HapX iron sensing in Aspergillus fumigatus involves the interaction with the monothiol glutaredoxin GrxD


Mareike Scheven 1,2 Matthias Misslinger 3 Peter Hortschansky 1 Thomas Krüger 1 Hubertus Haas 3 Axel A. Brakhage 1,2
1Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knöll Institute (HKI), Jena, Germany
2Institute of Microbiology, Friedrich Schiller University, Jena, Germany
3Division of Molecular Biology, Biocenter, Innsbruck Medical University, Innsbruck, Austria

Aspergillus fumigatus is a ubiquitous saprophytic mold, which causes life-threatening diseases in immunocompromised patients. During infection, sufficient iron supply is crucial for fungal growth. Iron is a vital nutrient, but can be harmful in excess by triggering the formation of cell damaging reactive oxygen species. As a result, A. fumigatus has evolved fine-tuned mechanisms to maintain iron equilibrium. Adaptation to iron limitation and iron excess are mediated by the bZIP transcription factor HapX, which functions via physical interaction with the heterotrimeric CCAAT-binding complex. During iron starvation, iron consuming pathways are repressed and iron uptake is activated. During iron overload, the cell is detoxified from iron by activation of vacuolar iron storage [1].

Currently, the molecular mechanisms of iron sensing by HapX are unknown and remain to be elucidated. As shown for iron regulators in other ascomycetes, A. fumigatus HapX senses the cellular iron status most likely by interaction with other regulators, like monothiol glutaredoxin (GrxD). We applied a co-immunoprecipitation approach for identification of possible GrxD as well as HapX interaction partners. VENUS-tagged GrxD and MYC-tagged HapX proteins were enriched from crude cell extracts by GFP-Trap and MYC-Trap, respectively. Immunoprecipitated proteins were identified by nano LC-MS/MS measurement. HapX co-precipitated during GrxDVENUS enrichment under iron starvation, sufficiency and excess. Vice versa, GrxD was co-enriched during MYCHapX immunoprecipitation. The interaction of GrxD and HapX was subsequently confirmed in vivo by bimolecular fluorescence complementation analysis. In line with the in vivo results, recombinant A. fumigatus GrxD and HapX proteins were also co-purified with an unknown Fe-S ligand in vitro from Escherichia coli. In summary, these data provide first evidence that HapX iron sensing in A. fumigatus involves the interaction with GrxD.

Reference

[1] Gsaller, F.; Hortschansky, P. et al. EMBO J 2014, 33, 2261-2276.