An iron homeostasis regulatory circuit with reciprocal roles in Candida albicans commensalism and pathogenesis

Abstract

The mammalian gastrointestinal tract and bloodstream are highly disparate biological niches that differ in concentrations of nutrients such as iron. However, some commensal-pathogenic microorganisms, such as the yeast Candida albicans, thrive in both environments. We report the evolution of a transcription circuit in C. albicans that controls iron uptake and determines its fitness in both niches. Our analysis of DNA-binding proteins that regulate iron uptake by this organism suggests the evolutionary intercalation of a transcriptional activator called Sef1 between two broadly conserved iron-responsive transcriptional repressors, Sfu1 and Hap43. Sef1 activates iron uptake genes and promotes virulence in a mouse model of bloodstream infection, whereas Sfu1 represses iron uptake genes and is dispensable for virulence but promotes gastrointestinal commensalism. Thus, C. albicans can alternate between genetic programs conferring resistance to iron depletion in the bloodstream versus iron toxicity in the gut, and this may represent a fundamental attribute of gastrointestinal commensal-pathogens.

INTRODUCTION

The unique chemical properties of iron underlie its broad utility as a cofactor for essential cellular processes as well as its toxicity (via hydroxyl radicals produced by the Fenton reaction) to proteins, lipids, and nucleic acids (Pierre et al., 2002). Virtually all organisms have evolved mechanisms to precisely regulate the uptake and storage of iron. This task is particularly challenging for commensal-pathogens such as the yeast, Candida albicans, that inhabit the mammalian gastrointestinal tract (Odds, 1988) but also enter the bloodstream (Edmond et al., 1999). Gastrointestinal commensals are bathed in comparatively high levels of iron (~15 mg or 0.27 mmol/day in humans) since the majority of dietary iron is not absorbed (McCance and Widdowson, 1938; Miret et al., 2003). In contrast, bloodstream pathogens face extraordinary iron depletion (~10⁻²⁴ M free Fe³⁺) because of active sequestration by the host (Martin et al., 1987). Only a handful of clinically important commensal-pathogenic microorganisms are adapted to both environments.

To survive in the bloodstream, C. albicans maintains at least three systems of iron extraction from the host (Figure 1). Iron is recovered from red blood cells by means of secreted hemolysins (Luo et al., 2001; Manns et al., 1994) and the Rbt5 system of hemoglobin utilization (Weissman and Kornitzer, 2004; Weissman et al., 2008). Iron bound to small molecule siderophores is imported via the Sit1 siderophore transporter (Ardon et al., 2001; Heymann et al., 2002; Hu et al., 2002). High-affinity uptake of free or chelated Fe³⁺ occurs through sequential reduction and oxidation-internalization steps mediated, respectively, by a family of cell surface ferric reductases (Hammacott et al., 2000; Knight et al., 2002) and complexes of the Ftr1 iron permease with any of several multicopper ferrous oxidases (Knight et al., 2002). The reductive system is also used in tissues to extract iron from host ferritin, in a process dependent on the cell surface adhesin Als3 (Almeida et al., 2008). In contrast to this detailed understanding of C. albicans adaptation to iron limitation in the bloodstream, comparatively little is known about how C. albicans defends against iron excess in the gut.

Figure 1. Iron acquisition in C. albicans.

Shown are key factors mediating the three known pathways of high-affinity iron uptake in this organism: reductive iron uptake, siderophore-iron uptake, and hemoglobin-iron uptake. Gene families encode ferric reductases and multicopper oxidases of the reductive pathway, and only a subset are expressed in an iron-dependent fashion. Ccc2 is a copper transporter required for proper assembly and function of Fet3/iron permease complexes. Genes for each depicted protein are directly activated by Sef1, and genes for Ftr1, Sit1, and Rbt5 are directly repressed by Sfu1.

In most ascomycetes and the basidiomycete, Cryptococcus neoformans, a simple switch regulates the expression of iron homeostasis genes (Haas et al., 1999; Hortschansky et al., 2007; Jung et al., 2010; Jung et al., 2006; Mercier et al., 2006; Pelletier et al., 2002). When environmental iron is replete, a GATA family transcription factor directly represses genes for iron acquisition as well as the gene encoding the regulatory component of the CCAAT-binding complex. When environmental iron is low, the CCAAT-binding complex directly represses the GATA factor gene as well as genes for noncritical iron-dependent processes. C. albicans was thought to share this system because its GATA factor ortholog, called Sfu1, can functionally substitute for the GATA factor of S. pombe (Pelletier et al., 2007), and deletion of SFU1 leads to inappropriate expression of iron acquisition genes when environmental iron is replete (Lan et al., 2004). Moreover, mutants affecting the C. albicans regulatory component of the CCAAT-binding complex exhibit multiple abnormalities on iron-depleted media, including defective growth (Baek et al., 2008; Homann et al., 2009; Hsu et al., 2011), failure to upregulate the FRP1 ferric reductase gene (Baek et al., 2008), and inappropriate expression of iron-utilization genes (Hsu et al., 2011). This hap43ΔΔ mutant is also defective for virulence in a mouse bloodstream infection model (Hsu et al., 2011), like mutants affecting orthologs in the fungal pathogens Aspergillus fumigatus (Schrettl et al., 2010) and C. neoformans (Jung et al., 2010).

We identified C. albicans SEF1, which encodes a predicted Zn(2)Cys(6) DNA-binding protein, in a screen for candidate virulence factors (Noble et al., 2010). Our discovery that SEF1 is also required for growth on iron-depleted media (Homann et al., 2009) suggested that it may transcriptionally activate genes involved in iron acquisition—but this hypothesis was at odds with the prevailing model of iron regulation, described above. We therefore determined the gene regulatory activities of Sef1, Sfu1, and Hap43 using whole-genome RNA expression and chromatin immunoprecipitation (ChIP) approaches. The results combined with systematic comparisons of mutants in C. albicans, S. pombe, and Saccharomyces cerevisiae suggest that C. albicans has evolved a unique, feed forward transcriptional circuit in which Sef1 is intercalated into the broadly conserved switch between the GATA factor and the CCAAT-binding complex. In C. albicans, Sfu1 (GATA factor) represses SEF1 and iron uptake genes, Sef1 activates HAP43 (CCAAT-binding complex) and iron uptake genes, and Hap43 represses SFU1 and iron utilization genes (i.e. genes for processes that require iron). We investigated the in vivo roles of SEF1 and SFU1 by profiling the respective deletion mutants in mouse models of virulence and commensalism. Only SEF1 was critical for virulence in the bloodstream, whereas SFU1 was selectively required for persistence in the gastrointestinal tract. These results suggest that the reciprocal abilities of C. albicans to activate iron uptake upon entry into the iron-depleted bloodstream, while efficiently restricting it in the potentially iron toxic environment of the gut, are critical to its success as a commensal-pathogen.

RESULTS

Sef1 activates the machinery for iron uptake in C. albicans

We identified C. albicans sef1ΔΔ in two separate genetic screens (note that C. albicans is an obligate diploid organism, necessitating the disruption of two copies of any gene): (1) an in vivo screen for genes required in competitive bloodstream infections (Noble et al., 2010) and (2) an in vitro screen for genes promoting growth under iron-limiting conditions (Homann et al., 2009). The hap43ΔΔ mutant was also sensitive to iron limitation in the latter study and previously (Baek et al., 2008; Homann et al., 2009; Hsu et al., 2011).

SEF1 encodes a predicted Cys(6)Zn(2) zinc binuclear cluster DNA binding protein without a clear ortholog in S. pombe; its S. cerevisiae ortholog has not been characterized. To test the hypothesis that Sef1 regulates iron homeostasis, we compared global RNA expression profiles of sef1ΔΔ and wild type C. albicans under iron-limiting conditions. Deletion of SEF1 resulted in downregulation of 170 genes and upregulation of 53 genes relative to wild type (minimum 2-fold change relative to wild type, 0.1% false discovery rate; Figure 2a and Table S1a). Morever, GO term analysis supported a significant association between Sef1-activated genes and iron homeostasis (6.5% of Sef1-activated genes vs. 0.5% in the genome, p=1×10⁻⁷). Indeed, the Sef1-activated gene set encodes every iron uptake factor depicted in Figure 1, as well as multiple components of the CCAAT-binding complex (Hap43, Hap2, and Hap3; Table 1).

Figure 2. Transcriptional regulatory activities of Sef1, Sfu1, and Hap43.

a) Gene sets controlled by Sef1, Sfu1, and Hap43, based on whole genome ORF microarray analysis of the respective knockout mutants. Activation was defined by a minimum 2-fold decrease of gene expression in the deletion mutant relative to wild type, and repression by a minimum 2-fold increase. sef1ΔΔ and hap43ΔΔ were assessed in low iron medium, and sfu1ΔΔ in iron replete medium. Expression of the lone Sfu1-activated gene (IRO1) was unaffected by mutation of SEF1 or HAP43.

b) Sef1 binds to the HAP43 promoter. ChIP enrichment profiles of duplicate Sef1-Myc extracts are plotted in dark and light blue, and results from untagged controls are in yellow and orange. Genes are transcribed from left to right above the baseline, and from right to left below.

c) Direct gene regulation by Sef1, Sfu1, and Hap43. Gene activation is indicated by a black line and repression by a grey line. Targets involved in iron uptake are shaded red, iron utilization targets are blue, transcription factors are grey, all others are yellow. Targets lacking common names are depicted by the unique numerical components of their standard names; i.e. “123” for orf19.123.

d) Simplified scheme of C. albicans iron homeostasis.

e) Sef1 and Hap43 DNA recognition motifs compared to consensus sequences of orthologs in other species

See also Figure S1 and Tables S1a and S1b.

Table 1. Sef1 activation targets that encode iron uptake factors or components of the CCAAT-binding complex.

Fold expression changes were calculated from microarray analysis of sef1ΔΔ and wild type strains grown in iron-limiting medium.

Systematic name	Gene name	Predicted protein	Expression sef1ΔΔ vs. WT
REDUCTIVE IRON UPTAKE
orf19.1263	CFL1	Putative ferric reductase	−4.8
orf19.1930	CFL5	Putative ferric reductase	−32.8
orf19.1264	CFL2	Putative ferric reductase	−7.1
orf19.3538	FRE9	Putative ferric reductase	−4.4
orf19.5634	FRP1	Putative ferric reductase	−5.8
orf19.7112	FRP2	Putative ferric reductase	−4.4
orf19.7219	FTR1	High-affinity iron permease	−2.9
orf19.7231	FTR2	High-affinity iron permease	−2.4
orf19.4211	FET3	Multicopper oxidase	−10.5
orf19.4328	CCC2	Copper-transporting P-type ATPase of Golgi that is required for wild-type iron assimilation	−3.5
SIDEROPHORE UPTAKE
orf19.2179	SIT1	Transporter of ferrichrome siderophores	−3.2
HEMOGLOBIN UPTAKE AND UTILIZATION
orf19.5636	RBT5	GPI-anchored cell wall protein involved in hemoglobin utilization	−2.4
orf19.5674	PGA10	Plasma membrane protein involved in heme-iron utilization	−3.4
orf19.6073	HMX1	Heme oxygenase	−4.5
orf19.2501	FLC1	Putative FAD transporter involved in uptake of heme	−2.1
CCAAT-BINDING COMPLEX
orf19.1228	HAP2	Component of the CCAAT-binding complex	−3.3
orf19.4647	HAP3	Putative component of the CCAAT-binding complex	−23.2
orf19.681	HAP43	Component of the CCAAT-binding complex required for iron-limitation response	−3.1

Sef1, Sfu1, and Hap43 regulate each other’s expression

Because C. albicans was previously understood to control iron homeostasis similarly to S. pombe and other fungi (Baek et al., 2008; Hsu et al., 2011; Lan et al., 2004; Pelletier et al., 2007), we analyzed RNA expression in mutants affecting C. albicans orthologs of the GATA factor and a CCAAT-binding complex component. Under iron-replete conditions, deletion of SFU1 (GATA factor) resulted in upregulation of SEF1, HAP43 (CCAAT-binding complex), and 25 other genes largely associated with iron homeostasis (37% of Sfu1-repressed genes vs. 0.5% in the genome, p=1.7×10⁻¹⁵; Figure 2a and Table S1a). Only the gene encoding Iro1 was consistently ~2-fold downregulated in the sfu1ΔΔ mutant. Although the molecular function of Iro1 is not known, ectopic expression of IRO1 complements the growth defect of a S. cerevisiae mutant with a defect in iron acquisition (Garcia et al., 2001).

Our results with Sfu1 differed from those of Lan et al. (2004), who previously profiled a different sfu1ΔΔ mutant. Similar to us, these investigators observed upregulation of 22 genes (as currently annotated) in their mutant, including SEF1, HAP43, and multiple genes involved in iron uptake; however, a much larger set of 97 genes was downregulated. To determine whether our microarrays had failed to detect bona fide Sfu1-activation targets, we used RT-PCR to reassess the expression of the 5 genes that were most strongly downregulated (9 to 14-fold) in the Lan et al. mutant. Comparison between RNA levels in our sfu1ΔΔ mutant and wild type confirmed virtually identical expression of four of the genes, whereas the fifth gene (orf19.1774) was 2-fold downregulated in the mutant (Figure S1a). These results validate our microarray studies, which likely identified the vast majority of Sfu1 targets but may have missed some weakly regulated genes, in keeping with the stringent 0.1% false discovery rate. An alternative explanation for the discrepant results may be that the “sfu1ΔΔ” mutant profiled by Lan et al. was actually a sfu1ΔΔiro1ΔΔ double mutant, since the 3′ half of IRO1 is absent from the parental strain (Garcia et al., 2001).

We evaluated the role of HAP43 under iron-limiting conditions, where deletion of this gene has been shown to impair cellular proliferation (Baek et al., 2008; Homann et al., 2009; Hsu et al., 2011), activation of FRP1 (Baek et al., 2008), and repression of 9 genes associated with iron-dependent processes (shown by RT-PCR of selected targets in Hsu et al., 2011). We observed upregulation of 286 genes in hap43ΔΔ relative to wild type (Figure 2a, Table S1a). These included SFU1, FRP1 and other iron homeostasis genes (2.8% of Hap43-repressed genes vs. 0.5% in the genome, p=0.023), as well as genes involved in a variety of iron-dependent processes (including the 9 identified by Hsu et al., 2011) such as aerobic respiration (7.7% vs. 0.9% in the genome, p=1.9×10⁻¹³), the respiratory electron transport chain (4.5% vs. 0.4%, p=2.2×10⁻⁸), heme biosynthesis (2.8% vs. 0.2%, p=4.6×10⁻⁶), and iron-sulfur cluster assembly (2.1% vs. 0.2%, p=0.013). Another 223 genes were downregulated in hap43ΔΔ (Figure 2a, Table S1a), with a slight bias towards those associated with cytokinesis (4.5% vs. 1.0% in the genome, p=0.020). In aggregate, these experiments suggest important roles for all three transcription factors (Sef1, Sfu1, and Hap43) in the regulation of iron homeostasis in this species.

DNA-binding analysis of Sef1, Sfu1, and Hap43 reveals a tightly knit circuit

To dissect the direct versus indirect regulatory roles of Sef1, Sfu1, and Hap43, we performed chromatin immunoprecipitation experiments using Myc epitope-tagged versions of the three transcription factors. Fusion proteins were fully (Sef1-Myc and Sfu1-Myc) or partially (Hap43-Myc) functional when expressed as the only copy in the cell (Figure S1b). Matched pairs of strains differing only by the presence of the epitope tag were grown in iron-depleted (Sef1, Hap43) or iron-replete (Sfu1) medium. Whole cell extracts were incubated with monoclonal antibodies to the Myc epitope, and immunoprecipitated DNAs were amplified, fluorescently labeled, and hybridized to C. albicans genomic tiling microarrays. Shown in Figure 2b is a representative peak of highly specific binding of Sef1-Myc to the HAP43 promoter. Dark and light blue plots depict two independent experiments using Sef1-Myc extracts, and orange and yellow plots depict results with the untagged controls. Similar strong peaks of specific binding were observed across the genome for Sef1-Myc and Hap43-Myc, whereas Sfu1-Myc produced somewhat lower signal to noise (Table S1b). Sfu1 targets were validated with four additional ChIP experiments, using qPCR to quantify levels of the 9 putative direct targets vs. 4 controls; every target but no control was at least 2-fold enriched in the Sfu1-Myc extracts (Figure S1c).

We defined a gene regulatory event to comprise: (1) a significant and specific peak of DNA association by a given transcription factor (Table S1b) and (2) significant dependence on the associated transcription factor for normal expression of the regulated gene (Table S1a). Gene regulatory events mediated by Sef1, Sfu1, and/or Hap43 are depicted in Figure 2c (full dataset in Table 2), where black lines indicate transcriptional activation and grey lines, repression. Interactions among Sef1, Sfu1, and Hap43 themselves are marked with arrows (activation) and bars (repression). Examination of Figure 2c reveals Sef1 to function primarily as a transcriptional activator, with a large direct regulon of 64 genes, whereas Hap43 and Sfu1 are primarily transcriptional repressors, with smaller direct regulons of 25 and 10 genes, respectively. Sef1 and Sfu1 control most of the iron uptake genes (red), whereas Hap43 controls genes involved in iron-utilizing processes (e.g. aerobic respiration, heme biosynthesis, etc.; blue). In addition, Sef1 and Hap43 each regulate at least one other transcription factor (grey), potentially accounting for much larger number of (directly plus indirectly) regulated genes observed in the RNA expression studies (Figure 2a and Table 1).

Table 2. Gene regulatory events mediated by Sef1-, Sfu1-, and Hap43.

Direct gene regulatory targets of Sef1, Sfu1, and/or Hap43 are presented with descriptions, published virulence associations, fold expression changes, and physically-associated transcription factor. sfu1ΔΔ was assessed under iron-replete conditions, and sef1ΔΔ and hap43ΔΔ under iron-depleted conditions. Citations for published virulence associations are: FTR1 (Ramanan and Wang, 2000), CFL2 (Noble et al., 2010), HAP43 (Hsu et al., 2011), orf19.4905 (Noble et al., 2010), SEF1 (Noble et al., 2010), HMX1 (Navarathna and Roberts), HEM3 (Kirsch and Whitney, 1991), CAT1 (Nakagawa et al., 2003), and ICL1 (Barelle et al., 2006; Lorenz and Fink, 2001; Ramirez and Lorenz, 2007).

REGULATED GENE					Wild type expression (replete vs. low iron)	C. albicans Transcription Factor
Systematic name	Gene name	Description	Cellular process	Role in virulence? (VIR)		Sfu1		Sef1		Hap43
						Expression (sfu1ΔΔ vs. WT)	Promoter binding (Sfu1)	Expression (sef1ΔΔ vs. WT)	Promoter binding (Sef1)	Expression (hap43ΔΔ vs. WT)	Promoter binding (Hap43)
orf19.539	LAP3	Putative ortholog of S.c. Lap3, a cysteine aminopeptidase that protects against homocysteine toxicity	Metabolism		−100.9			−2.63	Sef1	−2.5
orf19.7114	CSA1	Surface antigen on elongating hyphae and buds	Cell wall composition: protein		−64.6			−52.46	Sef1	−9.2
orf19.1263	CFL1	Putative ferric reductase, with sequence similarity to S.c. Fre2 which with Fre1 is a major cellular ferric reductase	Iron uptake: ferric reductase		−42.9	45.6		−4.81	Sef1	2.3
orf19.5636	RBT5	GPI-anchored cell wall protein involved in hemoglobin utilization	Iron uptake: heme utilization pathway		−41.1	14.1	Sfu1	−2.39	Sef1
orf19.3117	CSA2	Putative cell surface protein	Cell wall composition: protein		−39.5	6.3		−26.79	Sef1	−15.2
orf19.5634	FRP1	Putative ferric reductase, with sequence similarity to S.c. Fre5	Iron uptake: ferric reductase		−36.6	20.6	Sfu1	−5.78	Sef1	−3.2
orf19.1930	CFL5	Putative ferric reductase, with similarity to S.c. Fre3	Iron uptake: ferric reductase		−34.9	22.9		−32.80	Sef1	4.2
orf19.5952		Conserved hypothetical protein	Unknown		−32.5			−46.24	Sef1
orf19.2179	SIT1	Transporter of ferrichrome siderophores, but not ferrioxamine B; putative ortholog of S.c. Arn1	Iron uptake: siderophore transporter		−27.0	12.1	Sfu1	−3.16	Sef1	−4.1
orf19.4802	FTH1	Putative ortholog of S.c. Fth1 high affinity iron transporter for intravacuolar iron stores	Iron homeostasis: vacuolar transporter		−22.4	22.6	Sfu1
orf19.3538	FRE9	Putative ferric reductase with similarity to S.c. Fre3	Iron uptake: ferric reductase		−20.0	8.9	Sfu1	−4.44	Sef1	−2.2
orf19.7219	FTR1	High-affinity iron permease, putative ortholog of S.c. Ftr1	Iron uptake: reductive pathway	VIR	−17.6	14.4	Sfu1	−2.94	Sef1
orf19.4647	HAP3	Conserved hypothetical protein with similarity to S.c. Hap3, a subunit of the Hap2p/3p/4p/5p CCAAT-binding complex	Iron regulation: transcription factor		−17.5			−23.25	Sef1	−2.7
orf19.1264	CFL2	Putative ferric reductase with sequence similarity to S.c. Fre4	Iron uptake: ferric reductase	VIR	−17.3	21.2		−7.10	Sef1	2.3
orf19.5635	PGA7	Putative hyphal surface antigen with predicted GPI anchor	Cell wall composition: protein		−12.5	5.8	Sfu1	−4.70	Sef1	−2.9
orf19.334		Hypothetical protein, conserved in C.d., C.t.	Unknown		−9.7			−2.01	Sef1
orf19.4795		Conserved hypothetical protein	Unknown		−8.4			−2.85	Sef1	−2.3
orf19.7112	FRP2	Putative ferric reductase, with sequence similarity to S.c. Fre5	Iron uptake: ferric reductase		−7.2			−4.40	Sef1	−2.6	Hap43
orf19.4270		Putative mannosyltransferase	Cell wall composition: mannosylation		−5.2			−6.17	Sef1	−6.4
orf19.5801	RNR21	Putative ortholog of S.c. Rnr2, a component of ribonucleotide-diphosphate reductase that catalyzes the rate-limiting step in dNTP synthesis	Metabolism: biosynthesis of dNTPs		−5.0			−2.58	Sef1	−2.2
orf19.6793		Putative protein conserved in C.d. and C.t.	Unknown		−5.0			−4.35	Sef1	−4.7
orf19.681	HAP43	CCAAT-binding factor (CBF)-associated transcription factor required for iron-limitation response,	Iron regulation: transcription factor		−4.6	3.8		−3.06	Sef1	(−26.0)
orf19.1228	HAP2	Putative ortholog of S.c. Hap2, a component of CCAAT-binding factor	Transcription: transcription factor		−4.5			−3.33	Sef1	−2.2
orf19.5475		Hypothetical protein	Unknown		−4.4	3.2	Sfu1	−2.08		−2.2
orf19.4905		Putative MFS transporter	Transporter	VIR	−4.4			−5.39	Sef1	−2.1
orf19.3859		Putative ortholog of S.c. Ifa38, a microsomal beta-keto-reductase important for VLCFA synthesis	Metabolism: biosynthesis of lipids		−4.1			−2.43	Sef1	−2.1
orf19.7218	RBE1	Putative cell wall protein, no predicted GPI anchor	Cell wall composition: protein		−4.1			−4.03	Sef1	−4.0
orf19.4211	FET3	Multicopper oxidase with similarity to S.c. Fet3	Iron uptake: reductive pathway		−4.0	3.2		−10.47	Sef1
orf19.4735		Putative ornithine cyclodeaminase, putative ortholog of S.c. YGL159W	Metabolism: interconversion of amino acids		−3.9			−4.85	Sef1	−2.9	Hap43
orf19.2062	SOD4	Member of a family of superoxide dismutases, predicted GPI-anchor	Response to stress: oxidative, cell wall composition: protein		−3.7			−3.71	Sef1	−2.6
orf19.853	SAP99	Putative secreted aspartyl protease	Secreted enzyme		−3.7			−2.38	Sef1	−2.1
orf19.5779	RNR1	Putative ortholog of S.c. Rnr1, a regulatory component of ribonucleotide reductase	Metabolism: biosynthesis of dNTPs		−3.7			−5.25	Sef1	−3.1
orf19.411		Conserved hypothetical protein	Unknown		−3.7			−2.56	Sef1	−2.2
orf19.4328	CCC2	Copper-transporting P-type ATPase of Golgi that is required for wild-type iron assimilation, putative ortholog of S.c. Ccc2	Iron uptake: reductive pathway		−3.6	3.0		−3.53	Sef1
orf19.6482		Conserved hypothetical protein	Unknown		−3.6			−2.71	Sef1
orf19.2177		Hypothetical protein	Unknown		−3.6			−6.84	Sef1	−2.3
orf19.1932	CFL4	Putative ortholog of S.c. Fre3, a ferric reductase	Iron uptake: ferric reductase		−3.6	24.4	Sfu1			19.5
orf19.7445		Putative ortholog of S.c. Vid24, a peripheral membrane protein located at Vid (vacuole import and degradation) vesicles	Vacuolar function		−3.5			−2.79	Sef1
orf19.6832		Conserved hypothetical protein with K-Cl cotransporter domain	Transporter: ions		−3.3			−2.23	Sef1
orf19.4145	ZCF20	Putative zinc finger transcription factor	Transcription: transcription factor		−3.3			−2.63	Sef1
orf19.3753	SEF1	Putative transcription factor with zinc cluster DNA-binding motif, ortholog of S.c. Sef1	Iron regulation: transcription factor	VIR	−3.0	5.4	Sfu1	−8.89
orf19.335		Conserved hypothetical protein	Unknown		−2.9			−2.49	Sef1
orf19.6073	HMX1	Heme oxygenase, acts in utilization of hemin iron, putative ortholog of S.c. Hmx1	Iron uptake: heme utilization pathway	VIR	−2.9			−4.52	Sef1	−6.8
orf19.22		Conserved hypothetical protein with homology to peroxisomal membrane proteins	Peroxisomal function		−2.7			−3.16	Sef1
orf19.1588		Putative ortholog of S.c. Fmp21, a mitochondrial protein of unknown function	Mitochondrial function		−2.7			−2.72	Sef1
orf19.2059		Conserved hypothetical protein with homology to magnesium-dependent endonucleases and phosphatases involved in intracellular signalling	Signaling		−2.6			−2.70	Sef1
orf19.4743	AFG1	Conserved hypothetical protein with similarity to S.c. Afg1 mitochondrial ATPase	Mitochondrial function		−2.4			−3.19	Sef1
orf19.1942	SGE1	Putative MFS-MDR transporter	Transporter		−2.4			−2.52	Sef1
orf19.1926	SEF2	Putative zinc cluster protein	Transcription: transcription factor		2.2			−12.54	Sef1	9.0	Hap43
orf19.4869	SFU1	Transcriptional regulator of iron-responsive genes	Iron regulation: transcription factor		3.5	−17.1				5.9	Hap43
orf19.6550		Putative ortholog of S.c. Yor228c, a mitochondrial outer membrane protein	Mitochondrial function		3.7					2.9	Hap43
orf19.6948	CCC1	Putative vacuolar Fe2+/Mn2+ transporter	Iron storage: vacuolar, Transporter: ions		3.8					2.6	Hap43
orf19.1742	HEM3	Hydroxymethylbilane synthase (uroporphyrinogen I synthase)	Iron utilization: biosynthesis of heme	VIR	3.8			−2.67	Sef1	8.5	Hap43
orf19.7056		Putative amino acid permease	Transporter: amino acids		4.0	4.6				5.2	Hap43
orf19.6229	CAT1	Catalase; role in resistance to oxidative stress, neutrophils, peroxide	Resistance to stress: oxidation	VIR	4.2	2.0				10.6	Hap43
orf19.3034	RLI1	Putative ortholog of S.c. Rli1, an iron-sulfur protein required for ribosome biogenesis and translation initiation	Translation: initiation, ribosome biogenesis; iron utilization: iron-sulfur cluster protein		4.5					3.9	Hap43
orf19.5521	ISA1	Conserved hypothetical protein with strong similarity to S.c. Isa1, a mitochondrial matrix protein involved in biogenesis of iron-sulfur (Fe/S) cluster proteins	Iron utilization: biogenesis of iron-sulfur cluster proteins, mitochondrial function		4.9					10.1	Hap43
orf19.4099	ECM17	Putative ortholog of S.c. Met5, a sulfite reductase beta subunit	Metabolism: biosynthesis of amino acids		5.1			2.01		13.4	Hap43
orf19.6385	ACO1	Putative ortholog of S.c. Aco1, an aconitase required for the TCA cycle and maintenance of the mitochondrial genome	Metabolism: TCA cycle, mitochondrial function		5.4					49.9	Hap43
orf19.6257	GLT1	Putative ortholog of S.c. Glt1, an NAD(+)-dependent glutamate synthase	Metabolism: biosynthesis of glutamate		5.7					7.2	Hap43
orf19.1770	CYC1	Cytochrome c	Mitochondrial electron transport chain		8.4					16.4	Hap43
orf19.7498	LEU1	Putative ortholog of S.c. Leu1, an isopropylmalate isomerase that catalyzes the second step in the leucine biosynthesis pathway	Metabolism: biosynthesis of amino acids		8.7					20.4	Hap43
orf19.238	CCP1	Putative ortholog of S.c. Ccp1, a mitochondrial cytochrome-c oxidase	Mitochondrial function		9.0					26.9	Hap43
orf19.4040	ILV3	Putative ortholog of S.c. Ilv3, a dihydroxyacid dehydratase involved in biosynthesis of branched chain amino acids	Metabolism: biosynthesis of amino acids		9.6					6.2	Hap43
orf19.4674.1	CRD2	Metallothionein	Resistance to stress: copper		24.9					3.3	Hap43
orf19.4716	GDH3	Putative ortholog of S.c. Gdh3, a NADP(+)-dependent glutamate dehydrogenase that synthesizes glutamate from ammonia and alpha-ketoglutarate	Metabolism: biosynthesis of glutamate					−6.13	Sef1
orf19.1486		Conserved hypothetical protein	Unknown					−3.48	Sef1
orf19.2602	OPT1	Oligopeptide transporter; transports 3-to-5-residue peptides	Transporter: peptides					−3.34	Sef1	−4.6
orf19.4673	BMT9	Putative beta-mannosyltransferase	Cell wall composition: mannosylation					−3.26		−2.6	Hap43
orf19.1027	PDR16	Phosphatidylinositol transfer protein, putative ortholog of S.c. Pdr16 that controls levels of various lipids	Metabolism: regulation of lipids					−2.98	Sef1
orf19.3610		Conserved hypothetical protein with LgrB domain associated with murein hydrolase activity and penicillin tolerance	Unknown					−2.83	Sef1
orf19.2178		Putative ortholog of S.c. Mrs4, an iron transporter that mediates Fe2+ transport across the inner mitochondrial membrane under low iron conditions	Iron utilization: mitochondrial transporter					−2.73	Sef1
orf19.6548	ISU1	Putatvie ortholog of S.c. Isu1, mitochondrial matrix protein that performs a scaffolding function during assembly of iron-sulfur clusters	Iron utilization: biogenesis of iron-sulfur cluster proteins, mitochondrial function					−2.57	Sef1	2.0
orf19.3554	AAT1	Putative aspartate aminotransferase	Metabolism					−2.54	Sef1	−2.3
orf19.5137		Hypothetical protein	Unknown					−2.54	Sef1	−4.1
orf19.7244		Conserved hypothetical protein with homology to fumarylacetoacetate (FAA) hydrolases	Unknown					−2.50	Sef1	3.1
orf19.3040	EHT1	Putative ortholog of S.c. Eht1, an Acyl-coenzymeA:ethanol O-acyltransferase that plays a minor role in medium-chain fatty acid ethyl ester biosynthesis	Metabolism: biosynthesis of lipids					−2.50	Sef1
orf19.1077	ATM1	Putative ortholog of S.c. Atm1, a mitochondrial inner membrane ABC transporter	Mitochondrial function, transporter					−2.30	Sef1	2.9
orf19.1438		Conserved hypothetical protein with homology to NADH dehydrogenase, FAD-containing subunit	Unknown					−2.27	Sef1
orf19.2862	RIB1	Putative ortholog of S.c. Rib1, a GTP cyclohydrolase II that catalyzes the first step of the riboflavin biosynthesis	Metabolism: biosynthesis of cofactors					−2.12	Sef1
orf19.1927	SNM1	Conserved hypothetical protein with Rpr2 motif of Rnase MRP family members	Translation: rRNA processing					−2.04	Sef1	3.2	Hap43
orf19.4012	PCL5	Putative cyclin	Cell cycle					−2.02	Sef1	−2.5
orf19.6399	ATS1	Putative ortholog of S.c. Ats1, a protein required, with Elongator complex, Kti11p, and Kti12p, for modification of wobble nucleosides in tRNA	Translation: tRNA modification					−2.01	Sef1
orf19.6844	ICL1	Putative ortholog of S.c. Icl1, a key enzyme of the glyoxylate cycle	Metabolism: glyoxylate cycle	VIR				15.04	Sef1	32.9
orf19.4679	AGP2	Putative ortholog of S.c. Agp2, an amino acid permease	Transporter: polyamines							−2.1	Hap43
orf19.516		Putative ortholog of S.c. Rft1, a protein required for translocation of Man5GlcNac2-PP-Dol from the cytoplasmic side to the lumenal side of the ER membrane	Cell wall composition: N-glycosylation							2.1	Hap43
orf19.4676		Conserved hypothetical protein with homology to mitochondrial intermembrane space proteins	Mitochondrial function							2.2	Hap43
orf19.93		Putative ortholog of S.c. Mic17, a mitochondrial intermembrane space protein	Mitochondrial function							2.2	Hap43
orf19.4041		Putative ortholog of S.c. Pex4, a peroxisomal ubiquitin conjugating enzyme	Peroxisome function							2.2	Hap43
orf19.517	HAP31	Putative ortholog of S.c. Hap3, a subunit of the CCAAT-binding complex	Iron regulation: transcription factor							2.7	Hap43
orf19.2825		Putative ortholog of S.c. Dre2, a protein required for iron-sulfur cluster assembly and sister chromatid cohesion	Iron utilization: iron-sulfur cluster assembly							3.3	Hap43
orf19.2149		Putative ortholog of S.c. Ypr003c, a putative sulfate permease	Transporter: sulfate							3.3	Hap43

A simplified interaction network focused on iron homeostasis is presented in Figure 2d. Under iron-replete conditions, Sfu1 directly represses Sef1 and iron uptake genes. Under iron-limiting conditions, Sef1 directly activates Hap43 and iron uptake genes, and Hap43 directly represses Sfu1 and iron utilization genes. The intercalation of Sef1 into this network reconfigures the reciprocal switch between the GATA factor (Sfu1) and the CCAAT-binding complex (Hap43) that is found in most other ascomycetes; however, the overall logic is unchanged, with iron uptake genes being repressed and iron utilization genes expressed under iron-replete conditions, and the reverse when environmental iron is low.

Putative DNA binding motifs recognized by Sef1 and the Hap43-associated CCAAT-binding complex were determined by MEME analysis of the highest-confidence DNA binding targets. As shown in Figure 2e, the top MEME hits strongly resemble consensus sequences of orthologs in other species. The Sef1 motif contains three CGG repeats (one inverted). Such repeats are characteristic of fungal zinc binuclear finger proteins, with spacing that is specific to individual family members (MacPherson et al., 2006). The Hap43-associated motif contains the signature CCAAT sequence of the CCAAT-binding complex, as defined in multiple eukaryotic species (Chodosh et al., 1988). Although unbiased searches of putative Sfu1 targets did not reveal a unifying motif, the conserved HGATAR motif (H represents A,T, or G; (Scazzocchio, 2000)) appears at least once in 7 of the 9 targets (Table S1b).

Sef1 is uniquely important in C. albicans

We questioned whether the prominence of Sef1 in C. albicans iron homeostasis is unique to this species vs. existing unrecognized among other fungi. Reciprocal amino acid sequence comparisons revealed no clear Sef1 ortholog in the S. pombe fungal lineage (Figure 3a). To exclude the presence of a functional homolog that is divergent in sequence, we tested mutants affecting 29 of 31 predicted zinc binuclear finger proteins for sensitivity to iron depletion (deletions of SPBC1773.12 and SPCC965.10 were not recovered and may be inviable). None of the mutants was highly sensitive to iron depletion (Figure S2a), arguing against the presence of a functional Sef1 homolog.

Figure 3. Analysis of transcription factor orthologs in C. albicans, S. cerevisiae, and S. pombe.

a) Phylogeny of iron-related transcription factors, based on amino acid sequence. Transcription factors present in each boxed lineage are shown on the left. Notably, Sef1 and Aft factors were gained by the C. albicans and S. cerevisiae lineages, and the GATA factor was lost by the S. cerevisiae lineage. Although the Hap2, Hap3, and Hap5 components of the CCAAT-binding complex are conserved at the sequence level in all three lineages, orthologs of the “HapX” regulatory component could not be unambiguously identified based on amino acid sequence and were instead defined functionally (Baek et al., 2008; Forsburg and Guarente, 1989; Hortschansky et al., 2007; Mercier et al., 2006).

b) Phenotypic comparison of orthologous mutants in C. albicans, S. cerevisiae, and S. pombe. Strains were plated on low iron medium (with iron chelators), iron replete medium (standard), high iron medium (with ferrichrome), or high copper medium (with copper sulfate) and incubated at 30°C. Note that densely growing C. albicans is darkly pigmented on high copper medium.

c) Updated comparison of iron homeostasis among three model fungi. The schematic integrates published information with our current analysis of C. albicans, S. pombe, and S. cerevisiae. S. pombe Php4/CBP has been implicated in repression of iron utilization genes (Mercier et al., 2006), but direct regulation has not yet been demonstrated.

See also Figure S2. Strains and primers are described in Tables S2a and S2b, respectively.

In contrast to S. pombe, the S. cerevisiae lineage has maintained a Sef1 ortholog that is recognizable at the amino acid level (Figure 3a). However, this lineage (along with the C. albicans lineage, Figure 3a) also acquired Aft family transcription factors that are known to regulate iron uptake in S. cerevisiae (Courel et al., 2005; Yamaguchi-Iwai et al., 1995; Yamaguchi-Iwai et al., 1996).

To clarify the iron-related roles of orthologous transcription factors among C. albicans, S. pombe, and S. cerevisiae, we profiled knockout mutants in each species on media containing low, standard, or elevated levels of iron, as well as high copper medium (that promotes iron uptake through enhanced assembly of iron permease/oxidase complexes; phenotypes are presented in Figure 3b). On standard medium, all mutants grew similarly to wild type except for S. cerevisiae aft1Δ (Aft factor), which exhibited a mild defect. On iron-depleted media, S. cerevisiae aft1Δ, C. albicans sef1ΔΔ and hap43ΔΔ (CCAAT-binding complex), and S. pombe php4Δ (CCAAT-binding complex) exhibited substantial growth defects, indicating roles for the deleted genes in resistance to iron depletion. On ferrichrome-supplemented media (“high iron”), only C. albicans sfu1ΔΔ GATA factor and S. pombe fep1Δ (GATA factor) exhibited growth defects, suggesting roles for these factors in resistance to iron toxicity; these phenotypes were enhanced on copper-supplemented media.

These results emphasize the importance of the GATA factor and CCAAT-binding complex in S. pombe and C. albicans iron homeostasis (whereas Aft1 is the major player in S. cerevisiae). The GATA factor protects against toxicity when environmental iron is abundant, and the CCAAT-binding complex prevents deficiency when environmental iron is scarce. The central role of Sef1 under iron-limiting conditions appears restricted to C. albicans, since this factor is not conserved in S. pombe and since the S. cerevisiae ortholog is not required for growth (nor is S.c.SEF1 expression upregulated, Figure S2b) under such conditions. These observations are integrated into an updated comparison of the three fungal regulatory circuits in Figure 3c.

SEF1 and SFU1 are differentially expressed in the bloodstream and gut

Given the divergent roles of Sef1 and Sfu1 in protection from iron depletion vs. iron toxicity in vitro, we considered whether they might also play specialized roles in the host. We compared SEF1 and SFU1 expression in wild type C. albicans grown for 1 hour at 37°C in human plasma or propagated for 5 days in the murine gastrointestinal tract (see below for description of this model). RT-PCR revealed a 2-fold induction of SEF1 vs. 3-fold repression of SFU1 in the plasma relative to the gut (Figure 4a), with concomitant induction of iron uptake genes (Figure 4b, black bars). Disparate effects on the expression of iron uptake genes were seen in sef1ΔΔ and sfu1ΔΔ mutants grown in the same environments (Figure 4b). Deletion of SEF1 attenuated the expression of iron uptake genes primarily in plasma (white bars), whereas deletion of SFU1 enhanced expression especially in the gut (crosshatched bars). These results suggest that Sef1 activates iron uptake genes in iron poor niches such as the bloodstream, whereas Sfu1 restricts expression of these genes, particularly in iron replete niches such as the gut.

Figure 4. Analysis of C. albicans gene expression in host niches that differ in iron content.

a) SEF1 is induced in the bloodstream, and SFU1 is induced in the gut. RT-PCR was used to analyze RNA extracted from wild type C. albicans grown for 1 hour in human plasma or for 5 days in the mouse gastrointestinal infection model. Transcript levels were normalized to levels of 16S ribosomal RNA. Error bars indicate the standard deviation.

b) Expression of iron uptake genes in wild type, sef1ΔΔ, and sfu1ΔΔ after growth in human plasma or the murine gastrointestinal tract. P stands for plasma and G for gut. Numerical values (with standard deviation) are presented for results that exceed the scale of the chart.

Sef1 but not Sfu1 is required for virulence

To determine whether Sef1 or Sfu1 contributes to virulence, we performed intravenous infections of BALB/c mice with sef1ΔΔ, sfu1ΔΔ, or wild type C. albicans and determined the time to illness (Figure 5a). As shown in Figure 5b, sef1ΔΔ but not sfu1ΔΔ displayed a significant virulence defect (p<0.0001, log-rank test); the defect was complemented by restoration of a copy of wild type SEF1, confirming genetic linkage.

Figure 5. Roles of C. albicans Sef1 and Sfu1 in virulence and commensalism.

a) Virulence experiment. BALB/c mice were infected by tail vein injection with individual C. albicans strains (wild type, sef1ΔΔ, sfu1ΔΔ, or gene addback strains), and time to illness was monitored.

b) Sef1 but not Sfu1 is essential for virulence. Only the sef1ΔΔ mutant exhibited a significant decrease in virulence compared to wild type (asterisk indicates p<0.0001, log rank test).

c) Persistence experiment. BALB/c mice were infected by tail vein injection with 1:1 mixtures of wild type and sef1ΔΔ or sfu1ΔΔ). After mice developed clinical disease, the abundance of each strain in the inoculum (I) vs. mouse kidneys (R) was determined by qPCR.

d) Sef1 but not Sfu1 is required for persistence in host kidneys. Compared to wild type, sef1ΔΔ was significantly depleted from kidneys (p<0.0001, unpaired t-test), whereas sfu1ΔΔ was significantly enriched (p<0.0001).

e) Commensalism experiment. BALB/c mice were infected by gavage with 1:1 mixtures of wild type and sef1ΔΔ or sfu1ΔΔ. The abundance of each strain in the inoculum (I) and after recovery from fecal pellets (R) was determined by qPCR.

f) Sfu1 and Sef1 promote commensalism. sef1ΔΔ and sfu1ΔΔ were progressively depleted from fecal pellets relative to wild type (sef1ΔΔ: p=0.0017 at day 5, p=0.0047 at day 10, p=0.0012 at day 12; sfu1ΔΔ: p<0.002 at day 5, p<0.0003 at day 10, p<0.0001 at day 15; unpaired t-test). Comparison between the competitive indices of each mutant on day 15 indicated a more severe defect for sfu1ΔΔ (p=0.002, unpaired t-test).

We next investigated the abilities of the mutants to compete with wild type C. albicans for persistence in host tissues. BALB/c mice were infected intravenously with 1:1 mixtures of wild type and sef1ΔΔ or sfu1ΔΔ, followed by euthanasia when they developed signs of clinical illness. C. albicans was recovered from kidneys (the primary target organ in this model), and the relative abundance of each strain was determined by qPCR (Figure 5c). As shown in Figure 5d, sef1ΔΔ was significantly outcompeted by wild type C. albicans in mouse kidneys (p<0.0001, unpaired t-test), whereas sfu1ΔΔ displayed a significant competitive advantage (p<0.0001). These experiments indicate that C. albicans Sef1 but not Sfu1 is required for virulence and persistence in a mammalian bloodstream infection model.

Sef1 and Sfu1 promote commensalism

To assess the roles of Sef1 and Sfu1 in commensalism, we utilized a mouse model of gastrointestinal infection. In this model, infected mice remain healthy despite persistent, high-grade colonization with C. albicans (~10⁷ CFUs/g stool; data not shown). BALB/c mice were infected by gavage with 1:1 mixtures of wild type C. albicans and either sef1ΔΔ or sfu1ΔΔ, and the abundance of each strain in fecal pellets over 15 days was monitored by qPCR (Figure 5e). Both mutants exhibited competitive defects compared to wild type throughout the time course (p<0.002, unpaired t-test; Figure 5f). Comparison between the competitive indices of each mutant on Day 15 revealed sfu1ΔΔ to have the more substantial defect (p=0.002, unpaired t-test). These results indicate that both transcription factors contribute to commensalism, with Sfu1 perhaps playing the more prominent role.

DISCUSSION

C. albicans is a ubiquitous commensal of the human microbiome, as well as the most common cause of disseminated fungal infections. Our detailed analysis of the regulatory system governing iron homeostasis in this microorganism underscores its intense adaptation to the mammalian host. Unlike the bipartite system used by most ascomycetes, the C. albicans system hinges on three transcriptional regulators, Sef1, Sfu1, and the Hap43-associated CCAAT-binding complex. These compose a tightly wired network in which each component directly regulates the expression of another transcription factor in the circuit as well as genes for iron uptake (Sfu1 and Sef1) or iron utilization (Hap43). Functional profiling of sef1ΔΔ and sfu1ΔΔ mutants in mouse models of bloodstream and gastrointestinal infection revealed a tradeoff of importance between these regulatory components that depends on the microenvironment of the host.

The C. albicans system for regulating iron homeostasis (Figure 2d) was deduced from global RNA expression and chromatin immunoprecipitation experiments. What distinguishes this system from that of most ascomycetes is the intercalation of Sef1 between the GATA factor (Sfu1) and the CCAAT-binding complex (Hap43). In other fungi, orthologs of Sfu1 and Hap43 create a simple switch, in which each component represses a discrete set of genes as well as the other transcription factor. In C. albicans, Sfu1 regulates Hap43 only indirectly via Sef1, and Sef1 directly activates target genes that are also directly repressed by either Sfu1 or Hap43. Our comprehensive analysis of mutants affecting transcription factor orthologs in C. albicans, S. pombe, and S. cerevisiae supports the idea that Sef1 plays a unique role in C. albicans iron homeostasis and was not simply missed in the other fungi.

Despite substantial rewiring of this transcriptional circuit among the three species, the overall logic has been preserved. That is, high-affinity iron uptake genes are expressed only under circumstances of environmental iron depletion. Nevertheless, specific features of each system likely impart additional properties that may be adaptive for the organism. In engineering parlance, the C. albicans mode of iron homeostasis in which one transcription factor (Sfu1) regulates the expression of a second transcription factor (Sef1)—and both regulate a common target (iron uptake genes)—is known as a feed forward loop (Mangan and Alon, 2003). An emergent feature of such loops is that expression of coregulated genes is buffered against transient perturbations of the activating signal, such that the level of expression of Sef1-Sfu1 coregulated iron uptake genes should be stabilized relative to short-term fluctuations in environmental iron, and therefore guarded against “spurious” activation or repression. The ability to maintain continuous expression of survival factors or continuous repression of toxicity factors in the appropriate setting could conceivably be of selective advantage.

Our findings that Sef1 but not Sfu1 is critical for virulence and competitive infection, whereas Sfu1 has a more important role in commensalism, suggest that extreme contrasts in iron availability within the mammalian host may have helped to shape the C. albicans iron regulatory circuit. A requirement for upregulation of high-affinity iron uptake genes in the host bloodstream is a common and well-recognized feature of many bacterial pathogens, some of which also use iron depletion to cue the expression of virulence genes (Mey et al., 2005). In keeping with this precedent, Sef1 directly activates multiple genes with known or suspected roles in virulence (Table 2).

Far less is known about microbial adaptations to the mammalian gastrointestinal tract, yet the microbiome is a primary source of pathogens causing disseminated disease. Our studies are a starting point for understanding such adaptations in C. albicans. The findings that Sfu1 promotes competitiveness in the gut and resistance to iron toxicity in vitro suggest that iron toxicity is an important selective pressure on gut commensals. Iron depletion is likely also important, at least in certain microniches, since sef1ΔΔ was also defective in the commensal model. In more general terms, our results emphasize that different host niches differ dramatically in ways that must be sensed by microbes. We hypothesize that successful commensals, whether bacterial or fungal, have evolved signaling and regulatory mechanisms to promote success in the commensal habitat, whereas commensal-pathogens that also enter the bloodstream must be capable of rapid cellular reprogramming to survive in this environment. An understanding of these mechanisms will be needed to decipher the transition between the commensal and pathogenic lifestyles.

METHODS

Strains

Strains are described in Table S2a, and primers are listed in Table S2b. C. albicans mutants, complemented strains, and Myc-tagged alleles of Sef1, Sfu1, and Hap43 were created as described (Nobile et al., 2009; Noble et al., 2010; Noble and Johnson, 2005).

S. pombe deletion mutants affecting Fep1, Php4, and zinc binuclear finger proteins were created by homologous recombination in reference strain SP286 using gene disruption fragments containing KanMX6 (G418 resistance) flanked by 700–900 bp of DNA homologous to sequences upstream and downstream of the target ORFs. Colony PCR was used to screen G418-resistant transformants for the expected 5′ and 3′ recombination junctions, and absence of the targets ORFs was confirmed using primers internal to the disrupted ORFs.

Media

Liquid “iron replete” medium was YPD (Guthrie and Fink, 1991), and “low iron” medium was YPD plus 500 μM bathophenanthrolinedisulfonic acid (BPS). Solid “iron replete” medium for C. albicans and S. cerevisiae was SC/2% agar (Guthrie and Fink, 1991); “low iron” was SC/2% agar with 350 μM BPS; “high iron” was SC/2% with 25 μM ferrichrome; and “high copper” was YPD/2% agar with 6 mM (C. albicans) or 800 μM (S. cerevisiae) CuSO₄. For S. pombe, solid “iron replete” medium was YES/2% agar (Forsburg, 2003); “low iron” was YES/2% agar with 140μM 2,2′-dipyridyl (DIP); “high iron” was YES/2% agar with 25 μM ferrichrome; and “high copper” was YES/2% agar with 800 μM CuSO₄.

Gene Expression Analysis

Saturated overnight cultures of sef1ΔΔ (SN330), hap43ΔΔ (SN694), sfu1ΔΔ (SN515), and isogenic wild type C. albicans (SN250) were inoculated into YPD to OD₆₀₀=10⁻⁴ and incubated with shaking at 30°C. The next morning, logarithmically growing cells were diluted to OD₆₀₀ 0.01 in iron-replete (wild type and sfu1ΔΔ) or low iron (wild type, sef1ΔΔ, and hap43ΔΔ) medium and incubated at 30°C for 5–6 hours before harvesting at OD₆₀₀=0.5–0.6. 5–6 biological replicates were performed per strain per condition. Total RNA was prepared using a hot phenol method (Miller and Johnson, 2002). 10 μg of each RNA was treated with DNase I (Turbo DNA-free kit, Ambion) and reverse transcribed using aminoallyl-dUTP and Superscript II (Invitrogen) according to manufacturers’ instructions. cDNA was labeled with Cy3 and Cy5 (Amersham), and 0.5 μg of each channel was hybridized to custom Agilent C. albicans ORF arrays (15,000 spots/array, 70-mer probes).

Fluorescently labeled cDNAs from mutants were directly hybridized against those from wild type grown under the same conditions; dye flip controls were included. 6 additional arrays were performed using wild type grown under iron-replete vs. iron-limiting conditions. Arrays were scanned using a Genepix 4000A Axon scanner, and spots were filtered using GenePix Pro software. Data were normalized using Goulphar (LOWESS normalization) and subjected to One-class Significance Analysis of Microarrays (SAM) analysis with a median false discovery rate of 0.1%. Candidates meeting SAM criteria were also required to exhibit median 2-fold changes among 5–6 experiments. Primary data are available at the Geo Expression Omnibus (GEO) website (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE30593.

GO Term analysis was performed using the GO Term Gene Ontology Finder tool on the Candida Genome Database website (Skrzypek et al., 2010).

Whole Genome Chromatin Immunoprecipitation Analysis

Saturated cultures of untagged wild type C. albicans (SN250), Sef1-Myc (SN423), Hap43-Myc (SN840), and Sfu1-Myc (SN646) were inoculated into low iron (untagged, Sef1-Myc, Hap43-Myc) or iron replete (untagged, Sfu1-Myc) liquid medium to OD₆₀₀=0.05. Cultures were incubated with shaking at 30°C until OD₆₀₀ 0.4, when formaldehyde was added to 1% final (with shaking, room temperature, 15 minutes), followed by glycine to 125mM final (with shaking, room temperature, 5 minutes). Cells were collected by centrifugation at 4°C and washed twice with 20 mM Tris-HCl pH 7.5/150 mM NaCl, followed by freezing in liquid N₂ and storage at −80°C. Cell lysis, DNA shearing, and ChIP-Chip were performed as described (Nobile et al., 2009).

12 independent hybridization experiments were performed on 6 biological replicates of the untagged control and 2 biological replicates each of Sef1-Myc, Sfu1-Myc, and Hap43-Myc. Agilent Chip Analytics software v1.2 (Agilent Technologies) was used for initial data normalization and analysis (Tuch et al., 2008), followed by visualization and additional analysis using MochiView v.1.39 (http://johnsonlab.ucsf.edu/). High confidence regulatory events were associated with Agilent segment p-values of ≥4 (−log₁₀ p-value based on the enrichment statistic for each probe in the region) and minimum 2-fold (Sef1-Myc and Hap43-Myc) or 1.5-fold (Sfu1-Myc) enrichment in both biological replicates of the epitope-tagged strains (Table S1b). Genes with enrichment peaks in untagged controls were excluded. Primary data are available at the GEO website (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE30593.

Identification of DNA recognition motifs

MEME v.3.5.7 software (Bailey and Elkan, 1994) was applied to 250 bp sequences centered at the midpoints of the most significant binding peaks of Sef1, Sfu1, and Hap43. Analysis was performed using minw=6, maxw=20, and nmotif=3.

Phylogenetic analysis

Protein sequences of iron homeostasis regulators in C. albicans, S. pombe, and S. cerevisiae were BLASTed (Altschul et al., 1997) against a database of all fungal ORFs. Matching ORFs with E-values <10⁻⁵ were extracted from the database and multiply aligned with MUSCLE (Edgar, 2004), followed by inference of an NJ tree using ClustalW (Higgins and Sharp, 1988). The resulting gene tree was inspected and gain/loss/duplication events were mapped to a fungal species tree inferred previously (Tuch et al., 2008). The results of this analysis (summarized in Figure 2a) are consistent with those in the YGOB (Byrne and Wolfe, 2005) and CGOB (Fitzpatrick et al.) databases, which cover a narrower range of species but also account for conservation of gene synteny. Whereas Sfu1 and components of CBP clearly existed prior to divergence of the ascomycetes studied here, evidence for an early origin of Sef1 and Aft1/2 is lacking.

In vitro growth assays

Freshly streaked C. albicans, S. cerevisiae, and S. pombe strains were inoculated into YPD (C. albicans, S. cerevisiae) or YES (S. pombe) and incubated overnight at 30°C. Saturated cultures were diluted with sterile water to A₆₀₀=0.8, serial 10-fold dilutions were made, and 5–10 μl of each dilution series was applied to solid test media, followed by incubation at 30°C for 2–4 days.

In vivo assays

Procedures involving animals were approved by the UCSF Institutional Animal Care and Use Committee. Virulence analysis was conducted by tail vein injection of groups of 8–10 week old female BALB/c with 5 × 10⁵ CFUs of wild type (SN425), sef1ΔΔ (SN452), sef1ΔΔ/SEF1 (SN436), sfu1ΔΔ (SN668), or sef1ΔΔ/SFU1 (SN664). Mice were monitored twice daily and euthanized when morbidity criteria were met (weight loss>15%, hunched posture, inactivity).

Competitive bloodstream infections were performed with wild type (SN250), sef1ΔΔ (SN330), and sfu1ΔΔ (SN515) as previously described (Noble et al., 2010).

The mouse model of C. albicans commensalism was adapted from published protocols (Koh et al., 2008; White et al., 2007). Groups of 8–10 week female BALB/c mice received penicillin 1500 un/ml and streptomycin 2 mg/ml in their drinking water for 3–5 days prior to gavage with 10⁸ CFUs of a 1:1 mix of wild type (SN250) and sef1ΔΔ (SN330) or sfu1ΔΔ (SN515). Antibiotics were continued, and fecal pellets were collected at specified intervals. C. albicans recovery and quantification were performed as described (Noble et al., 2010).

Highlights.

• C. albicans uses a transcriptional circuit to regulate iron acquisition from the host.

• Sef1 activates iron uptake genes and promotes virulence in the bloodstream.

• Sfu1 represses iron uptake genes and promotes commensalism in the gut.