Figure 1.
Genome wide ChIP-seq Analysis Identifies Direct Transcriptional Targets of SrbA in Hypoxic Conditions.
(A). Genome scale view of ChIP-seq data for one 4-hour ChIP sample. Blue lines rising above background are peaks identified as an excess of sequence fragments aligning to the genome of A. fumigatus sequenced strain A1163. The genome-wide view of the ChIP-seq reads aligned to the A1163 genome reveals strong visible peaks in the SrbA antibody ChIP sample, with few strong peaks observed in the A1163 wild type input control sample. Grey lines demarcate genome scaffolds. (B). ChIP-seq peaks for selected genes: srbA, srbB, erg 11A and erg 25A were among the highest ChIP-seq peaks and have relatively high enrichment (lower panels) vs. input control (upper panels). alcC, hem13, niiA and AFUB_071500 show significant, though lower enrichment vs. input control. In each case the peaks are upstream of the translational start site of the indicated genes. Asterisks mark the relevant gene model within each 3 kb region. (C). ChIP qPCR was conducted to validate ChIP-seq results for select targets. Data are presented as the mean and standard error of two biological replicates. The actin promoter (actA(p)) was included as a non-specific target. SrbA was enriched on the promoters of all tested genes. (D). SrbA binding motif as identified using MEME. (E). GO pie chart.
Table 1.
Selected SrbA ChIP Peaks with flanking gene information.
Figure 2.
RNA-seq and nCounter Analyses of ΔsrbA Confirms SrbA Regulation of ChIP-seq Target Genes in vitro and in vivo during Invasive Pulmonary Aspergillosis.
(A). Enrichment of RNA-seq differentially expressed genes in GO/Funcat categories of up- and down-regulated genes in srbA cells under hypoxia 120 minutes versus WT (B). Analysis of transcript levels of 12 of the ChiP-seq target genes in vivo in a murine model of invasive pulmonary aspergillosis for wild type (CEA10) and in vitro under normoxic/hypoxic conditions for ΔsrbA and wild type in vivo samples were at 48–96 hours post-infection (grey, n = 16). in vitro samples were wild type normoxia (red, n = 2) and hypoxia (blue, n = 6) followed by ΔsrbA under normoxia (red, n = 2) and hypoxia (blue, n = 6). Time under hypoxia for both wild type and ΔsrbA ranged from 30 to 120 minutes. Expression values are represented as total number of normalized counts per transcript. Quantitation and normalization was as follows: Digital counts for 60 genes (ChIP targets, housekeeping genes and other genes of interest) were adjusted for binding efficiency with background subtraction using the included positive and negative controls from the manufacturer as per NanoString nCounter data analysis guidelines. Data sets were normalized to facilitate across sample comparisons using the geometric mean of 20 stably expressed genes.
Figure 3.
Temporal transcriptional induction of SrbA and SrbB in response to Hypoxia Reveals SrbB transcript levels are induced prior to SrbA.
Aspergillus fumigatus wild type was cultured in normoxia at 37°C for 18 hours and shifted to hypoxia for additional incubation for 5, 15, and 30 minutes. Data are presented as the mean and standard error of three biological replicates. Compared to normoxia (Hyp 0′), srbB expression was significantly induced in hypoxia 15 min, which was earlier than the initial srbA induction. The data were analyzed by two-way ANOVA followed by Bonferroni posttests.
Figure 4.
Loss of the Hypoxia Induced Transcriptional Regulator SrbB Results in a Significant Growth Defect and Red Pigmented Mycelia in Hypoxia.
(A). Growth of ΔsrbB in normoxia and hypoxia on solid media. Wild type, ΔsrbB, and srbB-reconstituted strains were incubated on GMM at 37°C for 3 days in normoxia or hypoxia. The number of conidia used for inoculation is illustrated by the plate image. Compared to wild type and the reconstituted strain, growth of ΔsrbB is restricted in hypoxia. (B). A biomass test with wild type, an srbB null mutant, and an srbB reconstituted strain in liquid cultures in normoxia or hypoxia. Mycelia of wild type, ΔsrbB and srbB-reconstituted (srbB-recon) strains in liquid cultures were harvested, dried and weighed for the biomass study. Data are presented as the mean and standard error of three biological replicates. No significant differences between wild type and srbB-recon biomass were observed in all conditions tested. When analyzed by two-way ANOVA followed by Bonferroni posttest, biomass of ΔsrbB was not different from wild type or srbB-recon in normoxia. However, biomass of ΔsrbB significantly decreases in hypoxia compared to wild type (p<0.001). (C). E-test strips were utilized to test susceptibility to VCZ. 105 conidia were overlaid on RPMI media, cultured at 37°C for 2 days. Minimal inhibitory concentrations (MIC, marked as an arrow) were measured. In both normoxia and hypoxia, ΔsrbB is slightly more tolerant to VCZ compared to wild type and the srbB reconstituted strain. MIC ratios of ΔsrbB to wild type are 1.76 and 3.42 in normoxia and hypoxia, respectively. (D). Conidia of each strain were cultured in LGMM at 37°C, 200 rpm for 2 days in hypoxia. ΔsrbB produces reddish mycelia compared to the wild type and reconstituted strain.
Figure 5.
SrbB is a transcriptional regulator of genes involved in carbon metabolism, lipid metabolism, and heme biosynthesis.
The FungiFun2 web server was utilized to assign FunCat and gene ontology enrichment in genes with transcript levels increased or decreased 4 fold in ΔsrbB compared to the wild type strain. Statistically significant (P≤0.05) FunCat categories are presented at the respective time points in hypoxia.
Figure 6.
Loss of srbB impairs heme biosynthesis and results in accumulation of heme intermediates.
(A–B). Amount of Protoporphyrin IX (PP IX) and intermediate compounds including uroporphyrin (C8), heptacarboxylporphyrin (C7), hexacarboxylporphyrin (C6), pentacarboxylporphyrin (C5), and coproporphyrin (C4) were analyzed using HPLC. Mycelia used for HPLC analysis were harvested from cultures in LGMM at 37°C for 2 days in hypoxia. Compared to wild type, ΔsrbB produces more PP IX and other intermediates in hypoxia. (A) is a chromatogram from HPLC analysis, and (B) is a graph to present the HPLC result with statistical analysis. Data are presented as the mean and standard error of three biological replicates, and analyzed by one-way ANOVA followed by a Tukey's multiple comparison test. (C). A thousand conidia were inoculated on GMM or GMM containing 5 µM hemin. In 2 days, radial growth of each strain in normoxia or hypoxia was observed. Addition of hemin improved ΔsrbB growth in hypoxia.
Figure 7.
A sub-set of SrbA ChIP Target Genes are co-regulated by SrbB.
The RNA-seq data for annotated genes corresponding to SrbA ChIP-seq peaks are shown as ratios of 30 and 120-minute wild-type hypoxia vs. wild-type normoxia, and gene deletion strains are shown as the deletion strain vs. the equivalent wild type hypoxia time point. Genes discussed and/or examined in detail in this manuscript are noted with asterisks. MeV analysis was performed using hierarchical clustering. Optimized gene and leaf ordering groups the wild type 30- and 120-minute hypoxic conditions together, with the 30-minute ΔsrbA sample more similar to wild type for the SrbA targets, using Pearson correlation with complete linkage clustering.
Figure 8.
Co-Regulation of SrbA target genes by SrbB.
Conidia of each strain were cultured in normoxia at 37°C, 250 rpm for 18 hours and shifted to hypoxia for additional incubation for 4 hours. Expression of SrbA target genes involved in ergosterol biosynthesis, nitrate assimilation, heme biosynthesis, and carbohydrate metabolism in the strains was studied using qRT-PCR. Data are presented as the mean and standard error of two biological replicates, and analyzed by one-way ANOVA followed by Bonferroni's posttests. Expression of erg1, erg25A, niiA, hem13, and alcC requires both SrbA and SrbB. In contrast, expression of erg3B, erg11A, and niaD are regulated by only SrbA. SrbB appears to have a dominant role over SrbA in regulation of hem13 and alcC expression. ΔΔsrbAsrbB: a double knock-out mutant of srbA and srbB, srbB-ove;ΔsrbA: ΔsrbA with restored srbB expression, srbA-ove;ΔsrbB: a srbA overexpression strain in ΔsrbAΔsrbB.
Figure 9.
Binding of SrbB to the promoter of specific SrbA target genes and binding of SrbA in ΔsrbB.
(A) SrbB tagged with GFP was expressed in A. fumigatus wild type. The resulting strain was cultured in normoxia at 37°C, 250 rpm for 18 hours and shifted to hypoxia for additional incubation for 4 hours. ChIP was conducted using GFP antibody followed by ChIP-qPCR to study SrbB enrichment on the promoters of SrbA target genes. Compared to wild type control, SrbB enrichment was significant in SrbB:GFP for srbA, srbB, erg25A, hem13, and alcC, which suggest SrbB directly binds on the promoter of these genes for transcriptional regulation. In contrast, SrbB enrichment on the promoters of erg11A and actA were not significant. Data are presented as the mean and standard error of two biological replicates, and analyzed by two-way ANOVA followed by Bonferroni posttest. (B) SrbA binding to the promoter of srbA, srbB, and erg11A in ΔsrbB was examined by ChIP-qPCR. Compared to wild type, SrbA enrichment on the gene promoters was not altered by disruption of SrbB. Data are presented as the mean and standard error of two biological replicates and analyzed by two-way ANOVA followed by Bonferroni posttests.
Figure 10.
Restoration of srbB transcript levels in ΔsrbA promotes growth in hypoxia.
(A). Using two promoters, flavA(p) and gpdA(p), srbB was expressed in ΔsrbA. Expression levels of srbB in TDC43.18 (flavA(p)) and TDC44.2 (gpdA(p)) in normoxia or hypoxia 2 h were verified using quantitative PCR. Data are presented as the mean and standard error. Compared to ΔsrbA, srbB transcript in TDC43.18 and TDC44.2 was more abundant by 5- and 3.7-fold in normoxia and 2.7- and 1.4-fold in hypoxia 2 h, respectively. This indicates these promoters function properly. (B). Sequentially diluted conidia (102–105) were inoculated on GMM plates and culture in normoxia or hypoxia at 37°C for 3 days. Increased expression of srbB using flavA(p) or gpdA(p) partially restores defective hypoxia growth in ΔsrbA. (C). Susceptibility of TDC43.18 and TDC44.2 to the triazole drug, voriconazole (VCZ) was tested. A million conidia were overlaid on GMM, and VCZ in DMSO was applied to the center of the plate. Cleared areas represent inhibited fungal growth in response to VCZ. Although growth in hypoxia was partially rescued in both strains as shown (B), increased susceptibility to VCZ in ΔsrbA compared to wild type was not affected by restoration of srbB expression.
Figure 11.
Loss of SrbB attenuates Aspergillus fumigatus virulence through reductions in pulmonary fungal burden.
(A). 6–8 week old immunosuppressed CD-1 mice (each group N = 20) inoculated via the intranasal route with 2×106 conidia of wild type, ΔsrbB and srbB-reconstituted strains. Comparing wild type and reconstituted strain Kaplan-Meier curves with the ΔsrbB strain shows a significant increase in survival for the animals inoculated with the ΔsrbB strain (Log rank test, p = 0.0027). All control PBS inoculated animals survived. (B) Triamcinolone mouse model was used for fungal burden analysis. Mice were infected with 106 conidia of each strain and lungs were collected on day +3. Data represented are the mean and standard error of 3–5 mice per group.
Figure 12.
Working models for transcriptional regulation of the hypoxia response by SrbA and SrbB in A. fumigatus.
A. fumigatus SREBPs, SrbA and SrbB have dependent and independent functions in regulation of hypoxic genes involved in ergosterol biosynthesis, carbohydrate metabolism, heme biosynthesis, and nitrate assimilation. In response to hypoxia, srbB transcription is induced earlier than srbA (15 minutes in hypoxia) possibly by an unknown factor (marked as ‘X’). SrbA regulates srbB and its own transcript abundance. Similarly, SrbB regulates srbA and its own transcript abundance. qRT-PCR and ChIP-qPCR data presented in this study show that abundance of erg1, erg25A, hem13, niiA, and alcC is regulated by both SrbA and SrbB. In contrast, abundance of erg11A, erg3B, erg5, and niaD is dependent on SrbA. SrbB appears to have a dominant function over SrbA in regulation of hem13 and alcC transcript abundance (indicated by dotted lines).