Moloney, Nicola (2017) Exploring the secreted proteome and metabolome of iron-starved Aspergillus fumigatus for tools to combat fungal infection. PhD thesis, National University of Ireland Maynooth.
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Abstract
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.
Item Type: | Thesis (PhD) |
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Keywords: | secreted proteome; metabolome; iron-starved; Aspergillus fumigatus; combat; fungal infection; |
Academic Unit: | Faculty of Science and Engineering > Biology |
Item ID: | 16805 |
Depositing User: | IR eTheses |
Date Deposited: | 05 Jan 2023 16:28 |
URI: | https://mural.maynoothuniversity.ie/id/eprint/16805 |
Use Licence: | This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here |
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