A Proteomic Approach to Investigating Gene Cluster Expression and Secondary Metabolite Functionality in Aspergillus fumigatus

Doyle S (2014) A Proteomic Approach to Investigating Gene Cluster Expression and Secondary Metabolite Functionality in Aspergillus fumigatus. PLoS ONE 9(9): e106942. doi:10.1371/journal.pone.0106942 A Proteomic Approach to Investigating Gene Cluster Expression and Secondary Metabolite Functionality in Aspergillus fumigatus Rebecca A. Owens 0 Stephen Hammel 0 Kevin J. Sheridan 0 Gary W. Jones 0 Sean Doyle 0 Kap-Hoon Han, Woosuk University, Republic of Korea 0 Department of Biology, National University of Ireland Maynooth , Maynooth, Co. Kildare , Ireland 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. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: 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. Competing Interests: The authors have declared that no competing interests exist. 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 [310]. 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]. MS-based or shotgun proteomics can adopt multiple approaches including, (i) direct LC-MS/MS, (ii) indirect LC-MS/MS and (iii) 2-D-LC-MS/MS (multidimensional protein identification technology, MudPIT) [12,13]. Direct LC-MS/MS involves the on-line separation of complex peptide mixtures using reversed phase nano-LC columns with extended acetonitrile gradients to effect peptide separation [14]. 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]. Subproteome strategies have also been implemented to investigate glutathione binding [4] and mitochondrial proteins [6]. Indeed, 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]. This study would have been difficult to perform using 2-D due to the incompatibility of hydrophobic proteins, and proteins with transmembrane (TM) regions, with detergents used in isoelectric focusing, the first separation stage of 2-D [17]. Quantitative MSbased proteomics, both label-free and using isobaric tagging for relative and absolute quantitation (iTRAQ), have been used to comparatively profile the stages of A. fumigatus germination [9,10]. Activity-based MS proteomics has also recently been developed to investigate A. fumigatus following incubation with human sera [18]. The application of MS-based proteomics to dissect the proteome of A. fumigatus has the potential to provide a global overview of the pathways and biological processes active under a set of conditions. In addition, (i) bioinformatic analysis can expand the characterisation of large datasets generated by MSbased proteomics, and (ii) shotgun proteomics offers the possibility of identifying the presence of either hypothetical proteins or proteins of unknown function, whose existence may either be unclear, or only previously demonstrated at the transcript level. Furthermore, (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]. Proteomic approaches may also have an application in characterizing the effect of exogenous SMs on A. fumigatus [20]. Indeed, despite the many advantages of shotgun proteomics, 2-D has been successfully deployed to inform on proteomic alterations in A. fumigatus under various conditions [2027]. Thus, a complementary strategy of shotgun and 2-D proteomics offers much in terms of the ability to reveal the nature of the proteome in pathogenic microorganisms, provide further insight into SM biosynthesis- and explore how apparently synergistic stressors may interact in unexpected ways. Interestingly, both gliotoxin and H2O2, separately, have been shown to result in numerous, growth inhibitory-associated, alterations to the proteome of A. fum (...truncated)