Abstract
A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism.
Highlights
Following the publication of A. fumigatus Af293 [1] genomic sequence and the sequencing of a second A. fumigatus strain, A1163 [2], extensive efforts have been undertaken to characterise the proteome of this opportunistic human pathogen [3,4,5,6,7,8,9,10]
Shotgun mass spectrometry identifies 414 proteins from A. fumigatus mycelia grown in Aspergillus minimal media (AMM)
Utilising a direct shotgun proteomics approach, a total of 1826 unique peptides were identified in A. fumigatus mycelial lysates, corresponding to 414 distinct A. fumigatus proteins from 405 protein groups (Table S1)
Summary
Following the publication of A. fumigatus Af293 [1] genomic sequence and the sequencing of a second A. fumigatus strain, A1163 [2], extensive efforts have been undertaken to characterise the proteome of this opportunistic human pathogen [3,4,5,6,7,8,9,10]. Traditional proteomic strategies have utilised 2-D separation with subsequent protein identification by MS. Shotgun MS-based proteomics has developed more recently and provides a complementary method to 2-D for proteome profiling [9,10], since 2-D can occasionally be limiting for the identification of particular subsets of proteins, especially hydrophobic proteins, membrane proteins, and proteins with large molecular mass or extreme pI [11]. Indirect LC-MS/MS is where complex peptide or protein mixtures are pre-fractionated off-line (e.g. by SDS-PAGE) before LC-MS/MS analysis [15]. The recent emergence of MS-based proteomics studies of A. fumigatus has been undertaken whereby 530 plasma membrane associated proteins were identified by utilising a combination of SDS-PAGE fractionation of total protein followed by peptide separation and identification by 2-D-LC-MS/MS [16]. (iii) shotgun MS-based proteomics has the potential to be used for the non-targeted identification of secondary metabolite (SM) cluster expression, which, coupled with subsequent metabolomics, could result in the identification of novel cluster products [19]
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