Homeostatic control of an iron repressor in a GI tract resident
- The Center for Microbes, Development and Health, Key Laboratory of Molecular Virology and Immunology, Unit of Pathogenic Fungal Infection & Host Immunity, Institut Pasteur of Shanghai, Chinese Academy of Sciences, China
- The University of Chinese Academy of Sciences, China
- Department of General Surgery, Shanghai General Hospital, China
- State Key Laboratory of Trauma, Burns and Combined Injury, Department of Wound Infection and Drug, Army Medical Center (Daping Hospital), China
- College of Life Science, China
Figures
Figure 1 with 2 supplements
Deletion of HAP43 significantly increases the commensal fitness of C. albicans in gastrointestinal (GI) tract of mice fed a high-Fe diet (HFD).
(A) As depicted in the schematics, mice were fed a normal Fe (NFD) or HFD for 3 d prior to C. albicans inoculation. The mice continuously received the same diet during the course of experiments. (B) …
Figure 1—figure supplement 1
Mutant lacking HAP43 exhibits no change in commensal fitness in normal Fe diet (NFD)-treated mice.
Mice (n = 8) fed a normal Fe diet (NFD) were inoculated by gavage with 1:1 mixtures of the wild-type (WT) and hap43Δ/Δ mutant cells (1 × 108 CFU per mice). The fitness value for each strain was …
Figure 1—figure supplement 2
A high-Pi diet significantly promotes the intestinal colonization of C. albicans.
(A) Cells of SC5314 strain (108 CFU/mouse) were separately inoculated into groups of mice fed normal Fe diet (NFD) or high-Fe diet (HFD) (n = 8) by gavage. The intestinal colonization of C. albicans …
Figure 2
High iron triggers Hap43 phosphorylation that is modulated by the protein kinase Ssn3.
(A) qRT-PCR analysis for HAP43 mRNA in WT strain grown under iron-replete (H, high iron) or iron-depleted (L, low iron) conditions. Transcript levels were normalized to the level of ACT1 mRNA. …
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Figure 2—source data 1
Uncropped images of gels and blots in Figure 2.
- https://cdn.elifesciences.org/articles/86075/elife-86075-fig2-data1-v2.zip
Figure 3 with 2 supplements
Ssn3-modulated phosphorylation induces cytoplasmic localization and protein degradation of Hap43 by ubiquitin-proteasome pathway.
(A) Left panels: indirect immunofluorescence of Hap43-Myc in WT and ssn3Δ/Δ mutant strains grown under iron-replete (H, high iron) or iron-depleted (L, low iron) conditions. DIC represents phase …
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Figure 3—source data 1
Uncropped images of gels and blots in Figure 3.
- https://cdn.elifesciences.org/articles/86075/elife-86075-fig3-data1-v2.zip
Figure 3—figure supplement 1
Chloroquine had no effect on Hap43 degradation under high iron conditions.
After treatment with doxycycline, the WT cells stably expressing doxycycline-inducible Myc-tagged Hap43 (TetO-Hap43-Myc) were harvested, washed, and treated with or without the lysosomal protease …
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Figure 3—figure supplement 1—source data 1
Uncropped images of gels and blots in Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/86075/elife-86075-fig3-figsupp1-data1-v2.zip
Figure 3—figure supplement 2
Immunoblots showing the induction of an epitope-tagged 3xHA-ubiquitin under the control of the doxycycline (DOX)-inducible promoter.
C. albicans cells co-expressing 3xHA-tagged ubiquitin and Hap43-Myc as well as Hap43-Myc cells were incubated in YPD supplemented with 50 μg/ml Dox for 6 hr. Log-phase cells were collected and …
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Figure 3—figure supplement 2—source data 1
Uncropped images of gels and blots in Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/86075/elife-86075-fig3-figsupp2-data1-v2.zip
Figure 4 with 5 supplements
The critical phosphorylation sites are essential for Hap43 stabilization.
(A) Schematic representation of C. albicans Hap43. Putative phosphorylation sites predicted by the Kinasephos 2.0 server and Cdk8-dependent phosphorylation sites are represented. (B) Immunoblots of …
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Figure 4—source data 1
Uncropped images of gels and blots in Figure 4.
- https://cdn.elifesciences.org/articles/86075/elife-86075-fig4-data1-v2.zip
Figure 4—figure supplement 1
The Hap43 mutants harboring serine/threonine-to-alanine substitutions in its one or two putative phosphorylation sites showed the WT-like degradation patterns of Hap43 under high iron conditions.
(A) Immunoblots of Hap43-Myc in strains expressing the indicated amino acid substitution allele of Hap43. Cells were treated at high iron conditions. (B) Strains expressing the indicated amino acid …
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Figure 4—figure supplement 1—source data 1
Uncropped images of gels and blots in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/86075/elife-86075-fig4-figsupp1-data1-v2.zip
Figure 4—figure supplement 2
The mutants harboring amino acid substitutions or fragment truncation showed no defects in vegetative growth.
Growth curve analysis of HAP43 mutant strain harboring 29-point mutations in YPD liquid medium supplemented with 250 μM or 500 μM the impermeable iron chelator bathophenanthroline disulfonate (BPS) …
Figure 4—figure supplement 3
The critical phosphorylation region is essential for Hap43 stabilization.
(A) Schematic diagram illustrating the Hap43 truncation proteins used as part of this study. The positions of the major domains identified in individuals with Hap43 are indicated. Numbers indicate …
Figure 4—figure supplement 4
The mutants harboring amino acid substitutions or fragment truncation showed no defects in vegetative growth.
Growth curve analysis of Hap43 truncation in YPD liquid medium supplemented with 250 μM or 500 μM the impermeable iron chelator bathophenanthroline disulfonate (BPS) at 30°C. OD600 readings were …
Figure 4—figure supplement 5
The HAP43 mutant-4 strain (HAP43m4/hap43Δ) harboring amino acid substitutions showed no defects in vegetative growth.
Growth curve analysis of indicated strains in SC liquid medium supplemented with 250 uM or 500 uM the impermeable iron chelator bathophenanthroline disulfonate (BPS) at 30°C. OD600 readings were …
Figure 5 with 1 supplement
Hap43 phosphorylation is important for alleviating Fenton reaction-induced reactive oxygen species (ROS) toxicity and for gastrointestinal (GI) colonization.
(A, B) Intracellular ROS production of C. albicans under different experimental conditions. C. albicans yeast cells were grown on YPD supplemented with indicated reagents. About 1 × 107 cells in …
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Figure 5—source data 1
Uncropped images of gels and blots in Figure 5.
- https://cdn.elifesciences.org/articles/86075/elife-86075-fig5-data1-v2.zip
Figure 5—figure supplement 1
Growth of the hap43Δ/Δ mutant under oxidative stresses.
Top panel: WT and hap43Δ/Δ mutant cells were spotted with tenfold serial dilutions onto YPD or YPD supplemented with 2 mM or 4 mM H2O2 and grown for 2 d at 30℃. Bottom panel: growth curve analysis …
Figure 6 with 2 supplements
Iron-induced phosphorylation and degradation of Hap43 leads to de-repression of antioxidant genes.
(A) Left panels: indirect immunofluorescence of Hap43-Myc in HAP43/hap43Δ and HAP43tr/hap43Δ strains grown under iron-replete conditions. DIC represents phase images, DAPI represents nuclear …
Figure 6—figure supplement 1
Iron-induced phosphorylation and degradation of Hap43 leads to de-repression of antioxidant genes.
Shown are the results about a comparison between WT and Hap43m29 mutant. (A) Left panels: indirect immunofluorescence of Hap43-Myc in HAP43/hap43Δ and HAP43m29/hap43Δ strains grown under …
Figure 6—figure supplement 2
Iron-induced phosphorylation and degradation of Hap43 leads to de-repression of antioxidant genes.
Shown are the results about a comparison between WT and Hap43m4 mutant. (A) Left panels: indirect immunofluorescence of Hap43-Myc in HAP43/hap43Δ and HAP43m4/hap43Δ strains grown under iron-replete …
Figure 7
Model for the role of post-translational medication of Hap43 in promoting gastrointestinal (GI) commensalism of C. albicans.
In the iron-rich environment such as GI tract, the iron-responsive regulator Hap43 is subject to covalent post-translational modifications, including phosphorylation and ubiquitination, and causes …
Author response image 1
(A) Left panels: Indirect immunofluorescence of Hap43-Myc in WT and ssn3Δ/Δ mutant strains grown under iron-replete conditions. DIC represents phase images, DAPI represents nuclear staining, FITC represents Hap43-Myc staining, and Merge represents the overlay of Hap43-Myc and nuclear staining. Right panels: Quantification of the cellular distribution of Hap43. Each bar represents the analysis of at least 100 cells. C representing >90% cytoplasmic staining, N >90% nuclear staining, and C+N a mixture of cytoplasmic and nuclear staining. Scale bar, 5 µm. (B) Quantification of fluorescence in images of A.
Author response image 2
(A) Immunoblots of Hap43-Myc recovered from indicated C. albicans cells under iron-replete conditions. a-tubulin, internal standard. (B) Strain expressing Hap43-TAP was inoculated to YPD medium and grown overnight at 30oC for protein purification. Cell extracts were sequentially immunoprecipitated with lgG Sepharose and calmodulin Sepharose. Proteins were identified by gel electrophoresis separation and silver staining. The arrow represents Hap43.
Additional files
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Supplementary file 1
Strains (a), plasmids (b), and primers (c) used in this study.
- https://cdn.elifesciences.org/articles/86075/elife-86075-supp1-v2.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/86075/elife-86075-mdarchecklist1-v2.docx
