Response of pathogenic and non-pathogenic yeasts to steroids

Journal of Steroid Biochemistry & Molecular Biology 129 (2012) 61–69

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Response of pathogenic and non-pathogenic yeasts to steroids Rajendra Prasad a,∗ , Frédéric Devaux b , Sanjiveeni Dhamgaye a , Dibyendu Banerjee a,1 a b

Membrane Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India Laboratoire de génomique des microorganismes, CNRS FRE3214, Université Pierre et Marie Curie, 15 rue de l’école de médecine, 75006 Paris, France

a r t i c l e

i n f o

Article history: Received 24 July 2010 Received in revised form 10 November 2010 Accepted 18 November 2010 Keywords: Saccharomyces cerevisiae Candida albicans ␤-Estradiol Progesterone Cellular stress Transcriptome

a b s t r a c t Steroids are known to induce pleiotropic drug resistance states in hemiascomycetes, with tremendous potential consequences on human fungal infections. The proteins capable of binding to steroids such as progesterone binding protein (PBP), estradiol binding proteins (ESP) are found in yeasts, however, the well 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 activation of 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 discuss that a fairly conserved response to steroids at the level of transcription and translation exists between pathogenic and non-pathogenic yeasts. Article from a special issue on steroids and microorganisms. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Human steroid hormones affect growth, morphogenesis and drug susceptibilities of pathogenic and non-pathogenic yeast cells. However, the molecular basis of steroids action and signaling remains unresolved [1–5]. Relevance of human steroids in yeast physiology has emerged from several observations, which showed that yeast cells such as pathogenic Candida albicans undergoes changes in its morphology and growth rate upon supplementation of the growth medium with steroids [3,4,6–9]. Another factor that relates steroids with the patho-physiology of C. albicans is the prevalence of vulvovaginal candidiasis (VVC), which most often occurs in women during the late luteal phase of the menstrual cycle, when estrogen and progesterone levels are elevated [10]. The presence of human steroid hormones in Candida’s host would also imply that these could act as environmental cues in controlling its virulence [6,9]. Taken together, though steroids are known to 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 that the well-characterized steroid receptor pathways found in higher eukaryotes are non-existent not only in Candida but also in other yeasts and fungi as well [2]. Interestingly, steroid binding proteins such as estradiol binding protein (EBP) [11] corticosteroid binding protein (CBP) [12] and progesterone binding protein (PBP) [13] do exist in Candida. However, they appear to be non-DNA binding proteins [11], and their role in steroid signaling in yeast is not very clear. There are very limited studies where steroid response in yeasts has been examined. Therefore, transcriptome and proteome responses to steroids are particularly discussed from our own and a few other studies [14–17]. Nonetheless, these limited studies already demonstrate that yeast cells are extremely responsive to steroids in affecting its morphology, growth and expression profile of several genes and proteins.

1.1. Steroids as substrates of MDR pump proteins Abbreviations: VVC, vulvovaginal candidiasis; EBP, estradiol binding protein; CBP, corticosteroid binding protein; PBP, progesterone binding protein; ABC, ATP-binding cassette; MF, major facilitator; R6G, rhodamine 6G; 3D-QSAR, three-dimensional quantitative structure activity relationship; SRR, steroid responsive region; DRE, drug responsive element; BRE, basal responsive element; PDR, pleiotropic drug resistance; MDR, multidrug resistance. ∗ Corresponding author. Tel.: +91 11 26704509; fax: +91 11 26741081. E-mail addresses: [email protected], [email protected] (R. Prasad). 1 Present address: Department of Radiation Oncology, University of Maryland, School of Medicine, Baltimore, MD 21201, United States. 0960-0760/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2010.11.011

C. albicans and other pathogenic species of Candida derive their importance not only from the severity of their infections but also for their ability to develop resistance against antifungals, such as azoles, in patients undergoing long-term or prophylactic treatment. Although several mechanisms which contribute to the azole-resistance in clinical isolates have been identified, upregulation of drug extrusion pump encoding genes belonging to the superfamily of either ABC (ATP-binding cassette, e.g. CaCDR1 and

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Fig. 1. Physiological response of steroids in yeasts.

CaCDR2) or MF (major facilitator, e.g. CaMDR1) represents one of the most prevalent mechanisms of drug resistance [18–20]. Steroids act as substrates of ABC proteins. For example, multidrug transporter CaCdr1p can specifically transport ␤-estradiol and corticosterone, which could be blocked by molar excesses of ␤-estradiol, corticosterone, ergosterol or dexamethasone. Notably, progesterone which generates good response in yeasts, is not a substrate of CaCdr1p [21]. Interestingly, some of the drugs such as cycloheximide, chloramphenicol, fluconazole and o-phenanthroline, to which an overexpression of CaCDR1 confers resistance, could also prevent efflux and enhance accumulation of ␤-estradiol implying common binding site sharing between steroids and drugs [22]. In conclusion, human steroid hormones are the substrates for CaCdr1p and the energy dependent transport mediated by it is specific for ␤-estradiol and corticosterone (Fig. 1). Unlike CaCdr1p, the MFS multidrug transporter CaMdr1p does not transport steroids. Kolaczkowski et al. [23], earlier demonstrated that ABC transporter ScPdr5p (homologue of CaCdr1p) of baker’s yeast Saccharomyces cerevisiae mediates resistance to progesterone and deoxycorticosterone by showing hyper susceptibility of Scpdr5 strain to both of these steroids. They also observed that both progesterone and deoxycorticosterone competitively inhibit Pdr5p mediated rhodamine 6G (R6G) transport, which strongly suggested that they are indeed transport substrates of ScPdr5p. Mahe et al. [24], have monitored (3 H)-estradiol accumulation in Scpdr5 and Scsnq2 null strains and observed that it was increased by 3-fold in Scpdr5 as compared to wild type strain, however, Scsnq2 nulls did not show much difference. The subsequent deletion of both Scpdr5 and Scsnq2 led to even higher accumulation of (3 H)estradiol implying that ScSnq2p works in concert with ScPdr5p to contribute for ␤-estradiol export in S. cerevisiae cells. Steroids are well known substrates of MDR proteins of higher eukaryotes as well. For example, human ABC transporter Pglycoprotein (Pgp) which is present in various tumor cells and imparts drug resistance to them can export various steroids. Using three-dimensional quantitative structure activity relationship (3DQSAR), cortisol, aldosterone, dexamethasone, 11-deoxycortisol and

corticosterone were identified as substrates while pregnanedione and progesterone were strong inhibitors of human Pgp [25]. Recently, a steroid-binding element has been identified in the membrane domain of ABCG2 an ATP binding cassette transporter that confers drug resistance to cancer cells. Steroid binding to this element results in modulation of ABCG2 activity [26]. 1.2. Steroid as growth and morphogenesis regulator Morphological switching from yeast to hyphal form has been regarded as one of the important virulence traits [27]. This switch depends on various environmental cues inside the host. Steroid presents one such environmental cue that affect hyphal development of C. albicans. White’s group demonstrated the effect of mammalian hormones viz. estradiol, testosterone, cholesterol on clinical isolates of C. albicans and showed that there was a noted delay in the ability of steroid stripped serum to facilitate the germination, which was partially restored upon the addition of estradiol [7]. Cheng et al. [15] focused on the effect of 17-␤ estradiol, ethynyl estradiol and estriol on several C. albicans strains and observed an increase in germ tube length in a dose and strain dependent manner. This study has further revealed a potential relationship between 17-␤-estradiol and the upregulation of CaPDR16 and CaPLD1, which promote hyphae formation in C. albicans. Recently, in a transcriptome based study, Banerjee et al. [16] observed differential regulation of genes associated with hyphal development and showed that progesterone is a morphogenetic regulator influencing 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 resistant (AR) clinical isolates, in the presence and absence of the human steroid hormones progesterone and ␤-estradiol revealed a consistent increase in MIC80 for the drugs like fluconazole and

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ketoconazole [16]. Recently, azole susceptible clinical isolates of C. albicans have been shown to acquire transient drug resistance in presence of steroids ␤-estradiol and progesterone. The raised levels of R6G efflux in progesterone and ␤-estradiol induced AS strains and their ability to withstand higher doses of azole drugs as compared with basal levels, confirmed that steroid induction imparts resistance to otherwise susceptible clinical isolates [28]. 1.4. Steroids transcriptionally activate MDR genes of yeasts Recent efforts have revealed that the CaCDR1 promoter not only responds to multitude of drugs but also to other environmental stimuli such as heat shock, heavy metals and human steroids [29–31]. Karnani et al. [32] have identified presence of steroid responsive region (SRR) that confers ␤-estradiol and progesterone inducibility to CaCDR1 promoter. SRR spans a region between −696 and −521 bp upstream of transcription start site. SRR was further divided into two progesterone responsive sequences (−628 to −594 and −683 to −648) and one ␤-estradiol responsive sequence (−628 to −577). Even more deletions in the SRR delimited it to two distinct elements that were called SRE1 and SRE2. Both SREs are specific for steroids, SRE1 respond only to progesterone whereas SRE2 respond to both progesterone and ␤-oestradiol. de Micheli et al. [33], also identified the presence of cis regulatory elements in the promoter of CaCDR1 and CaCDR2 responsive to estradiol. To identify the regulatory elements, the promoter deletions of both these genes were cloned in frame with Renilla luciferase reporter gene and exposed to estradiol for transient induction. This led to the identification of two regulatory elements i.e. BEE (basal expression element) and DREI (drug responsive element) in the CaCDR1 promoter; BEE is required for basal expression and DRE for estradiol responsiveness. However, only one regulatory element was found in CaCDR2 called DREII that was responsive for estradiol responsiveness. DREI and DREII share a 21 base pair consensus sequence that is not common to any known eukaryotic element. Unlike SRE1 and SRE2, which are exclusively responsive to steroids, DREs are responsive to both steroids and drugs. 1.5. The transcriptome response of steroids mimics stress responses Whole genome cDNA microarray analyses in response to the human steroid hormone progesterone were performed in C. albicans and in S. cerevisiae, using a supra-physiological concentration (1 mM) of progesterone and a 30 min time-point, which gave the highest expression response of CaCDR1 and CaCDR2 in C. albicans [16]. Of note, it was reported that using a physiological or nearphysiological concentration of oestrogen had very few effects on gene expression in C. albicans cells [15]. In S. cerevisiae, the overall scenario of differentially regulated genes suggested that the short-term progesterone treatment leads to a stress response in yeast. For example, transport facilitation genes, which are involved in drug and other xenobiotic efflux, comprise a large percentage of up-regulated genes (12%). These genes have been previously shown to be up-regulated by transcription factors ScPDR1 and ScPDR3, heat shock, H2 O2, and other stressful conditions [34–36], (http://www.transcriptome.ens.fr/ymgv/). Additionally, several genes belonging to the cell cycle control (ScAPC11, ScCIS1, ScCDC34, ScPCL6, ScMID2, ScCTF13, ScNUF2, and ScTRF4) and to the biogenesis of cell wall were induced by progesterone exposure. Interestingly these genes also respond to various other cellular stresses (http://www.transcriptome.ens.fr/ymgv/) [37]. Notably, ScMID2, which encodes a potential cell wall stress sensor and upstream activator of cell integrity pathway [38], was significantly up-regulated by progesterone exposure. The genes involved in carbohydrate utilization, regulation and transport

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(ScYAL061W, ScTDH1, ScFBP1, ScGAL80, ScSHR5, ScYOL007C, ScHXT6, ScHXT7), which are induced by various stresses [37], also exhibited up-regulation by progesterone treatment. The STREs (having core consensus AG4 or C4 T) have been demonstrated to occur in the upstream promoter region of a number of genes responsive to stress signals [39–42]. The analysis of steroid affected genes revealed that several affected gene promoters had STRE sequences present at least two-times or more. The high doses of progesterone which are perceived as a stress response by the S. cerevisiae cells was further evident from among the down-regulated genes. During stressful conditions, when the cell needs to devote more of its resources to diffuse the stress, ribosomal protein synthesis is shut down [43] Notably, a number of genes encoding ribosomal proteins (ScRPS3, ScRPS5, ScRPS4B, ScRPS6B, ScRPL4B, and ScRPL17B) were significantly down-regulated by steroids. Similarly, in C. albicans, many of the genes induced by progesterone belong to drug resistance and its associated gene categories. Some hyphae-associated genes like CaGPX1 (CaEfg1p, CaRfg1p, CaCyr1p, CaTup1p and CaNrg1p regulated), CaDUR1,2 (CaNrg1p regulated), CaMIG1 (CaTUP1 dependent and independent), CaGDH3, CaHSP90 (hyphal surface localization), CaGLG2 (hyphal induced), CaCDC19 (mutation affects filamentation), CaPDC11 (found only in hyphal cells) were also induced. The hyphae-associated genes affected by progesterone reflect the similarity between steroid response and dimorphic transition regulation [16]. The upregulated categories of genes included stress-associated genes like CaDDR48 (gene consistently up-regulated under conditions favoring filamentation), CaPRB1 (heat regulated, GlcNAc induced), CaSSA4 (heat shock protein), CaKAR2 (similar to chaperone of Hsp70 family; expression greater in high iron), CaHSP90, CaHSP12 and CaHSP60 (heat shock chaperone proteins) and CaIPF6629 (oxidative stress response). Notably, C. albicans CaHsp90p can provide steroid dependent activation of a mammalian steroid receptor when both proteins are expressed in S. cerevisiae [34]. Interestingly, the SRR consensus sequences are present in many responsive genes. It also includes an inverted CCAAT box which in combination with other conserved sequences is attributed towards human CaMDR1 responsiveness to cellular stresses [44–46]. Thus, yeast cells perceive high doses of steroids as a cellular stress. Several similarities can be observed between yeast cells exposed to human steroid hormones and those exposed to other 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 to be a very good inducer of filamentation in C. albicans. Several genes were 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 mM down to 1 nM) of the human steroid hormone progesterone was conducted. Some interesting insights into the patho-physiological similarities and differences between the two yeast species emerged from that study. In S. cerevisiae, two pathways are mainly responsible for multidrug-resistance phenotypes: the PDR pathway, controlled by the ScPdr1p and ScPdr3p transcription factors, and the ergosterol biosynthesis pathway [50–52]. Gene ontology and DNA regulatory motif mining of microarray data indicated that progesterone specifically and extensively activated these two pathways. The DNA consensus motif bound by ScPdr1p and ScPdr3p (named PDRE) was found to be significantly correlated with progesterone induction. These genes represent about 80% of the ScPdr1p/ScPdr3p targets defined previously. Such a complete PDR response had been only observed in the case of constitutively multidrug-resistant strains harboring ScPDR1 or ScPDR3 gain of

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function alleles [50,53–56]. This makes progesterone the most efficient inducer of PDR pathway of S. cerevisiae. The early PDR response of progesterone exposure was followed by the induction of genes involved in the ergosterol biosynthesis. As a consequence, the ergosterol metabolism pathway was the main GO (gene ontology) category to be significantly enriched if cells were exposed to progesterone for longer time (∼90 min). These genes encode enzymes from the ERG pathway (ScERG25, ScERG11, ScERG5, ScERG4, ScERG3 and ScERG28), the ScPdr16p protein, etc. Interestingly, the transcriptional activator of the ERG genes, ScUpc2p, was itself similarly induced. Moreover, the global expression of ScUpc2p DNA binding motif-containing genes was significantly correlated with progesterone induction [17]. ScUpc2p is reported to confer resistance to antifungal drugs [57] and thus may play a role in drug export and resistance. Noteworthy, the PDR and ERG response to progesterone was very stable, being observed up to 7 h after the beginning of the treatment. 1.7. The PDR response of steroids is partially conserved in C. albicans In C. albicans, our knowledge of MDR regulation is rather limited. Multidrug resistance can be due to the up-regulation of multidrug transporters belonging to the ABC (ATP-binding cassette) transporter family (e.g. CaCDR1 and CaCDR2), which is the equivalent of the PDR pathway of budding yeast, or of the major facilitator family (e.g. CaMDR1), which is the equivalent of the YAP1/FLR1 pathway in S. cerevisiae. In one transcriptome-based study, CaCDR1 and CaCDR2 genes were not induced by MIC50 concentrations of drugs like amphotericin B, caspofungin and fluorocytosine and only ketoconazole could induce both CaCDR1/2 [58]. Of note, only fluphenazine had been shown to induce a large CaCDR1/CaCDR2 response in C. albicans. Remarkably, progesterone induced many genes known to be co-regulated with CaCDR1/CaCDR2 in C. albicans [59,60]. This included CaCDR1, CaCDR2, CaIFU5, CaRTA3 and CaHSP12. The levels of transcription factor CaMMR1 which exclusively regulates CaMDR1, did not change in response to progesterone treatment. This would support the fact that the regulation of CaMDR1 is different from those of ABC transporters. The CaCDR1 gene expression profile was similar to the ScPDR5 expression profile, indicating that the mechanisms involved in the progesterone induction of these genes may be conserved from S. cerevisiae to C. albicans. Remarkably, this is in spite of the fact that the promoters of the two genes harbor different regulatory elements. To achieve a global qualitative and quantitative estimation of the conservation of the PDR and ERG responses to progesterone in a non-pathogenic and a pathogenic yeast species, two independent hierarchical clustering of the PDR and ERG genes induced by progesterone in S. cerevisiae and of their closest homologues in C. albicans were made and directly compared the gene expression profiles of the homologous gene pairs using Pearson correlation distances. No conservation of the ERG response identified in S. cerevisiae and in C. albicans was evident. In contrast, a full conservation of the induction of the oxidoreductase (Gre2p-like) and of the putative flippase (Rsb1p-like) encoding genes involved in the PDR response, two homologous genes being similarly induced in both species, was observed. However, in the case of the ABC transporter family, only a partial conservation was seen, with just two Candida genes (CaCDR1 and CaCDR2) being up-regulated by progesterone while four of their homologues (ScPDR5, ScPDR15, ScPDR10 and ScSNQ2) were induced in S. cerevisiae [17] (Fig. 2). Notably, about 20 ScPdr1/ScPdr3 target genes get induced in S. cerevisiae, when only 9 homologues of these genes were similarly regulated in C. albicans. One simple explanation would be that the MDR pathways in C. albicans involve genes which are not clear homologues of the S. cerevisiae PDR genes but which play a similar

role. Actually, this is the case of the C. albicans MDR transcriptional regulator, CaTac1p, which is not strictly homologous to the S. cerevisiae ScPdr1p and ScPdr3p but which belongs to the same family of Gal4p-like transcription factors, and exhibited a similar expression profile in response to progesterone. Many known putative CaTac1p targets were induced by progesterone (discussed in Section 1.9) [17]. 1.8. Several transcription factors are induced in response to progesterone As mentioned above, yeast and fungi are responsive to steroid exposure but are not known to have their receptors as in higher eukaryotes. The Candida transcription factors induced by progesterone in the microarray experiments and thus tentatively involved in steroid response are: CaTAC1 (transcriptional activator of drug-responsive genes including CaCDR1 and CaCDR2), CaGAT2 (mutation affects filamentous growth), CaCZF1 (hyphal growth regulator), CaZNC1 (Zn(2)–Cys(6) binuclear cluster, regulated by CaGcn2p and CaGcn4p), CaZCF39 (filament induced), CaIPF2822 (Zn-finger TF) and CaCAS5 (cell wall damage response; downregulated in core stress response). The down-regulated TFs were CaCBF1 (sulfur amino acid biosynthesis; mutant defective in morphology), CaFCR1 (Zn-cluster TF; negative regulator of fluconazole, ketoconazole, brefeldin-A resistance; transposon mutation affects filamentous growth), CaCRZ2 (homozygous Cacrz1 null mutation suppresses 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 sterol uptake; binds CaERG2 promoter). Interestingly, ScUPC2 and its target genes were clearly induced in S. cerevisiae, revealing a strong divergence 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 set of target genes and recognize the same DNA consensus sequence (PDRE) in their promoters. The ScPDR3 gene itself has been shown to be regulated by ScPdr3p and ScPdr1p and was sensitive to progesterone. Most of the PDR genes were insensitive to progesterone in a Scpdr1/Scpdr3 strain. Remarkably, ScPdr1p was dispensable for the regulation of ScPDR3 in these conditions, although it constitutively binds to its promoter, suggesting that the progesterone induction of ScPDR3 occurs through autoregulation [53]. Progesterone was used to investigate the respective physiological roles of ScPdr1p and ScPdr3p in the PDR response to drug and confirmed that ScPDR3 has a dispensable role in drug response. Based on their ScPDR3 dependency in a Scpdr1 background, one could distinguish between three different groups of PDR targets. ScPdr1p and ScPdr3p have identical activity on the genes encoding most of the main transporters involved in pleiotropic drug export (ScPDR5, ScPDR15, ScSNQ2, and ScTPO1), they have an overlapping effect with a predominance of ScPdr1p on lipid metabolism genes, and some co-regulated proteins of unknown function (ScRSB1, ScPDR16, ScYGR035c, and ScYLR346c) and ScPdr1p specifically regulates genes which (for most of them), are sensitive to other stress response pathways (ScGRE2, YPL088w, YLL056c, ScICT1, etc.) (Fig. 3). The authors found a correlation between these groups and the nature and number of PDRE present in the corresponding promoters, suggesting that the DNA binding affinity of ScPdr1p and ScPdr3p may explain these different gene behaviors [17]. In C. albicans, CaTac1p is the only clear regulator of the PDR response identified till date [60]. CaTac1p belongs to the same family of Gal4p-like transcription factors as ScPdr1p and ScPdr3p and the progesterone induction profile of CaTAC1 was very similar to

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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 progesterone treatment in microarray experiment. Color coding indicates the main PDR gene families in both species: blue, ABC transporters; red, oxidoreductases from the Gre2 family; brown, putative flippases from the RTA family; gray, ICT1; green, putative aryl alcohol dehydrogenases; black, others. An arrow connects two homologues when the Pearson correlation distance between their expression profiles is significantly low (d < 0.4) (Banerjee et al. [17]). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

that of ScPDR3. Noteworthy, CaTAC1 may also be subjected to positive autoregulation, as suggested by the binding of CaTac1p to the CaTAC1 promoter [62]. A deletion of CaTAC1 is enough to decrease the basal level of expression of CaCDR1 and CaCDR2. This is differ-

ent from the situation in S. cerevisiae, where the double deletion of ScPDR1 and ScPDR3 was needed to observe the same effect. CaTac1p has been shown to recognize the DRE DNA consensus motif present in their promoters, which is different from the S. cere-

Log2(fold induction) 3,5

WT pdr1Δ pdr1Δpdr3Δ pdr3Δ

3 2,5 2 1,5 1 0,5 0 -0,5

GRE2

YOR1 YPL088w

ICT1

GROUP I

YLL056c PDR10

RSB1

YGR035c YLR346c PDR16

GROUP II

PDR5

PDR15

SNQ2

TPO1

YAL061w

PDR3

GROUP III

Fig. 3. Progesterone induction of PDR genes depending on expression levels in microarray study in ScPDR1 deletant, ScPDR3 deletant and ScPDR1/ScPDR3 deletant strains in S. cerevisiae. Group I represents genes exclusively dependent on ScPdr1p, Group II represents genes in which absence of ScPdr1p is partially complemented by ScPdr3p and Group III represents genes in which absence of ScPdr1p is fully complemented by ScPdr3p (taken from Banerjee et al. [17]).

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S. cerevisiae

C. albicans

GRE2, YOR1, YPL088w, ICT1, YLL056c, PDR10 RSB1, YLR346c, YGR035c, PDR16 PDR5, PDR15, SNQ2, TPO1, YAL061w

??

Pdr1p ??

PROGESTERONE

Pdr3p

??

Tac1p

IFU5 CDR1 CDR2 RTA3

Fig. 4. Comparison of effect of Progesterone on PDR network in S. cerevisiae and C. albicans. Plain arrows show regulatory interaction, dashed arrows show the regulation is dispensable for its progesterone induction (taken from Banerjee et al. [17]).

visiae PDRE [33,60]. However, the definition of the CaTAC1 regulon in C. albicans is not clear-cut. A genome-wide study has identified binding of CaTac1p at 37 promoters, but only 17 of the corresponding genes have their expression modulated in strains harboring CaTac1p gain of function mutants while, 25 CaTac1p bound promoters do not contain DRE [62,63]. To date, 5 genes (CaCDR1, CaCDR2, CaIFU5, CaRTA3, CaPDR16) are unambiguously considered as direct CaTAC1 targets. Remarkably, they were all induced by progesterone, which made steroid response a good model to explore the CaTac1p-related transcriptional pathway (Fig. 4). Surprisingly, only CaCDR1, CaCDR2, CaRTA3, CaMET15 and CaHSP70 were dependent on CaTac1p for their steroid induction. These results suggest that other, yet unknown, transcription factors may second CaTac1p in steroid response, like ScPdr3p seconds ScPdr1p in S. cerevisiae. At least three ScPDR1/ScPDR3 homologues, named CaFCR1-3 have been identified in C. albicans, with apparently no role in the basal expression of CaCDR1 and CaCDR2 [64], but which are obvious candidates to be tested for their role in steroid response. It is interesting to note as to how much the CaCDR1/ScPDR5, CaCDR2/ScPDR15 and the CaRTA3/ScRSB1 expression patterns have been conserved, when the transcription factors, the DNA regulatory motifs and the structure of the corresponding transcriptional networks have significantly diverged between the two species. The role of the other putative TFs also needs to be explored. The fact that PDR was the most sensitive pathway to progesterone suggests that progesterone acts directly on the signal(s) that trigger the PDR response. These signals are unknown to date and their discovery is a key challenge in the combat against fungal infections. Some inferences can be drawn from the existing literature. Progesterone, as an ergosterol analogue, may alter the lipid composition and the properties of the plasma membrane. This is suggested by the induction of ERG genes in S. cerevisiae, which occurs later than the PDR response. However, specific inhibitors of the ergosterol biosynthesis pathway like azoles, although able to induce a clear ERG response, are poor inducers of the PDR genes [22]. This suggests that the PDR and ERG response would not be necessarily linked. Of note, in Candida, the response of ERG genes to steroid is ambiguous, CaERG1 and CaERG4 being induced while most of the ERG genes are repressed, even when a clear CaCDR1/CaCDR2 induction is measured. Alternatively, progesterone could act on PDR by directly modifying the activity and properties of some PDR ABC transporters. A feedback may exist between the activity of the PDR transporters and the transcriptional regulation of the corresponding genes, since a knock-out of ScPDR5 has been shown to increase the expression of ScSNQ2 and ScYOR1, this effect being dependent on ScPdr1p. The inhibition by progesterone of ScPdr5p, ScPdr15p and other transporter activity may thus cause ScPdr1p/ScPdr3p activation. Although it was shown that progesterone is a good substrate of ScPdr5p and CaCdr1p, it is unlikely that progesterone acts just by competing “natural” ScPdr5p or CaCdr1p substrates

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 inducer of the PDR response [65] and (3) very low (1 ␮M) doses of progesterone are enough to trigger an efficient ScPDR5/CaCDR1 induction [17]. Recently, estradiol-derivatives have been shown to efficiently inhibit the drug transport and ATPase activities of ScPdr5p and CaCdr1p [66]. This suggests that human steroids act directly on the activity of the PDR transporters through a mechanism which is yet to be determined, but which has important and long-term implications 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. cerevisiae and in Candida glabrata, ScPdr1p is able to directly bind drugs like ketoconazole, which stimulates its activity at the promoter of MDR genes. This sensor-like property of ScPdr1p, which is also harbored by ScPdr3p in S. cerevisiae, is reminiscent of the mammalian nuclear receptors PXR, which control the expression of human MDR genes [67]. Although the protein domains involved in this interaction need to be more precisely identified and the functional consequences of xenobiotic transcription factor binding investigated in more details, this work opens the interesting possibility that steroids could be directly sensed by ScPdr1p, ScPdr3p or CaTac1p with a high affinity. This would explain the impressive sensitivity and the very fast response of PDR to progesterone in S. cerevisiae, although S. cerevisiae strains rarely meet steroids in their environment. Therefore, it is tempting to speculate that ScPdr1p, ScPdr3p and CaTac1p would act as bona fide steroid nuclear receptors in yeasts. 1.10. Proteomic response of C. albicans to steroid treatment Banerjee et al., [61] used nucleo-cytoplasmic extracts and identified changes in the proteome of C. albicans in response to progesterone treatment. An attempt was made to find out if the transcriptional response to progesterone that was evident within 30 min gets converted into the translational response. A comparison of the gels led to the identification of a limited number of spots on the 2D-gels that were over expressed in the progesterone treated protein samples. These protein spots were then analyzed by MALDI-TOF–TOF and the differentially over expressed proteins were identified. The proteomic analysis of progesterone response showed an over expression of proteins belonging to ribosomal protein synthesis machinery (CaRPL12, CaRPL28.3), transcription (CaIPF670), translational machinery (CaIPF277), glycolysis pathway (CaPGK1, CaFBA1), TCA cycle (CaPDB1), azole resistance associated proteins (CaIPF11153, CaPDB1, CaFBA1, CaADH1, CaATP3), mitochondrial F0F1-ATPase (CaATP2, CaATP3, CaSHM1), GTPase activity (CaYRB1),

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Table 1 Proteins over-expressed in response to progesterone. Gene name

Common name

Description

CA1691

CaPGK1

CA5714

CaIPF2431

CA5339

CaIPF885

CA5180

CaFBA1

CA4765

CaADH1

CA5460 CA1662

CaIPF277 CaRPL28.3F

CA5998 CA4817

CaIPF670 CaPOM152

CA4362

CaATP2

CA1489

CaATP3

CA4001

CaRPL12

CA3773 CA2582

CaIPF11153 CaTAL1

CA2162 CA0433

CaPDB1 CaSHM1

CA5822

CaYRB1

CA5528

CaRPN7

Phosphoglycerate kinase; enzyme of glycolysis; localizes to cell wall and to cytoplasm; antigenic during murine or human systemic infection; biofilm, Hog1p, GCN-induced; down-regulated upon phagocytosis; induction by symbiont of host defense response, interaction with host. Protein of TSA/alkyl hydroperoxide peroxidase C (AhPC) family; similar to thiol-dependent peroxidases, roles in oxidative stress signaling; immunogenic; on hyphal surface, nucleus; yeast-form nucleus, cytoplasm; neutrophil, peroxide induced. Protein described as similar to glucan 1,3-beta-glucosidase; regulated by Nrg1p, Tup1p; possibly regulated by Tac1p; induced upon biofilm formation; induced by nitric oxide; induced during cell wall regeneration. Putative fructose-bisphosphate aldolase; enzyme of glycolysis; antigenic in murine or human infection; regulated on yeast-hyphal switch; induced by Efg1p, Gcn4p, Hog1p, biofilm growth, or fluconazole; phagocytosis-repressed; fungal-specific. Alcohol dehydrogenase; at surface of yeast-form cells but not hyphae; soluble in hyphae; immunogenic in human or mouse; complements S. cerevisiae adh1 adh2 adh3 mutation; regulated by growth phase, carbon source; fluconazole-induced. Translation Machinery Associated. Putative ribosomal protein; Plc1p-regulated; genes encoding cytoplasmic ribosomal subunits, translation factors, and tRNA synthetases are down-regulated upon phagocytosis by murine macrophage; translation, cytosolic large ribosomal subunit. Nucleosome disassembly, Ada2/Gcn5/Ada3 transcription activator complex. 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. 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. Predicted ORF from Assemblies 19 and 20; flucytosine induced; caspofungin repressed; macrophage/pseudohyphal-induced. 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 murine macrophage. Predicted ORF in Assemblies 19 and 20; regulation correlates with clinical development of fluconazole resistance. Predicted ORF from Assembly 19; oxidative stress-induced via Cap1p; induced by nitric oxide in yhb1 mutant; transaldolase activity, pentose-phosphate shunt, cytoplasm. Protein described as similar to pyruvate dehydrogenase; fluconazole-induced. 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. 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 or by dimorphic transition. Predicted ORF from Assemblies 19 and 20; regulated by Gcn2p and Gcn4p; ubiquitin-dependent protein catabolic process, proteasome regulatory particle.

nucleo-cytoplasmic transport proteins (CaYRB1, CaPOM152), stress related proteins like CaTAL1 (oxidative stress), proteosome regulatory protein (CaRPN4), etc. Interestingly, many of the proteins that were found up-regulated in the 2D-gels were either the same as the genes up-regulated in the microarray experiments or belonged to similar pathways. Table 1 lists the identified proteins, which were over expressed in response to progesterone treatment. Although, this is a single study by Banerjee et al., where proteomic approach has been employed to examine steroid/progesterone response, it amply demonstrates that upon steroid exposure of pathogenic yeast, the up-regulation of genes that is seen at the transcriptional level, is also relevant at the translational level and proteins can be seen up-regulated even at short progesterone exposure time-point. Notably, since only nucleo-cytoplasmic extracts was used to analyze differential protein expression in response to progesterone treatment, the most induced membrane bound MDR genes such as CaCDR1/CaCDR2 could not be detected in that proteomic study. Indeed, a detailed proteomic analyses is required to examine global translation response to progetesrone in Candida cells. The implementation of proteomics in the post-genomic era of this important human fungal pathogen can provide important information about its biological complexity and pathogenic traits. Proteomics is an important tool in C. albicans research, particularly to address problems that cannot be solved by genomic studies such as protein post-translational modifications that can affect pathogenicity and host-response. It is expected that in the near future, the results

from proteomic experiments will lead to novel approaches for the management of candidiasis. 2. Conclusions Studies so far amply demonstrate that yeast cells respond to human steroid hormones and affect their cell growth, morphology and virulence. The response can be detected at both transcriptional and translation levels. Notably, these responses presented by yeast upon exposure to steroids lack the involvement of any receptor mediated signaling cascade, which commonly occurs in higher eukaryotes. Steroid exposure to baker’s yeast S. cerevisiae and pathogenic yeast C. albicans show reasonably conserved transcriptome and proteome response. The up regulation of MDR genes upon progesterone exposure in yeasts is most noteworthy as it induces both a wider spectrum of PDR genes and induces them to higher levels of expression, as compared to well known inducer drugs. Steroids also act as substrates of MDR transporters and transiently induce levels of MDR transporter encoding genes making resistant the otherwise susceptible Candida strains to azoles. Progesterone response in S. cerevisiae helped uncovering PDR targets based on their PDR3 dependency in a pdr1 background one could detect otherwise undistinguishable three different groups of PDR targets. A correlation between PDR target groups and the nature and number of PDRE present in the promoters of the corresponding genes, suggested that the DNA binding affinities of ScPdr1p and ScPdr3p may be different. The response towards PDR and ERG gene in S. cerevisiae strains

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appeared to be only partially conserved in pathogenic C. albicans.

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