Figure 1.
The hap3Δ, hap5Δ and hapXΔ mutants grow poorly on low-iron media supplemented with hemin.
Ten-fold serial dilutions of cells (starting at 104 cells) were spotted onto solid YPD medium, solid YNB medium (control), low-iron medium (YNB+100 µM BPS), low-iron medium supplemented with 100 µM FeCl3, 10 µM Hemin or 10 µM Feroxamine. The bottom panel shows a comparison of the growth of the ferroxidase mutant cfo1Δ with double mutants lacking the CFO1 gene and the HAP3, HAP5 or HAPX gene. For the double mutants, two independent strains of each mutant were tested as indicated. The plates were incubated at 30°C for two days.
Figure 2.
Overview of differentially-regulated genes in response to different iron sources.
A histogram shows the number of genes whose expression changed by at least two-fold with statistical significance (q<0.05). The numbers represent differentially expressed genes in the mutants versus WT in response to different iron sources and also in the WT strain grown under iron-replete versus iron-limited conditions.
Figure 3.
Enriched GO terms for differentially-regulated genes in the mutants and WT strain in response to different iron sources.
The figure shows the GO terms identified by Gene Score Re-sampling with the microarray data mining tool ermineJ for differentially expressed genes in the mutants versus WT in response to different iron sources. The terms are also shown for WT in iron-replete versus iron-limited conditions. For each comparison, GO terms with statistical significance (p<0.05) are indicated by colored boxes. The numbers within the colored boxes represent the GO term rankings within each comparison. Two GO terms (indicated by asterisk) from the ermineJ annotation file are updated on the GO website (http://www.geneontology.org/): GO:0042775 is described as mitochondrial ATP synthesis coupled electron transport and GO:0006118 has been made obsolete and replaced by GO:0055114 oxidation reduction.
Table 1.
HapX and Hap3 negatively regulate the expression of genes encoding iron-dependent functions.
Table 2.
HapX positively regulates the expression of genes encoding iron acquisition functions.
Figure 4.
HapX positively regulates the levels of the CIR1 and RIM101 transcripts.
Quantification of HAPX, CIR1 and RIM101 transcripts by quantitative RT-PCR in WT, hapXΔ and cir1Δ strains under low-iron (top) and iron-replete (bottom) conditions. The data were normalized using 18S rRNA as an internal control and are presented as relative expression in comparison to the WT value set at 1. The data are from three biological and two technical replicates, and the bars represent standard deviations.
Figure 5.
The hap3Δ and hap5Δ mutants have a smaller capsule than WT or hapXΔ cells.
A. Cells were cultured in low-iron medium (LIM) at 30°C for 48 h. Capsule formation was assessed by India ink staining for the WT strain, the hap3Δ, hap5Δ, hapXΔ, cfo1Δ, hap3Δ cfo1Δ, hap5Δ cfo1Δ, and hapXΔ cfo1Δ mutants, as well as the reconstituted strains. Bar = 10 µ. B. Fifty cells from each strain were measured to determine cell diameter and capsule radius. The capsule sizes of hap3Δ, hap5Δ, hap3Δ cfo1Δ (both #1 and #2) and hap5Δ cfo1Δ (both #54 and #61) cells were statistically different (t-test, p<0.001) from the sizes for cells of the WT strain, the hapXΔ cfo1Δ mutants and the reconstituted strains. Each bar represents the average of 50 measurements with standard deviations.
Figure 6.
The hap3Δ and hap5Δ mutants exhibit enhanced growth at host temperature.
Ten-fold serial dilutions of WT and mutant cells (starting at 104 cells) were spotted onto solid YPD medium with no addition or with 100 µM Hemin and incubated at the indicated temperatures for two days.
Figure 7.
The hap3Δ and hap5Δ mutants exhibit poor growth on acetate, ethanol and sucrose.
Ten-fold serial dilutions of the WT, hap3Δ, hap5Δ and hapXΔ mutants and the reconstituted strains (starting at 104 cells) were spotted on YNB supplemented with the indicated carbon sources (all at 2%). Plates were incubated at 30°C for two (on glucose and sucrose) or three (on acetate and ethanol) days.
Figure 8.
The hapXΔ mutant is modestly attenuated for virulence in a mouse inhalation model of cryptococcosis.
A. Ten female BALB/c mice were infected intranasally with 5×104 cells of each of the strains indicated (H99 (WT), cfo1Δ, hap3Δ, hap3Δ::HAP3, and hap3Δ cfo1Δ) and the survival of the mice was monitored twice per day. Assessment of the p-values for survival did not reveal significant differences between WT-, hap3Δ::HAP3- and hap3Δ-infected mice, or for comparisons of survival for the cfo1Δ mutant versus the hap3Δ cfo1Δ double mutant. The WT and cfo1Δ strains show differences in virulence as previously reported [18]. B. Parallel virulence tests were performed for the hapXΔ, hapXΔ::HAPX, and hapXΔ cfo1Δ strains. Note that the same data for the WT (H99) strain and the cfo1Δ mutants are presented in both figures to allow comparisons with the hap mutants. Assessments of survival revealed significant differences between WT or the hapXΔ::HAPX strain and hapXΔ (p<0.05); however, no significant difference was found upon comparison of the survival for the cfo1Δ mutant versus the hapXΔ cfo1Δ double mutant.
Figure 9.
Acidic pH rescues the growth of double mutants on ferric chloride and hemin.
Ten-fold serial dilutions of cells (starting at 104 cells) were spotted onto solid YPD medium, low-iron medium (YNB+100 µM BPS) at either pH 5.0 or 7.0, and low-iron medium supplemented with 100 µM FeCl3, 10 µM Hemin or 10 µM Feroxamine at each of the two pH conditions. The plates were incubated at 30°C for two days.