saccharomyces cerevisiae, FEMS Yeast Res. 7 (2007)254–264.
52] W.S. Moye-Rowley, Regulation of the transcriptional response to oxidativestress in fungi: similarities and differences, Eukaryot. Cell 2 (2003) 381–389.
53] A. Delahodde, T. Delaveau, C. Jacq, Positive autoregulation of the yeast tran-scription factor Pdr3p, which is involved in control of drug resistance, Mol.
Cell. Biol. 15 (1995) 4043–4051.
54] E. Carvajal, H.B. Van Den Hazel, A. Cybularz-Kolaczkowska, E. Balzi, A. Gof-feau, Molecular and phenotypic characterisation of yeast PDR1 mutants thatshow hyperactive transcription of various ABC multidrug transporter genes,Mol. Gen. Genet. 256 (1997) 406–415.
mistry
[
[
[
[
[
[
[
[
[
[
[
[
R. Prasad et al. / Journal of Steroid Bioche
55] A. Nawrocki, S.J. Fey, A. Goffeau, P. Roepstorff, P.M. Larsen, The effects oftranscription regulating genes PDR1, pdr1-3 and PDR3 in pleiotropic drug resis-tance, Eur. J. Mass Spectrom. 7 (2001) 195–205.
56] M.S. Tuttle, D. Radisky, L. Li, J. Kaplan, A dominant allele of PDR1 alterstransition metal resistance in yeast, J. Biol. Chem. 278 (2003) 1273–1280.
57] S. MacPherson, B. Akache, S. Weber, D. De, X.M. Raymond, B. Turcotte, Candidaalbicans zinc cluster protein Upc2p confers resistance to antifungal drugs and isan activator of ergosterol biosynthetic genes, Antimicrob. Agents Chemother.49 (2005) 1745–1752.
58] T.T. Liu, R.E. Lee, K.S. Barker, R.E. Lee, L. Wei, R. Homayouni, P.D. Rogers,Genome-wide expression profiling of the response to azole, polyene,echinocandin, and pyrimidine antifungal agents in Candida albicans, Antimi-crob. Agents Chemother. 49 (2005) 2226–2236.
59] A.T. Coste, V. Turner, F. Ischer, J. Morschhauser, A. Forche, A. Selmecki, J. Berman,J. Bille, D. Sanglard, A mutation in Tac1p, a transcription factor regulatingCDR1 and CDR2, is coupled with loss of heterozygosity at Chromosome 5 tomediate antifungal resistance in Candida albicans, Genetics 172 (2006) 2139–
2156.
60] A.T. Coste, M. Karababa, F. Ischer, J. Bille, D. Sanglard, TAC1, transcriptionalactivator of CDR genes, is a new transcription factor involved in the regulationof Candida albicans ABC transporters CDR1 and CDR2, Eukaryot. Cell 3 (2004)1639–1652.
[
& Molecular Biology 129 (2012) 61– 69 69
61] D. Banerjee, Transcriptome analysis of steroid response in yeasts, JNU, Ph.D.thesis, 2008.
62] T.T. Liu, S. Znaidi, K.S. Barker, L. Xu, R. Homayouni, S. Saidane, J. Morschhauser, A.Nantel, M. Raymond, P.D. Rogers, Genome-wide expression and location anal-yses of the Candida albicans Tac1p regulon, Eukaryot. Cell 6 (2007) 2122–2138.
63] S. Znaidi, D. De, X.S. Weber, T. Rigby, A. Nantel, M. Raymond, The zinc clustertranscription factor Tac1p regulates PDR16 expression in Candida albicans, Mol.Microbiol. 66 (2007) 440–452.
64] D. Talibi, M. Raymond, Isolation of a putative Candida albicans transcriptionalregulator involved in pleiotropic drug resistance by functional complementa-tion of a pdr1 pdr3 mutation in Saccharomyces cerevisiae, J. Bacteriol. 181 (1999)231–240.
65] V. Fardeau, G. Lelandais, A. Oldfield, H. Salin, S. Lemoine, M. Garcia, V. Tanty,C.S. Le, C. Jacq, F. Devaux, The central role of PDR1 in the foundation of yeastdrug resistance, J. Biol. Chem. 282 (2007) 5063–5074.
66] G. Conseil, J.M. Perez-Victoria, J.M. Renoir, A. Goffeau, P.A. Di, Potent competi-tive inhibition of drug binding to the Saccharomyces cerevisiae ABC exporterPdr5p by the hydrophobic estradiol-derivative RU49953, Biochim. Biophys.
Acta 1614 (2003) 131–134.
67] J.K. Thakur, H. Arthanari, F. Yang, S.J. Pan, X. Fan, J. Breger, D.P. Frueh, K. Gulshan,D.K. Li, E. Mylonakis, K. Struhl, W.S. Moye-Rowley, B.P. Cormack, G. Wagner,A.M. Naar, A nuclear receptor-like pathway regulating multidrug resistance infungi, Nature 452 (2008) 604–609.
