HapX-mediated adaption to iron starvation is crucial for virulence of Aspergillus fumigatus

Abstract

Iron is essential for a wide range of cellular processes. Here we show that the bZIP-type regulator HapX is indispensable for the transcriptional remodeling required for adaption to iron starvation in the opportunistic fungal pathogen Aspergillus fumigatus. HapX represses iron-dependent and mitochondrial-localized activities including respiration, TCA cycle, amino acid metabolism, iron-sulfur-cluster and heme biosynthesis. In agreement with the impact on mitochondrial metabolism, HapX-deficiency decreases resistance to tetracycline and increases mitochondrial DNA content. Pathways positively affected by HapX include production of the ribotoxin AspF1 and siderophores, which are known virulence determinants. Iron starvation causes a massive remodeling of the amino acid pool and HapX is essential for the coordination of the production of siderophores and their precursor ornithine. Consistent with HapX-function being limited to iron depleted conditions and A. fumigatus facing iron starvation in the host, HapX-deficiency causes significant attenuation of virulence in a murine model of aspergillosis. Taken together, this study demonstrates that HapX-dependent adaption to conditions of iron starvation is crucial for virulence of A. fumigatus.

Figure 1. HapX affects iron regulation.

(A) Mutual transcriptional control between HapX and SreA. (B) Examples for negative (cycA) and positive (aspF1) impact of HapX in iron regulation. (C) HapX is required for transcriptional activation of the siderophore system. A. fumigatus wt, Δ sreA and Δ hapX were grown in shake flask cultures under iron-replete (+Fe) and depleted (−Fe) conditions. Total RNA was isolated and subjected to Northern analysis of genes selected from the genome-wide expression profiling (Fig. S1 and S2). As a control for quality and quantity, RNAs were hybridized with the ß-tubulin encoding tubA gene.

Figure 2. During iron depleted but not iron-replete conditions, HapX-deficiency impairs growth in liquid and solid media, colony formation from single conidia, conidiation, resistance to zinc and tetracycline, and causes reddish pigmentation.

(A) Growth on solid media with and without tetracycline: conidia were point-inoculated on plates reflecting harsh iron starvation (−Fe +BPS), iron starvation (−Fe) and iron-replete conditions (+Fe). Radial growth was recorded after 48h of growth at 37°C and normalized to that of wt grown under the same condition. (B) Colony formation from single conidia: approximately 100 conidia were plated and photographs were taken after growth for 48h at 37°C. (C) Conidiation: conidia production by 1 cm² of mycelia after growth for 120h of at 37°C was recorded and normalized to that of wt under the same condition. (D) Hyphal pigmentation: photographs of liquid cultures were taken after 24h of growth at 37°C. (E) Growth in liquid media with and without limitations or zinc excess: biomass production of 10⁸ conidia inoculated in 100 ml media was scored after 24 h of growth at 37°C and normalized to that of wt in +Fe. For limitation of carbon (−C), nitrogen (−N), zinc (−Zn) and copper (−Cu), the growth medium contained 0.1% glucose, 2 mM glutamine, 1 mM ZnSO₄ and no added copper, respectively, which decreased the biomass production of the wt to about the same extent as iron limitation. High zinc-medium (hZn) contained 0.5mM ZnSO₄. The data in (A), (C), and (E) represent the means ± standard deviations from three independent experiments.

Figure 3. HapX-deficiency decreases production of TAFC and FC but increases cellular accumulation of PpIX.

(A) Quantification of siderophore production after growth for 24 hours at 37°C under −Fe conditions normalized to that of wt. (B) Quantification of the PpIX content after growth for 24h at 37°C under iron-replete (+Fe) and depleted (−Fe) conditions. The data represent the mean ± standard deviation of three individually performed experiments.

Figure 4. Iron starvation transcriptionally up-regulates biosynthesis of ornithine in a HapX-dependent manner.

(A) Schematic representation of ornithine/arginine metabolism in A. fumigatus. Ornithine biosynthesis takes place in mitochondria (mito). Ornithine and citrulline are shuttled to the cytoplasm (cyto) and serve as precursors for arginine, siderophores and polyamines. Enzymatic steps within the pathways are numbered and corresponding to the Northern analysis in (B): 1, acetylglutamate synthetase (Afu2g11490) 2, acetylglutamate kinase and glutamate-5-semialdehyde dehydrogenase (Afu6g02910); 3, acetylornithine aminotransferase (Afu2g12470); 4, arginine biosynthesis bifunctional enzyme (Afu5g08120); 5, carbamoylphosphate synthase(Afu5g06780); 6, ornithine carbamoyltransferase (Afu4g07190); 7, arginase (Afu3g11430); 8, ornithine aminotransferase (Afu4g09140); 9, ornithine decarboxylase (Afu4g08010); 10, pyrroline carboxylate dehydrogenase (Afu6g08750); 11, ornithine transporter (Afu8g02760). Red and green arrows mark enzymatic steps transcriptionally up and down-regulated, respectively, by iron starvation in the wt as shown in (B); Red and green circles mark genes, which are transcriptionally up- and down-regulated, respectively, in a ΔhapX strain as shown in (B). (B) For Northern analysis, wt, ΔsidA and ΔhapX strains were grown for 24h at 37°C in under iron-replete (+Fe) and depleted (−Fe) conditions, respectively.

Figure 5. HapX-deficiency results in attenuation of A. fumigatus virulence.

Survival of leucopenic mice (A) and mice immunosuppressed with cortisone acetate (B) after infection with ΔhapX, the complemented strain ΔhapX^C, or wt. (C) Histopathology using PAS staining (hyphae stain pink) of leucopenic mice infected with ΔhapX, ΔhapX^C, or wt.