Exploring the secreted proteome and metabolome of iron-starved Aspergillus fumigatus for tools to combat fungal infection

Aspergillus fumigatus is a prevalent airborne fungal pathogen with the potential to cause Invasive Aspergillosis (IA) in susceptible hosts. Limited treatment and diagnostic options largely underlie the high mortality associated with IA. Like many human pathogens, A. fumigatus possesses sophisticated mechanisms for iron acquisition that are crucial to its virulence. Comparative proteomics revealed that iron starvation induced significant (p < 0.05) remodelling of the A. fumigatus secretome. A cell wall remodelling response was induced with significantly (p < 0.0001) decreased glucanase activity during iron starvation. The secretomic remodelling was relevant to host recognition, evident in the increased abundance of many immunogenic proteins. ELISA analysis also indicated significantly (p < 0.0001) increased immunoreactivity against proteins isolated from iron-starved growth using sera from the general population. This highlights the importance of iron-starved A. fumigatus in host interactions. During iron starvation A. fumigatus utilises extracellular siderophores, fusarinine C (FSC) and triacetylfusarinine C (TAFC), for iron uptake. Fluorescently derivatised siderophores were taken up by A. fumigatus during iron-starved growth, evidenced by the vacuolar fluorescence observed exclusively therein. In addition to informing on the fate of siderophores following uptake, this demonstrated an aminebased derivatisation strategy for A. fumigatus siderophores with other applications. Using this strategy, FSC and TAFC immunogens were synthesized for murine immunisations. These mice were then used to generate hybridomas producing antisiderophore antibodies. Antibody production was verified using a novel competitive FSC or TAFC ELISA. An anti-FSC IgM was isolated and characterised. This IgM demonstrated the proteinaceous inhibition of siderophore-mediated iron uptake in A. fumigatus in vitro. An anti-TAFC IgG was also isolated and characterised. This IgG could detect low concentrations of TAFC within clinically relevant ranges in a competitive ELISA, indicative of its potential for implementation in a diagnostic assay. Using a proteogenomic strategy, putative N- and C-terminal sequences associated with this IgG were identified by MS to permit cDNA sequencing for further expression. Overall this work has comprised an application-driven exploration of iron-starved A. fumigatus and successfully yielded output for future diagnostic and therapeutic strategies against A. fumigatus infection