ChIP-Seq and In Vivo Transcriptome Analyses of the Aspergillus fumigatus SREBP SrbA Reveals a New Regulator of the Fungal Hypoxia Response and Virulence

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs\u27 complex role in infection site adaptation and fungal virulence

ChIP-seq and in vivo transcriptome analyses of the Aspergillus fumigatus SREBP SrbA reveals a new regulator of the fungal hypoxia response and virulence.

The Aspergillus fumigatus sterol regulatory element binding protein (SREBP) SrbA belongs to the basic Helix-Loop-Helix (bHLH) family of transcription factors and is crucial for antifungal drug resistance and virulence. The latter phenotype is especially striking, as loss of SrbA results in complete loss of virulence in murine models of invasive pulmonary aspergillosis (IPA). How fungal SREBPs mediate fungal virulence is unknown, though it has been suggested that lack of growth in hypoxic conditions accounts for the attenuated virulence. To further understand the role of SrbA in fungal infection site pathobiology, chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq) was used to identify genes under direct SrbA transcriptional regulation in hypoxia. These results confirmed the direct regulation of ergosterol biosynthesis and iron uptake by SrbA in hypoxia and revealed new roles for SrbA in nitrate assimilation and heme biosynthesis. Moreover, functional characterization of an SrbA target gene with sequence similarity to SrbA identified a new transcriptional regulator of the fungal hypoxia response and virulence, SrbB. SrbB co-regulates genes involved in heme biosynthesis and demethylation of C4-sterols with SrbA in hypoxic conditions. However, SrbB also has regulatory functions independent of SrbA including regulation of carbohydrate metabolism. Loss of SrbB markedly attenuates A. fumigatus virulence, and loss of both SREBPs further reduces in vivo fungal growth. These data suggest that both A. fumigatus SREBPs are critical for hypoxia adaptation and virulence and reveal new insights into SREBPs' complex role in infection site adaptation and fungal virulence

RNA-seq and nCounter Analyses of Δ<i>srbA</i> Confirms SrbA Regulation of ChIP-seq Target Genes <i>in vitro</i> and <i>in vivo</i> during Invasive Pulmonary Aspergillosis.

<p>(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 <i>in vivo</i> in a murine model of invasive pulmonary aspergillosis for wild type (CEA10) and <i>in vitro</i> under normoxic/hypoxic conditions for Δ<i>srbA</i> and wild type <i>in vivo</i> samples were at 48–96 hours post-infection (grey, n = 16). <i>in vitro</i> samples were wild type normoxia (red, n = 2) and hypoxia (blue, n = 6) followed by Δ<i>srbA</i> under normoxia (red, n = 2) and hypoxia (blue, n = 6). Time under hypoxia for both wild type and Δ<i>srbA</i> 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.</p

Loss of SrbB attenuates <i>Aspergillus fumigatus</i> virulence through reductions in pulmonary fungal burden.

<p>(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, Δ<i>srbB</i> and <i>srbB</i>-reconstituted strains. Comparing wild type and reconstituted strain Kaplan-Meier curves with the Δ<i>srbB</i> strain shows a significant increase in survival for the animals inoculated with the Δ<i>srbB</i> 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.</p

Binding of SrbB to the promoter of specific SrbA target genes and binding of SrbA in Δ<i>srbB</i>.

<p>(A) SrbB tagged with GFP was expressed in <i>A. fumigatus</i> 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 <i>srbA</i>, <i>srbB</i>, <i>erg25A</i>, <i>hem13</i>, and <i>alcC</i>, which suggest SrbB directly binds on the promoter of these genes for transcriptional regulation. In contrast, SrbB enrichment on the promoters of <i>erg11A</i> and <i>actA</i> 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 <i>srbA</i>, <i>srbB</i>, and <i>erg11A</i> in Δ<i>srbB</i> 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.</p

Selected SrbA ChIP Peaks with flanking gene information.

<p>Selected SrbA ChIP Peaks with flanking gene information.</p

SrbB is a transcriptional regulator of genes involved in carbon metabolism, lipid metabolism, and heme biosynthesis.

<p>The FungiFun2 web server was utilized to assign FunCat and gene ontology enrichment in genes with transcript levels increased or decreased 4 fold in Δ<i>srbB</i> compared to the wild type strain. Statistically significant (P≤0.05) FunCat categories are presented at the respective time points in hypoxia.</p

Restoration of <i>srbB</i> transcript levels in Δ<i>srbA</i> promotes growth in hypoxia.

<p>(A). Using two promoters, <i>flavA</i>(p) and <i>gpdA</i>(p), <i>srbB</i> was expressed in Δ<i>srbA</i>. Expression levels of <i>srbB</i> in TDC43.18 (<i>flavA</i>(p)) and TDC44.2 (<i>gpdA</i>(p)) in normoxia or hypoxia 2 h were verified using quantitative PCR. Data are presented as the mean and standard error. Compared to Δ<i>srbA</i>, <i>srbB</i> 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 <i>srbB</i> using <i>flavA</i>(p) or <i>gpdA</i>(p) partially restores defective hypoxia growth in Δ<i>srbA</i>. (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 Δ<i>srbA</i> compared to wild type was not affected by restoration of <i>srbB</i> expression.</p

A sub-set of SrbA ChIP Target Genes are co-regulated by SrbB.

<p>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 Δ<i>srbA</i> sample more similar to wild type for the SrbA targets, using Pearson correlation with complete linkage clustering.</p