Post-Transcriptional Regulation of the Sef1 Transcription Factor Controls the Virulence of Candida albicans in Its Mammalian Host

The yeast Candida albicans transitions between distinct lifestyles as a normal component of the human gastrointestinal microbiome and the most common agent of disseminated fungal disease. We previously identified Sef1 as a novel Cys6Zn2 DNA binding protein that plays an essential role in C. albicans virulence by activating the transcription of iron uptake genes in iron-poor environments such as the host bloodstream and internal organs. Conversely, in the iron-replete gastrointestinal tract, persistence as a commensal requires the transcriptional repressor Sfu1, which represses SEF1 and genes for iron uptake. Here, we describe an unexpected, transcription-independent role for Sfu1 in the direct inhibition of Sef1 function through protein complex formation and localization in the cytoplasm, where Sef1 is destabilized. Under iron-limiting conditions, Sef1 forms an alternative complex with the putative kinase, Ssn3, resulting in its phosphorylation, nuclear localization, and transcriptional activity. Analysis of sfu1 and ssn3 mutants in a mammalian model of disseminated candidiasis indicates that these post-transcriptional regulatory mechanisms serve as a means for precise titration of C. albicans virulence.


Introduction
Candida albicans is a ubiquitous component of the mammalian microbiome [1] as well as the most common fungal pathogen of humans [2,3,4,5]. As this organism transits between its commensal niches (mammalian skin and gastrointestinal tract) and those of virulence (bloodstream and internal organs), it experiences profound shifts in the levels of nutrients, the physical environment, and immune surveillance. We previously demonstrated that a novel C. albicans transcriptional regulatory circuit is required for survival in at least two distinct habitats, the host bloodstream and gastrointestinal tract [6], where levels of bioavailable iron differ by more than 20 orders of magnitude [7,8]. In the bloodstream, where iron is tightly sequestered by host transferrin [7], C. albicans defends against iron deficiency through expression of Sef1, a Cys 6 Zn 2 transcriptional activator of iron uptake genes and an indirect suppressor of the gene for Sfu1 [6]. In the gastrointestinal tract, where iron is abundant thanks to diet and sloughed cells [8,9], C. albicans defends against iron toxicity through the expression of Sfu1 [6], a GATA family transcriptional repressor that inhibits both SEF1 and genes for iron uptake [6,10]. Remarkably, the opposing roles of Sef1 and Sfu1 in iron homeostasis extend to differing relationships with the host, with Sef1 promoting virulence and Sfu1 promoting commensalism in animal models [6]. However, the details of how these transcriptional regulators are themselves regulated by iron remain to be elucidated. Sfu1 is broadly conserved among ascomycetes, and orthologs from multiple species have been shown to play a negative role in iron homeostasis through repression of iron uptake genes [10,11,12,13,14,15]. The best-characterized ortholog is Fep1 from Schizosaccharomyces pombe that, like Sfu1, is subject to repression at the transcriptional level when environmental iron is limiting [16,17]. In this species, protein activity is also regulated by iron, since only iron-bound Fep1 can associate with DNA [18]. By contrast, orthologs of Sef1 have not been extensively characterized, in part because the genomes of only a handful of species in the Saccharomyces and Candida lineages encode this protein [6]. Moreover, C. albicans Sef1 appears to function differently from its S. cerevisiae ortholog, since iron homeostasis in the latter species is controlled by Aft family proteins [19,20,21] and is not dependent on Sef1 [6].
Here we describe studies that reveal an unexpected, transcription-independent role of C. albicans Sfu1 in inhibiting Sef1 function, as well as a role for a predicted protein kinase, Ssn3, in Sef1 activation. Specifically, we find that, under iron-replete conditions, Sfu1 physically associates with Sef1 and sequesters it in the cytoplasm, where it is destabilized. In contrast, under irondepleted conditions, Sef1 forms an alternative complex with Ssn3, resulting in Sef1 phosphorylation, nuclear localization, and the transcriptional activation of iron uptake genes. These posttranscriptional regulatory events are of direct consequence to C. albicans virulence, since either overexpression of SFU1 or deletion of SSN3 results in attenuated virulence in a mammalian model. We hypothesize that these multiple, opposing mechanisms for Sef1 regulation, including a surprising protein-protein interaction with its own transcriptional inhibitor, enable this obligate mammalian parasite to fine-tune its interactions with the host on a spectrum from commensalism to virulence.

Iron-dependent transcriptional repression by Sfu1 accounts for the observed regulation of SEF1
Given the important role of Sef1 in promoting C. albicans virulence [6], we speculated that it would be a prime target for regulation. We and others had previously shown that, under ironreplete conditions, transcription of SEF1 is repressed by Sfu1 [6,10], the C. albicans structural and functional ortholog of S. pombe Fep1 [22]. To determine whether additional regulators contribute to SEF1 gene expression, we used RT-qPCR to compare SEF1 transcript levels in a wild-type strain vs. an isogenic strain lacking the SFU1 gene. The result was that deletion of SFU1 was sufficient to fully derepress SEF1, independent of the iron content of the growth medium (Figure 1a, compare the level of SEF1 in wildtype cells grown in iron-depleted medium [bar 2, derepressing condition] to that in the sfu1DD strain, grown in either iron-replete [bar 3] or iron-depleted medium [bar 4]); numerical values and statistical analysis are provided in Table S1. These results suggested that iron-dependent transcriptional repression by Sfu1 is sufficient to account for SEF1 transcript levels in wild-type cells.
To determine whether forced overexpression of SFU1 could further suppress SEF1 gene expression, we created a strain in which the endogenous promoter of SFU1 was replaced with the strong, constitutively active TDH3 promoter (SFU1 OE ); increased levels of SFU1 RNA and protein were confirmed by RT-qPCR and immunoblot analysis, respectively (Figure S1a and S1b). Overexpressed Sfu1 did not substantially diminish the level of SEF1 mRNA under iron-replete or iron-depleted conditions (Figure 1a, compare bar 1 with bar 5 and bar 2 with bar 6). The failure of overexpressed SFU1 to inhibit the transcription of SEF1 under iron-depleted conditions suggested that C. albicans Sfu1 might, like its S. pombe ortholog [18], require iron as a cofactor for binding to DNA and transcriptional repression.

Sfu1 promotes Sef1 localization in the cytoplasm
Although SEF1 mRNA levels were normal in the SFU1overexpression strain (Figure 1a), this strain demonstrated hypersensitivity to treatment with the iron chelator, bathophenanthroline disulfonic acid (BPS; Figure S2), suggestive of a potential defect in iron acquisition. Addition of FeCl 3 to the BPS-treated medium was sufficient to reverse the growth defect ( Figure S2), confirming the specificity of the iron-chelation phenotype. To determine whether Sef1 protein levels were affected in the SFU1 OE strain, we utilized an epitope-tagged version of Sef1 in which 13 copies of the Myc epitope were fused in-frame at the C-terminus; this fusion protein is fully functional [6]. Surprisingly, the steady state level of Sef1-Myc was substantially reduced, particularly under iron-depleted conditions ( Figure S3). The observations that overexpression of SFU1 does not affect SEF1 mRNA levels but strongly decreases Sef1 protein levels raised the possibility that Sfu1 may have a second function in the post-transcriptional regulation of Sef1.
To determine whether Sef1 localization is regulated, we used indirect immunofluorescence to visualize Sef1-Myc in wild-type cells exposed to varying concentrations of iron. Under iron-replete conditions, Sef1-Myc was localized primarily in the cytoplasm (Figure 1b, WT strain, H; note the absence of green Sef1-Myc signal in the FITC channel in areas that correspond to red DNA signal in the DAPI channel; a negative control showing minimal staining of an isogenic strain that lacks the Myc epitope is shown in Figure S4a). Under iron-depleted conditions, however, Sef1-Myc was primarily nuclear, with prominent areas of yellow overlap when the FITC and DAPI channels were merged. Notably, examination of Sef1-Myc in an sfu1DD mutant revealed constitutive nuclear localization, even under iron-replete conditions (sfu1DD strain, Figure 1b). Conversely, overexpression of SFU1 resulted in substantial cytoplasmic localization of Sef1-Myc even under iron-depleted conditions in which it is usually nuclear (SFU1 OE strain, Figure 1b). By comparison, an Sfu1-Myc fusion protein was found to be distributed between the nucleus and cytoplasm in wild-type cells propagated under iron-replete conditions and primarily cytoplasmic under iron-limiting conditions ( Figure S4b).
These results established that Sef1 localization varies as a function of iron, that Sfu1 promotes Sef1 localization in the cytoplasm, and that the protein localizing activity of Sfu1-unlike its transcriptional repression activity ( Figure 1a)-does not inherently require iron.

Ssn3 promotes Sef1 phosphorylation and nuclear localization under iron-depleted conditions
Immunoblot analysis of Sef1-Myc recovered from wild-type cells grown under iron-replete vs. iron-depleted conditions demonstrated an inverse relationship between Sef1 protein abundance and iron levels ( Figure 2a, lanes 1 and 2), which was expected based on the known, iron-dependent inhibitory activity of Sfu1 on SEF1 gene expression. An unexpected finding was that the electrophoretic mobility of Sef1 also varied in an irondependent fashion. This subtle but reproducible decrease in Sef1 mobility under iron-depleted conditions was observed not only in wild-type cells, but also in an sfu1DD deletion mutant (lanes 3 and 4), arguing against a role for Sfu1 in this process.
We hypothesized that the lower mobility form of Sef1 might result from covalent phosphorylation. To test this hypothesis, we

Author Summary
Candida albicans is a fungus that resides on the skin and in the gastrointestinal tract of humans and other mammals. However, this commensal organism is also capable of proliferating and causing disease in people who have received antibiotics, who are immunocompromised, or who have suffered injury to epithelial layers. We previously identified a novel transcription factor called Sef1 that promotes C. albicans virulence by activating the expression of iron uptake genes in iron-poor environments, such as the host bloodstream. However, in iron-replete environments such as the gastrointestinal niche, the SEF1 gene is repressed by a second transcription factor called Sfu1. Here, we report our discovery of a series of posttranscriptional regulatory events that determine the intracellular localization, stability, and activity of Sef1 protein. Mutants that disrupt these post-transcriptional events alter C. albicans virulence in a mammalian model of disseminated infection. The existence of multiple levels of regulation speaks to the importance of Sef1 in C. albicans virulence and suggests that close titration of Sef1 activity is important for adaptation to distinct microenvironments within the mammalian host. Note that C. albicans gene deletion mutants are represented as DD because two alleles of target genes must be disrupted in this obligate diploid species. a) RT-qPCR results for SEF1 mRNA in wildtype (WT), sfu1DD, and SFU1-overexpression (SFU1 OE ) strains grown under iron-replete (H, high iron) or iron-depleted (L, low iron) conditions. Error used a tandem affinity purification strategy to recover TAP-tagged Sef1 from C. albicans grown under iron-replete or iron-depleted conditions. Purified TAP-tagged Sef1 exhibited an iron-dependent mobility shift similar to that of Sef1-Myc, with protein from the iron-depleted cells running with slightly lower mobility (Figure 2b, compare lanes 1 and 3). Treatment of the purified proteins with lambda phosphatase, a broad specificity enzyme with activity on phospho-serine, phospho-threonine, and phosphotyrosine residues, resulted in conversion of the lower mobility form of Sef1-TAP to the higher mobility form (Figure 2b, compare lane 4 to lanes 1 and 2), in support of our hypothesis.
To identify the kinase responsible for low-iron-dependent phosphorylation of Sef1, we tested the 31 available homozygous knockout mutants affecting predicted kinases for sensitivity to BPS. Our reasoning was that, if phosphorylation of Sef1 is required for full induction of iron uptake genes, then a mutant lacking the relevant kinase might be hypersensitive to iron depletion, that is, phenotypically similar to sef1DD itself [6,23]. Our screen identified the ssn3DD mutant as being hypersensitive to iron depletion ( Figure S2). Further, an immunoblot of Sef1-Myc recovered from the ssn3DD strain revealed persistence of the higher mobility form under iron-depleted conditions (Figure 2c), consistent with a role for Ssn3 in phosphorylation of Sef1. The identical result was obtained when Sef1-Myc was examined in a strain encoding a predicted kinase-dead allele of Ssn3 (Ssn3 D325A , Figure S5a).
Although C. albicans Ssn3 has not yet been characterized, its S. cerevisiae ortholog is a cyclin-dependent kinase with two known functions: first, it is a component of the Mediator complex with inhibitory activity on RNA polymerase II [24]; second, it phosphorylates a number of specific transcription factors to regulate their activity, nuclear-cytoplasmic localization, and/or stability [25,26,27]. To determine whether C. albicans Ssn3 influences the localization of Sef1, we performed indirect immunofluorescence on Myc-tagged Sef1 in the ssn3DD mutant. As shown in Figure 2d, deletion of SSN3 resulted in constitutive cytoplasmic localization of Sef1-Myc under both iron-replete and iron-depleted conditions; similar mislocalization was observed in a strain containing Ssn3 D325A ( Figure S5b). Unlike the case with SFU1, however, overexpression of SSN3 via the TDH3 promoter (SSN3 OE ) had no obvious effect on Sef1-Myc localization ( Figure  S5b), perhaps indicating that the nuclear localization activity of Ssn3 is restricted to low iron conditions.

Sfu1 and Ssn3 each physically interact with Sef1 but play opposing roles
The preceding results were suggestive of a model in which Sfu1 and Ssn3 have opposite and competing roles in Sef1 localization, with Sfu1 promoting cytoplasmic localization and Ssn3 promoting nuclear localization. To test this model, we utilized the SFU1overexpression strain that mislocalizes Sef1-Myc to the cytoplasm bars correspond to the standard deviation among quintuplicate (H) or septuplicate (L) biological samples. Numerical values and statistical analysis are provided in Table S1). b) Indirect immunofluorescence of Sef1-Myc in WT, sfu1DD, and SFU1 OE strains grown in iron-replete or iron-depleted medium. DIC represents phase images, FITC represents Sef1-Myc staining, DAPI represents DNA staining, and Merge represents the overlay of Sef1-Myc and DNA staining. Quantification of 100 cells in each experiment is shown on the right, with C representing .90% cytoplasmic staining, N .90% nuclear staining, and C+N a mixture of cytoplasmic and nuclear staining. Scale bar, 5 mm; all images were obtained at the same magnification. doi:10.1371/journal.ppat.1002956.g001 Figure 2. Ssn3 promotes phosphorylation and nuclear localization of Sef1 under iron-depleted conditions. a) Immunoblot of Sef1-Myc and alpha tubulin (internal standard) in wild-type vs. sfu1DD cells propagated under iron-replete (H) or iron-depleted (L) conditions. B) Immunoblot of purified Sef1-TAP protein either treated (+) or not treated (2) with lambda phosphatase. To accumulate sufficient quantities of the higher mobility form of Sef1-TAP (lanes 1 and 2), the protein was purified from an sfu1DD mutant grown under iron-replete conditions. The lower mobility form (lanes 3 and 4, prior to phosphatase treatment) was purified from wild-type cells grown under iron-depleted conditions. * indicates a presumed Sef1-Myc C-terminal proteolysis product that is observed only when Sef1 is recovered from nondenaturing extracts, such as those used for TAP purification; this fragment is not seen when denaturing extracts are used as in (a) above and (c) below. c) Immunoblot of Sef1-Myc and alpha tubulin recovered from wild-type or ssn3DD cells under iron-replete or iron-depleted conditions. d) Indirect immunofluorescence of Sef1-Myc in the ssn3DD mutant under iron-replete or iron-depleted conditions. Scale bar, 5 mm; all images obtained at the same magnification. doi:10.1371/journal.ppat.1002956.g002 under iron-depleted conditions (Figure 1b). We predicted that, if Ssn3 competes with Sfu1 for localization of Sef1, then overexpression of SSN3 might rescue this Sef1 mislocalization phenotype. Indeed, a strain in which both genes are driven by the strong TDH3 promoter exhibits substantial restoration of nuclear Sef1-Myc under iron-depleted conditions, with normal cytoplasmic localization under iron-replete conditions (Figure 3a). These results indicate that Sfu1 and Ssn3 exert opposing roles on Sef1 localization, but only under iron-depleted conditions (when Sef1 is phosphorylated).
To determine whether Sef1 physically associates with Sfu1 and/ or Ssn3, we created a series of double epitope-tagged strains, each containing a Myc-tagged version of one of the three potentially interacting proteins and a TAP-tagged version of another; the TAP epitope consists of a calmodulin binding domain fused to a TEV cleavage site and a Protein A domain ( Figure S6a; [28,29]). Co-immunoprecipitation experiments were performed using whole cell extracts prepared from cells grown under iron-replete or iron-depleted conditions. Extracts were incubated with IgGsepharose, which binds to the Protein A component of the TAP epitope, followed by extensive washing of the immunoprecipitated complexes and protein electrophoresis under denaturing conditions (SDS-PAGE; see Figure S6b for a schematic of the protocol). Finally, immunoblots were probed with anti-Myc antibodies to determine the presence or absence of a Myc-tagged putative binding partner. Specificity of IgG-sepharose for the TAP tag was confirmed by performing experiments with strains containing Myc-tagged fusion proteins and an unfused TAP tag ( Figure S6c), and specificity of the anti-Myc antibodies for the Myc epitope was confirmed using cells containing only the TAPtagged fusion proteins (Figure 3b).
Shown in Figure 3b are the results with Sfu1-Myc and Sef1-TAP. Sfu1-Myc was efficiently co-immunoprecipitated with Sef1-TAP when cells were propagated in iron-replete medium (lane 3, IP), but not when iron-starved cells were used (lane 4, IP). On the other hand, when the epitope tags were reversed, co-immunoprecipitated Sef1-Myc was poorly visualized using extracts of ironreplete cells (Figure 3c, lane 3) but was easily seen using ironstarved cells (Figure 3c, lane 4). Together, these results suggest that Sef1 and Sfu1 interact physically in a manner that is independent of iron levels, whereas the sensitivity of our biochemical assay is a function of the relative abundance of the Myc-tagged protein in the extract.
Co-immunoprecipitation experiments combining either Sef1-Myc or Sfu1-Myc with Ssn3-TAP revealed a robust interaction between Ssn3 and Sef1, but no detectable interaction between Ssn3 and Sfu1 (Figure 3d). That is, Sef1-Myc was efficiently coimmunoprecipitated with Ssn3-TAP from an extract of irondepleted cells (Figure 3d note that Ssn3-Myc is relatively abundant under both conditions. These results suggest that Sef1 physically associates with Ssn3 as well as Sfu1, but these appear to represent alternative complexes since Ssn3 and Sfu1 do not associate with each other.

Sef1 is likely destabilized in the cytoplasm
To learn whether the stability of Sef1 varies with its intracellular localization, we determined the half-life of Myc-tagged Sef1 in wild-type C. albicans and in mutants in which Sef1 is stably localized in either the nucleus or the cytoplasm. Under ironreplete conditions, Sef1 is predominantly cytoplasmic in wild-type C. albicans but is mislocalized to the nucleus in sfu1DD (Figure 1b). To obtain sufficient Sef1 protein for the analysis and to uncouple the role of Sfu1 in Sef1 localization from its effects on SEF1 transcription, we replaced the endogenous SEF1 promoter with a constitutively active TDH3 promoter in both wild-type and sfu1DD strains; overexpressed Sef1-Myc exhibited the same pattern of iron-dependent nuclear vs. cytoplasmic localization as Sef1-Myc expressed from its endogenous promoter ( Figure S7). The strains were propagated to mid log phase in iron-replete medium, followed by addition of cycloheximide to block further translation, and serial sampling for measurements Sef1-Myc abundance. Shown in Figure 4a is a quantitative immunoblot of Sef1-Myc and alpha tubulin, which was used as an internal control for protein loading. Under these iron-replete conditions, the calculated half-life of cytoplasmic Sef1-Myc was ,80 minutes (wild type, R 2 = 0.94) and that of nuclear Sef1-Myc was ,160 minutes (sfu1DD, R 2 = 0.92). Next, we examined Sef1-Myc stability under iron-depleted conditions, in which the protein is predominantly nuclear in wild-type cells (Figure 1b) but mislocalized to the cytoplasm in the ssn3DD mutant (Figure 2d). Wild-type and ssn3DD strains expressing SEF1-MYC from the endogenous SEF1 promoter were propagated in iron-depleted medium to mid-log phase, then treated with cycloheximide and visualized as above (Figure 4b). Under these iron-depleted conditions, the calculated half-life of nuclear Sef1-Myc (,150 minutes in wild type; R 2 = 0.98) was once again more stable than that of cytoplasmic Sef1-Myc (,40 minutes in ssn3DD; R 2 = 0.96). The most parsimonious explanation for these results is that Sef1 is degraded more rapidly in the cytoplasm than in the nucleus; however, we cannot exclude the possibility that Ssn3 and Sfu1 exert independent effects on Sef1 degradation that are unrelated to its intracellular localization.
Our current model of Sef1 regulation, which integrates these results with previously published findings [6,10,22], is depicted in Figure 4c. According to the model, Sef1 is subject to two distinct forms of Sfu1-mediated repression when environmental iron is replete: 1) transcriptional repression of the SEF1 gene, through direct binding and repression of transcriptional initiation; and 2) post-translational inhibition of Sef1 protein, through direct binding and retention in the cytoplasm, where Sef1 is more rapidly degraded. Alternatively, under iron-limiting conditions, when Sfu1 protein is depleted, Sef1 is bound by Ssn3, phosphorylated, and localized in the nucleus, where it activates expression of iron uptake genes. Our recent observation that Sef1-Myc is constitutively cytoplasmic in an sfu1DD/ssn3DD double mutant strain ( Figure S8) suggests that Ssn3 may play actively promote the nuclear localization of Sef1, beyond merely extricating Sef1 from Sfu1.

Nuclear localization of Sef1 is associated with virulence
We previously demonstrated that SEF1 gene expression is induced in the iron-limiting environment of the host bloodstream and that SEF1 is required for virulence in a murine model of bloodstream candidiasis [6]. Conversely, we showed that SFU1 is not required for virulence but rather that the sfu1DD mutant exhibits increased competitive fitness relative to wild-type C. albicans, presumably because of an enhanced ability to take up extracellular iron [6]. Our current results suggest that the negative effect of Sfu1 on C. albicans virulence likely results from mislocalization of Sef1 to the cytoplasm rather than from repression of SEF1 gene expression, since only the former activity is observed under conditions of iron depletion (compare Figure 1a and Figure 1b). We tested this hypothesis by examining the virulence of mutants with moderate (SFU1 OE , Figure 1b, low iron condition) to severe (ssn3DD, Figure 2d, low iron condition) defects in Sef1 nuclear localization. As shown in Figure 4d and 4e, both mutants were significantly attenuated in the murine bloodstream infection model, such that mice infected with either mutant survived longer than mice infected with wild type. Note also that the strength of the virulence defects paralleled the . Sfu1 and Ssn3 each physically interact with Sef1 but play opposing roles in Sef1 localization. a) Overexpression of SSN3 restores nuclear localization of Sef1 to a strain that also overexpresses SFU1. Indirect immunofluorescence of Sef1-Myc in a strain that constitutively overexpresses both SFU1 and SSN3, shown for cells cultured in iron-replete (H) and iron-depleted (L) conditions. Scale bar, 5 mm; all images obtained at the same magnification. b) Sfu1-Myc is co-immunoprecipitated with Sef1-TAP. Wild-type strains containing only Sef1-TAP or both Sef1-TAP and Sfu1-Myc were grown under iron-replete or iron-depleted conditions. Whole cell extracts were prepared under nondenaturing conditions, and IgGsepharose was used to immunoprecipitate Sef1-TAP and associated proteins. Pellets were subjected to immunoblot analysis, using anti-Myc monoclonal antibodies to identify Sfu1-Myc. c) Sef1-Myc is co-immunoprecipitated with Sfu1-TAP. * indicates a presumed Sef1-Myc C-terminal proteolysis product that is observed only when Sef1 is recovered from nondenaturing extracts, such as those used for TAP purification. d) Sef1-Myc but not Sfu1-Myc is co-immunoprecipitated with Ssn3-TAP. e) Ssn3-Myc is co-immunoprecipitated with Sef1-TAP. * Presumed Sef1-Myc C-terminal proteolysis product, see above. doi:10.1371/journal.ppat.1002956.g003 strength of the Sef1 mislocalization defects of the two mutants, with those of ssn3DD being worse, although contributions from additional misregulated targets of Ssn3 cannot be excluded.

Discussion
Human pathogenic microorganisms encounter a dearth of iron in the host bloodstream and internal organs [30,31,32], and specialized systems for iron acquisition have been demonstrated to be essential for the virulence of numerous bacterial, fungal, and parasitic pathogens, e.g. [33,34,35,36,37,38,39,40]. Meanwhile, commensal-pathogens such as C. albicans face the additional challenge of potential iron toxicity (from free radicals generated by the Fenton reaction [41]) in niches such as the gastrointestinal tract, where iron is relatively abundant [8,9]. We previously showed that the C. albicans transcription factors Sef1 and Sfu1 are key components of an iron homeostasis regulatory circuit that permits adaptation to these widely divergent host niches [6]. Sef1 protects against iron deficiency in the bloodstream through the induction of iron uptake genes and repression of SFU1, whereas Sfu1 protects against iron toxicity in the gut through repression of iron uptake genes as well as SEF1. Still unanswered are the questions of how the activities of Sef1 and Sfu1 are themselves tied to iron levels and whether additional regulatory inputs are involved. Here, we define a system for post-transcriptional, irondependent regulation of Sef1 protein that precisely controls the virulence of this obligate commensal-pathogen.
A novel post-transcriptional regulatory mechanism controls iron homeostasis in C. albicans Sef1 plays a central role in C. albicans pathogenesis through promoting the expression of virulence factors as well as iron uptake genes, whereas Sfu1 is essential for commensalism [6]. Given its role in virulence and, perhaps, in the choice between commensal and virulent lifestyles, we hypothesized that Sef1 would be a prime target for regulation beyond transcriptional repression by Sfu1. Indeed, our analysis of Myc-tagged Sef1 in wild-type C. albicans has revealed multiple levels of iron-dependent regulation, including nuclear vs. cytoplasmic localization, phosphorylation, and differential protein stability. In wild-type cells, Sef1 protein is nuclear, phosphorylated, stable, and competent for transcriptional activa- . Sfu1 and Ssn3 mediate opposing effects on Sef1 stability and virulence. a) Sef1 protein is stabilized in an sfu1DD mutant. Wildtype or sfu1DD strains containing Sef1-Myc were propagated in iron-replete medium containing 2 mg/ml cycloheximide. Samples were recovered at the indicated time points, and Sef1-Myc was visualized using monoclonal antibodies against the Myc epitope, followed by incubation with secondary antibodies that were coupled to infrared dyes and quantified using a Li-Cor instrument. Note that a single band corresponding to higher mobility (unphosphorylated) Sef1 is present in both strains. b) Sef1 protein is destabilized in the ssn3DD mutant. The experiment was performed as above, except that cells were propagated in iron-depleted medium containing cycloheximide. Note that phosphorylated Sef1 recovered from wild-type cells runs with slower mobility. c) Model for Sef1 regulation by Sfu1 and Ssn3 under iron-replete vs. iron-depleted conditions. Note that, even under ironreplete conditions when nuclear Sfu1 functions as a transcriptional repressor, a cytoplasmic pool of Sfu1 is available ( Figure S4) that could participate in Sef1 sequestration. d) Overexpression of SFU1 leads to attenuated C. albicans virulence in a murine bloodstream infections model. * signifies p,0.02, log-rank test. E) Deletion of SSN3 leads to attenuated C. albicans virulence, and restoration of one copy of wild-type SSN3 complements the defect. * signifies p,0.0001. doi:10.1371/journal.ppat.1002956.g004 tion only under iron-depleted conditions such as those encountered in the bloodstream.
Our analysis of Sef1 in C. albicans mutants has shed further light on the mechanisms of Sef1 regulation. Surprisingly, in the sfu1DD mutant, Sef1-Myc is constitutively nuclear, whereas in an SFU1overexpression strain it is predominantly cytoplasmic. These results clearly suggested a role for Sfu1 in the cytoplasmic localization of Sef1. Our screen of C. albicans mutants affecting predicted kinases exposed a role for Ssn3 in promoting cellular resistance to iron depletion as well as phosphorylation of Sef1. Coimmunoprecipitation experiments indicating that Ssn3 forms a physical complex with Sef1 supported a direct role for Ssn3 in Sef1 phosphorylation. Our finding that Sef1-Myc is constitutively cytoplasmic in the ssn3DD mutant suggested that Ssn3 might oppose Sfu1 by promoting the nuclear localization of Sef1. This hypothesis was validated by the ability of overexpressed SSN3 to overcome the cytoplasmic Sef1-mislocalization phenotype (under low iron conditions) of an SFU1-overexpression strain. Finally, our observations that Sfu1 and Ssn3 were both detectable in complexes with Sef1, but that neither could be found associated with the other, suggested that the functional antagonism between Sfu1 and Ssn3 occurs in part through competitive binding to Sef1 protein. Meanwhile, the observation that Sef1 is constitutively cytoplasmic in an sfu1DD/ssn3DD double mutant argues that Ssn3 plays at least one additional role in Sef1 nuclear localization.
These studies led to a revised model of Sef1 regulation (Figure 4c). According to the model, under iron-replete conditions, Sfu1 utilizes two distinct mechanisms to inhibit the function of Sef1: 1) Transcriptional repression, via direct binding to the SEF1 promoter, and 2) Post-transcriptional repression, via binding to Sef1 protein and forced localization in the cytoplasm, where Sef1 is unstable and unable to participate in transcription. To our knowledge, this would be the first example of a regulatory factor that regulates it target by both transcriptional and posttranscriptional mechanisms. Under iron-limiting conditions, Sfu1 protein is depleted, and Sef1 associates with the predicted protein kinase, Ssn3. Ssn3 most likely phosphorylates Sef1 directly, and either the complex or free Sef1 is transported to the nucleus, where Sef1 functions as a transcriptional activator. A key goal of future studies will be to understand how iron regulates these newly described activities of Sfu1 and Ssn3.

Impact of Sef1 post-transcriptional regulation on virulence
The findings that Ssn3 and Sfu1 post-transcriptionally regulate Sef1, an important virulence factor, raised the question of whether these regulatory events impact C. albicans virulence. Previously, we observed that deletion of SFU1 leads to hypervirulence in the murine bloodstream infection model, with the sfu1DD mutant significantly better at colonizing host kidneys than wild-type C. albicans [6]. We attributed this enhanced fitness to derepression of SEF1 and iron uptake genes in the mutant, resulting in an increased capacity for iron acquisition. In light of our current results showing that Sfu1 requires iron for transcriptional repression activity, a more likely explanation for the fitness advantage of sfu1DD is that Sef1 is constitutively nuclear (and therefore transcriptionally active) in this strain, whereas in wild type some fraction of Sef1 is retained in the cytoplasm and degraded. Our current observations with SFU1 OE and ssn3DD mutants dovetail with these findings by showing the converse, i.e. that mutants with incremental defects in the nuclear localization of Sef1 have proportional defects in virulence. Together, these results strongly support the hypothesis that C. albicans iron acquisition (and therefore virulence) can be modulated up or down, respectively, through the activities of Ssn3 or Sfu1 on Sef1 localization and stability. We hypothesize that the evolution of such fine-tuned regulation of a potent transcription factor is particularly advantageous to an obligate commensal-pathogen, such as C. albicans, which must continuously adapt to differing iron concentrations among the various microenvironments of its mammalian host, while avoiding excessive expression of pathogenicity genes during its usual role as a commensal.

Ethics statement
All procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of California San Francisco and were carried out according to the National Institutes of Health (NIH) guidelines for the ethical treatment of animals.

Plasmid and strain construction
All C. albicans strains used in this study are described in Table  S2, primers are listed in Table S3, and plasmids are listed in Table S4. Construction of C. albicans knockout mutants, complemented (gene addback) strains, and strains containing Myc-tagged fusion proteins was performed as previously described [6,42,43,44].
For introduction of TAP epitopes at the C-terminus of Sef1, Sfu1, and Ssn3, a series of plasmids was constructed using PCR and homologous recombination in S. cerevisiae [45]. The vector was pRS316 [46], and the insert consisted of (59 to 39): a PmeI restriction site; 350-450 bp of target ORF sequence up to, but not including, the stop codon; the TAP tag coding sequence [29]; a SAT1 (dominant selectable marker)-flipper cassette [47]; 350-450 bp of sequence downstream of the target ORF; and a second PmeI restriction site. Plasmids were called pSN150 (Sef1-TAP), pSN228 (Sfu1-TAP), and pSN219 (Ssn3-TAP). PmeI-digested plasmids were transformed into wild-type C. albicans reference strain SN250 [42], and nourseothricin-resistant C. albicans transformants were screened by colony PCR to verify the correct orientation of the C-terminal TAP tag and SAT1-flipper cassette. Strains expressing both Myc-and TAP-tagged fusion proteins were constructed by transforming strains already expressing the Myc-tagged protein with the appropriate PmeI-digested TAP-tag integration fragment, as described above.
Overexpression strains for SEF1and SSN3 were created by replacing portions of the endogenous promoters with the highly active TDH3 promoter. PCR and homologous recombination in S. cerevisiae [45] were used to create plasmids containing (59 to 39): a PmeI restriction site; 350-450 bp of sequence homology ending ,500 bp upstream of the target ORF; the SAT1 gene (dominant selectable marker); the TDH3 promoter; 350-450 bp of sequence homology beginning with the start codon of the target ORF; and a second PmeI site. The vector was pRS316 [46], the source of NAT1-TDH3 promoter was pCJN542 [48], and the resulting plasmids were named pSN147 (SEF1 OE ) and pSN229 (SSN3 OE ). Correct integration of the inserts in nourseothricin-resistant transformants was verified by colony PCR, and overexpression of SEF1 and SSN3 was confirmed by RT-qPCR.
The SFU1 overexpression strain (SN742) was created using an analogous method. pSN141 was engineered to contain (59 to 39): a PmeI site; 350-450 bp of sequence upstream of the C. albicans LEU2 ORF; the C. dubliniensis ARG4 gene (selectable marker); the TDH3 promoter; the SFU1 ORF; 350-450 bp sequence downstream of the LEU2 ORF; and a second PmeI restriction site. After digestion with PmeI, the plasmid was transformed into SN515 (sfu1DD). Correct integration of the insert in Arg + transformants was verified by colony PCR, and overexpression of SFU1 was confirmed by RT-qPCR.
The Ssn3 D325A kinase-dead mutant (SN977) was created in a similar manner to that of the SFU1 OE strain. First, PCR and primers SNO1394 through SNO1397 (Table S3) were used to create a D325A-encoding variant of the SSN3 ORF. Next, plasmid pSN239 was engineered to contain (59 to 39): a PmeI site; 350-450 bp of sequence upstream of the C. albicans LEU2 ORF; C. dubliniensis ARG4 (selectable marker); the TDH3 promoter; the mutant SSN3 ORF; 350-450 bp sequence downstream of the LEU2 ORF; and a second PmeI restriction site. A Myc-tagged version of Ssn3 D325A (SN987) was created using a plasmid (pSN273) that contains (59 to 39): a PmeI site; 350-450 bp of SSN3 ORF sequence up to, but not including, the stop codon; sequence encoding 136Myc; a SAT1-flipper cassette [47]; 272 bp of sequence downstream of the SSN3 ORF; 350-450 bp of sequence downstream of the LEU2 ORF; and a second PmeI restriction site. PmeI-digested plasmid was transformed into SN977, and correct integration in nourseothricin-resistant transformants was verified by colony PCR. Sequences of all PCR products were verified by DNA sequencing.

Protein extraction and immunoblotting
C. albicans protein extracts were prepared under denaturing condition using a protocol adapted from a previously described method [50]. Lysates corresponding to 1 OD 600 of cells were analyzed by SDS-PAGE and immunoblotted with either anti-c-Myc (9E10, Covance Research) for Myc-tagged proteins or antiperoxidase soluble complex antibody (Sigma, P2416) for TAPtagged proteins. Immunoblots were also probed with anti-alpha tubulin antibody (Novus Biologicals, NB100-1639) as a loading control.

Quantification of Sef1 half-life
C. albicans strains were grown on YPD medium (''iron-replete'') or YPD medium supplemented with the specific iron chelator 2,29dipyridyl (DIP) at a final concentration of 0.5 mM (''irondepleted''). A sample of 1-1.5 OD 600 cells was taken immediately (zero time point) before addition of cycloheximide to a final concentration of 2 mg/ml. At the indicated times, 1 OD value of cells was collected and harvested for protein preparations and immunoblotting. Semiquantitative detection of protein levels was performed using the LiCor Odyssey Infrared Imager (Lincoln, NE). Integrated fluorescence intensities of individual bands were measured and background subtracted using the Odyssey Application software. The signal from Sef1-Myc bands was normalized to that of alpha tubulin. Calculations of half-life were performed as previously described [51].

TAP pull-down analysis
Cells expressing TAP-tagged Sef1, Sfu1 or Ssn3 were grown in YPD medium to OD 600 = ,0.3-0.35 and centrifuged for 5 min at 3,000 rpm. Cell pellets were resuspended in ''iron-replete'' medium (pre-warmed YPD) or ''low iron'' medium (pre-warmed YPD supplemented with DIP), and grown for an additional 4 hours in the dark. Cells were collected by centrifugation, washed three times with ice-old water, and resuspended in 1 ml of lysis buffer (20 mM Tris, pH 7.4, 100 mM KCl, 5 mM MgCl 2 , 20% glycerol) with protease and phosphatase inhibitors (Roche). Cells were lysed using a Bead Beater and one-third volume of glass beads. Cell lysates were centrifuged for 2620 minutes at 14,000 rpm at 4uC. Protein concentration of the supernatants was measured by the Bradford assay. 3 mg of proteins was used for immunoprecipitation with 50 ml of immunoglobulin G-Sepharose resin (IgG Sepharose 6 Fast Flow, GE Healthcare). After 24 h of protein binding with rotation at 4uC, the resin was washed 4 times with lysis buffer and 2 times with tobacco etch virus (TEV) protease cleavage buffer (10 mM Tris-HCl, pH 8, 150 mM NaCl, 0.5 mM EDTA, 0.1% Tween-20). TEV protease (100 U) cleavage was performed in 1 ml buffer at 4uC overnight. The TEV eluate was collected and proteins were recovered by TCA (trichloroacetic acid) precipitation.

RNA extraction and RT-qPCR analyses
Total RNA was prepared using a hot-phenol method [52] and treated with DNaseI using the Turbo DNA-free kit (Ambion). Ten micrograms of RNA was used in standard RT reactions using oligo [(dT)20-N] primers. cDNAs were quantified by qPCR with the primers listed in Table S3 and normalized against ACT1.

Virulence analysis
As previously described [6], groups of 10 female (8-to 10-weekold) BALB/c mice (Charles Rivers) were injected by tail vein with 5610 5 CFUs of wild type (SN425), SFU1 OE (SN742), ssn3DD (SN982), or ssn3DD/SSN3 (SN978). Mice were monitored twice daily and euthanized when morbidity criteria were met (weight loss .15%, hunched posture, inactivity). Figure S1 Fusion to TDH3 promoter results in overexpression of SFU1 RNA and protein. Results are shown for strains propagated under iron-replete conditions. a) RT-qPCR analysis of SFU1 RNA in wild-type and SFU1 OE strains. b) Immunoblot of Sfu1-Myc protein in wild-type and SFU1 OE strains. (EPS) Figure S2 BPS-sensitivity of C. albicans mutants. Dilutions of equivalent numbers of wild-type and mutant cells were plated on iron-replete medium (YPD), iron-depleted medium (YPD +300 mM BPS), and iron-depleted medium with supplemental iron (YPD +300 mM BPS +100 mM FeCl 3 ) and incubated at 30uC. Note that the BPS-related growth defects of SFU1 OE , sef1DD, and ssn3DD were complemented by FeCl 3 supplementa-tion, indicating that the phenotypes resulted from iron limitation (vs. chelation of an alternative divalent cation). (EPS) Figure S3 Immunoblot of Sef1-Myc and alpha tubulin in wild type vs. SFU1 OE . Cells were propagated in iron-replete (H) or iron-depleted (L) liquid medium, and proteins from cell equivalents were visualized in lanes 1, 2, 4, and 5. To better visualize Sef1-Myc extracted from cells grown under iron-depleted conditions, proteins from J cell equivalents were visualized in lanes 3 and 6.