Opportunistic infections have significantly increased due to advances in medical procedures in immunocompromised patients. Immunosuppression also increases dramatically the risk of invasive fungal infections, mainly aspergillosis. Aspergillosis is commonly caused by Aspergillus fumigatus – a saprophytic fungus typically residing in soil and in the air. Upon infection, A. fumigatus synthesizes transporters proteins essential for virulence called siderophores. These low molecular mass iron chelators are secreted by most fungal species for scavenging iron.
Siderophores can also be used for imaging and diagnostics of aspergillosis in animal models. Moreover, siderophore transport might enable fungal-specific uptake of antifungal compounds by conjugation since siderophore-iron chelates uptake is mediated by transporters. A. fumigatus has been shown to be able to utilize the endogenous siderophores (triacetylfusarinine C, fusarinine C and ferricrocin) as well as siderophores produced by other organisms, so called xenosiderophores (e.g. ferrioxamine B, ferrioxamine E, and coprogen).
A. fumigatus possesses five potential siderophore transporters. Optimization of the afore mentioned applications of siderophore transport requires the characterization of the substrate specificities of these potential siderophore transporters. Therefore, A. fumigatus mutant strains with deletions in individual, as well as multiple siderophore transporters, will be generated using different selection markers and recyclable marker cassettes. Gene deletions will be performed in A. fumigatus wild type strains as well as mutants strains lacking high-affinity iron acquisition (lacking both reductive iron assimilation and siderophore biosynthesis). The latter approach allows assaying of siderophore uptake by growth assays because the mutant is only able to grow in the presence of usable siderophores. In addition, siderophore uptake will be assayed by short-term uptake of siderophores labelled with gallium68. In addition to the generation of siderophore lack-of function mutants, siderophore transporters will be tagged with GFP to enable subcellular localization and elucidate further understanding in the molecular characterization of fungal siderophores.
