Post-transcriptional regulation of the Sef1 transcription factor controls the virulence of Candida albicans in its mammalian host

Abstract

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 Cys(6)Zn(2) 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.

Figure 1. Sfu1 regulates SEF1 gene expression and localization of Sef1 protein.

Note that C. albicans gene deletion mutants are represented as ΔΔ because two alleles of target genes must be disrupted in this obligate diploid species. a) RT-qPCR results for SEF1 mRNA in wild-type (WT), sfu1ΔΔ, and SFU1-overexpression (SFU1 ^OE) strains grown under iron-replete (H, high iron) or iron-depleted (L, low iron) conditions. Error 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, sfu1ΔΔ, 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 µm; all images were obtained at the same magnification.

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. sfu1ΔΔ cells propagated under iron-replete (H) or iron-depleted (L) conditions. B) Immunoblot of purified Sef1-TAP protein either treated (+) or not treated (−) 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 sfu1ΔΔ 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 ssn3ΔΔ cells under iron-replete or iron-depleted conditions. d) Indirect immunofluorescence of Sef1-Myc in the ssn3ΔΔ mutant under iron-replete or iron-depleted conditions. Scale bar, 5 µm; all images obtained at the same magnification.

Figure 3. 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 µm; 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 IgG-sepharose 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.

Figure 4. Sfu1 and Ssn3 mediate opposing effects on Sef1 stability and virulence.

a) Sef1 protein is stabilized in an sfu1ΔΔ mutant. Wild-type or sfu1ΔΔ 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 ssn3ΔΔ 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 iron-replete 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.