Owens, Rebecca A. and Hammel, Stephen and Sheridan, Kevin J. and Jones, Gary W. and Doyle, Sean (2014) A Proteomic Approach to Investigating Gene Cluster Expression and Secondary Metabolite Functionality in Aspergillus fumigatus. PLoS ONE, 9 (9). e106942. ISSN 1932-6203

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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.

Item Type:	Article
Additional Information:	© 2014 Owens et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. This work was funded in part by a Science Foundation Ireland Principal Investigator Award to SD (PI/11/1188). RAO and SH were recipients of Irish Research Council for Science Engineering and Technology Embark PhD Fellowships. LC-MS facilities were funded by a competitive award from the Irish Higher Education Authority. Funding from the 3U Partnership (DCU/NUIM/RCSI) is also acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Keywords:	Proteomics; Proteomic Approach; Gene Cluster Expression; Secondary Metabolite Functionality; Aspergillus fumigatus;
Academic Unit:	Faculty of Science and Engineering > Biology
Item ID:	5645
Identification Number:	https://doi.org/10.1371/journal.pone.0106942
Depositing User:	Dr. Sean Doyle
Date Deposited:	06 Jan 2015 15:22
Journal or Publication Title:	PLoS ONE
Publisher:	Public Library of Science
Refereed:	Yes
Funders:	Science Foundation Ireland (SFI), Irish Research Council for Science Engineering and Technology (IRCSET), Higher Education Authority (HEA)
URI:	http://www.plosone.org/
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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