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. 2010 Apr 1;6(4):e1000846.
doi: 10.1371/journal.ppat.1000846.

SREB, a GATA transcription factor that directs disparate fates in Blastomyces dermatitidis including morphogenesis and siderophore biosynthesis

Affiliations

SREB, a GATA transcription factor that directs disparate fates in Blastomyces dermatitidis including morphogenesis and siderophore biosynthesis

Gregory M Gauthier et al. PLoS Pathog. .

Abstract

Blastomyces dermatitidis belongs to a group of human pathogenic fungi that exhibit thermal dimorphism. At 22 degrees C, these fungi grow as mold that produce conidia or infectious particles, whereas at 37 degrees C they convert to budding yeast. The ability to switch between these forms is essential for virulence in mammals and may enable these organisms to survive in the soil. To identify genes that regulate this phase transition, we used Agrobacterium tumefaciens to mutagenize B. dermatitidis conidia and screened transformants for defects in morphogenesis. We found that the GATA transcription factor SREB governs multiple fates in B. dermatitidis: phase transition from yeast to mold, cell growth at 22 degrees C, and biosynthesis of siderophores under iron-replete conditions. Insertional and null mutants fail to convert to mold, do not accumulate significant biomass at 22 degrees C, and are unable to suppress siderophore biosynthesis under iron-replete conditions. The defect in morphogenesis in the SREB mutant was independent of exogenous iron concentration, suggesting that SREB promotes the phase transition by altering the expression of genes that are unrelated to siderophore biosynthesis. Using bioinformatic and gene expression analyses, we identified candidate genes with upstream GATA sites whose expression is altered in the null mutant that may be direct or indirect targets of SREB and promote the phase transition. We conclude that SREB functions as a transcription factor that promotes morphogenesis and regulates siderophore biosynthesis. To our knowledge, this is the first gene identified that promotes the conversion from yeast to mold in the dimorphic fungi, and may shed light on environmental persistence of these pathogens.

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Conflict of interest statement

Dr. Bruce S. Klein serves on the editorial board for PLoS Pathogens.

Figures

Figure 1
Figure 1. Phenotype of insertional mutant 3-15-1 and conserved motifs in SREB.
(A) Insertional mutant 3-15-1 failed to convert from yeast to mold after 14 days of incubation at 22°C. Parent strain T53-19 converted from yeast to mycelia within 7 days of shifting the temperature from 37°C to 22°C (Scale bar equals 20 μm). (B) Mutant 3-15-1 developed a yellow pigmentation and discolored the surrounding 7H10 medium, which contains 150 μM FeSO4. In contrast, T53-19 grew as white-colored yeast and did not pigment the medium. (C) The predicted amino acid sequence of B. dermatitidis SREB is aligned with A. nidulans SREA, P. chrysogenum SREP, N. crassa SRE, U. maydis URBS1, S. pombe FEP1, C. albicans SFU1, H. capsulatum SRE1, and C. neoformans CIR1. SREB contained several conserved domains including two zinc fingers (ZF #1, ZF #2) separated by a cysteine-rich region (CRR) and a conserved C-terminus (CCT) with a predicted coiled-coil domain. Conserved cysteine residues in ZF #1, ZF #2, and CRR are highlighted. ClustalW (1.81) was used to align the amino acid sequences. GeneBank accession numbers include AAD25328 (SREA), AAC49628 (SREP), AAC64946 (SRE), AAB05617 (URBS1), AAM29187 (FEP1), XP_723364 (SFU1), ABY66603 (SRE1), and ABG21303 (CIR1).
Figure 2
Figure 2. Complementation of insertional mutant 3-15-1.
(A) Under iron-replete conditions (10 μM FeSO4), mutant 3-15-1 and empty vector controls fail to suppress siderophore biosynthesis as demonstrated by a 2.5–3.0 fold higher absorbance at 425 nm compared to parental strain T53-19 and complemented strains A5 and D5 (p<0.01 as indicated by asterisk). Data from three independent experiments were analyzed. Siderophore production was measured using the ferric perchlorate assay. (B) Mutant 3-15-1 and empty vector control A4 were pigmented yellow and discolored the surrounding media when grown on 7H10 medium, which contains 150 μM FeSO4. In contrast, parental control T53-19 and complemented strains A5 and D5 were colored white and did not pigment the medium. All strains were incubated for 14 days at 37°C. (C) Mutant 3-15-1 and empty vector control A4 fail to convert to mycelia upon shifting the incubation temperature from 37°C to 22°C. Similar to the wild-type isolate, complemented strains A5 and D5 grew as mycelia at 22°C incubation. (D) Northern blot hybridization demonstrated reduction in transcript abundance in the insertional mutant when compared to the parent strain. Complemented strains A5 and D5 overexpress SREB. Transformation of 3-15-1 with an empty vector failed to restore transcript abundance in strain A4.
Figure 3
Figure 3. Phenotype of the SREB null mutant.
(A) When grown on 7H10 medium containing 150 μM FeSO4, the null mutant (SREBΔ) grew as yellow-orange pigmented colonies that discolored the medium (37°C; 15 days incubation). In contrast the wild-type strain (ATCC 26199) grew as white colored yeast and did not pigment the medium. At 37°C, SREBΔ and wild-type isolates grew as budding yeast. Following a shift in temperature from 37°C to 22°C, SREBΔ failed to complete the conversion from yeast to mycelia (17 days; HMM medium). (B) The null mutant (SREBΔ) does not accumulate significant biomass or expand by radial growth when compared to the wild-type isolate. For each strain, ×ばつ104 yeast were spotted on HMM medium and incubated at 22°C for 14 days. (C) The null mutant (SREBΔ) and wild-type isolates have a similar growth rate when they are cultured as yeast at 37°C incubation. Culture density was measured in triplicate at A600. Cultures were grown in liquid HMM supplemented with 10 uM FeSO4. The data were from two independent experiments.
Figure 4
Figure 4. Southern and Northern blot analyses of the SREB null mutant.
(A-D) Southern analysis of 26199 wild-type (WT) and null mutant SREBΔ (KO). Genomic DNA from WT and KO were digested with PciI alone or in combination with MluI. The hygromycin resistance cassette, but not SREB, contains a MluI restriction site. The blots were probed against hygromycin, Hyg probe (A); SREB, SREB probe (B); the 5′ flank, 1663 probe (C); and 3′ flank, 1747 probe (D). Arrows indicate hybridizing fragments and dashes depict molecular weight markers (MWM). The Hyg and SREB probes failed to hybridize to digested WT or KO DNA, respectively (A, B). The 1663 and 1747 probes, which flank SREB, gave the expected size restriction fragments, which indicated a clean replacement of SREB sequence with the hygromycin resistance cassette (C, D). (E) Schematic illustrating the location of the restriction sites in the wild-type and null mutant (SREBΔ), hybridization sites for probes Hyg, SREB, 1663, and 1747, and expected size of the restriction fragments. Yellow, red, and blue indicate the SREB coding region, hygromycin resistance cassette, and sequence flanking SREB, respectively. (F) Northern analysis of 26199 wild-type and SREB-null mutant when grown under iron-poor (−) and iron-replete (+; 10 μM FeSO4) conditions. SREB transcript is detectable in the wild-type isolate under iron-replete conditions and absent in SREBΔ.
Figure 5
Figure 5. Complementation of the SREB null mutant.
(A) Complemented strains C#6 and C#25 grew as white-colored yeast, similar to the wild-type isolate, on 7H10 slants (150 μM FeSO4). In contrast, the null mutant (SREBΔ) and empty vector strain EV-D grew as yellow-colored yeast and discolored the medium. Cultures were grown at 37°C for 14 days. (B) Complemented strains C#6 and C#25 were able to repress the biosynthesis of siderophores when grown-under iron-replete (10 μM FeSO4) conditions as measured by the ferric perchlorate assay. In contrast, the null mutant and empty vector (EV-D) continued to produce siderophores (p<0.01). Data from three independent experiments were analyzed. (C) Wild-type and complemented strains C#6 and C#25 convert to mycelia within 17 days of incubation at 22°C. In contrast, the null mutant and empty vector EV-D strains fail to complete the conversion to mycelia. (D) Northern blot hybridization demonstrated restoration of SREB transcript abundance in complemented strains C#6 and C#25, when compared to the null mutant.
Figure 6
Figure 6. Northern analysis of candidate genes in the SREB regulon involved with siderophore biosynthesis and uptake.
Wild-type B. dermatitidis strain 26199 upregulated the expression of SIDA, AMCA, MIRB, MIRC, and HAPX when grown in iron-poor medium (—); expression of these genes was repressed when iron was abundant (10 and 50 μM FeSO4). Deletion of SREB resulted in de-repression of SIDA, AMCA, MIRB, MIRC, and HAPX under iron-replete conditions.
Figure 7
Figure 7. Identification and characterization of siderophores in B. dermatitidis.
(A-C) Culture supernatant harvested from wild-type B. dermatitidis grown under iron-poor conditions was applied to a column packed with XAD-2 resin. Following a water wash, compounds bound to the resin were eluted with methanol and analyzed by LC/MS. The chromatogram revealed the presence of several compounds in the eluate (A). Analysis of these compounds using mass spectroscopy revealed that two of the peaks (4.16 and 7.26 minutes) had masses consistent with dimerum acid (538.2) and coprogen (822.2), respectively (B, C). (D) Culture supernatants harvested from wild-type B. dermatitidis grown under iron-poor and iron-replete conditions were compared to siderophore standards using HPLC. During conditions of iron-limitation, the wild-type isolate produced and secreted dimerum acid and coprogen. When iron was abundant, no siderophores were detected. In contrast, the null mutant SREBΔ produced and secreted dimerum acid and coprogen under iron-replete conditions.
Figure 8
Figure 8. Expression of candidate genes in the SREB regulon at 37°C and 22°C.
Quantitative RT-PCR (qRT-PCR) was performed to validate the expression of a subset of genes identified by bioinformatic and gene expression analyses. RNA was extracted from the wild-type (WT) strain and null mutant (KO) at 37°C and at 22°C (48 hours following the downward temperature shift). Deletion of SREB resulted in altered expression of genes involved with lipid metabolism and transport (lipid transfer protein, peroxisomal dehydratase, acetoacetyl-CoA synthase) as well as a glycosyl hydrolase that is predicted to have endo-1,3-β-glucanase activity. Data was generated from two biological replicates. qRT-PCR fluorescence was normalized to α-tubulin.

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