Functional transitions of the Aspergillus fumigatus iron regulator HapX are governed by conserved domains cooperatively binding [2Fe-2S] clusters

Accurate sensing of cellular iron levels is vital, as this metal is essential but toxic in excess. The iron-sensing transcription factor HapX is crucial for virulence of Aspergillus fumigatus, the predominant human mold pathogen. Its absence impairs growth under iron limitation and excess, but not under moderate iron availability, suggesting that HapX switches between three states to adapt to varying iron availability. This study suggests that the HapX state transitions are regulated by different propensities of four phylogenetically conserved cysteine-rich regions (CRRs) to coordinate [2Fe-2S] clusters resulting in cumulative occupancies depending on iron availability. The iron starvation state features no [2Fe-2S] clusters in any of the CRRs, the iron sufficiency/”neutral” state features clusters in CRR-B and/or -C, and the iron excess state has clusters in CRR-A, -B, and -C, while CRR-D plays a minor role. Combinatorial mutation of CRR-B and -C inhibited growth by locking HapX in the iron starvation state, leading to uncontrolled iron uptake and repression of iron-consuming pathways and iron detoxification. This growth defect was partially rescued by removing the C-terminal 27 amino acids, which are crucial for the iron starvation state and contain a degron. Noteworthy, the HapX iron starvation state induced several gene clusters encoding secondary metabolites. Overall design: RNA-seq profiling of fungal strains encoding the native hapX and the hapXA/B/C/D allele, which is characterized by the simultaneous mutation of all four cysteine-rich regions (CRR); both alleles are under control of the xylose-inducible xylanase promoter (xylP); fungal liquid cultures were grown at 37 °C under iron sufficient conditions without xylose supplementation for 17 h, followed by 1 h incubation in the presence of 0.1% (w/v) xylose for xylP induction;