title: Correlation of phenotypic profiles using targeted proteomics identifies mycobacterial esx-1 substrates.
abstract: The Esx/WXG-100 (ESAT-6/Wss) exporters are multiprotein complexes that promote protein translocation across the cytoplasmic membrane in a diverse range of pathogenic and nonpathogenic bacterial species. The Esx-1 (ESAT-6 System-1) system mediates virulence factor translocation in mycobacterial pathogens, including the human pathogen Mycobacterium tuberculosis. Although several genes have been associated with Esx-1-mediated transport and virulence, the contribution of individual Esx-1 genes to export is largely undefined. A unique aspect of Esx-1 export is that several substrates require each other for export/stability. We exploited substrate "codependency" to identify Esx-1 substrates. We simultaneously quantified changes in the levels of 13 Esx-1 proteins from both secreted and cytosolic protein fractions generated from 16 Esx-1-deficient Mycobacterium marinum strains in a single experiment using MRM/SRM targeted mass spectrometry. This expansion of measurable Esx-1 proteins allowed us to define statistical rules for assigning novel substrates using phenotypic profiles of known Esx-1 substrates. Using this approach, we identified three additional Esx-1 substrates encoded by the esx-1 region. Our studies begin to address how disruption of specific genes affects several proteins in the Esx-1 complex. Overall, our findings illuminate relationships between Esx-1 proteins and create a framework for the identification of secreted substrates applicable to other protein exporters and pathways.
authors.affiliation: Department of Chemistry and Biochemistry, ‡Department of Biological Sciences, §Eck Institute for Global Health, and ∥Center for Rare and Neglected Diseases, University of Notre Dame , Notre Dame, Indiana 46556, United States.
authors.forename: Matthew M
authors.initials: MM
authors.lastname: Champion
authors.forename: Emily A
authors.initials: EA
authors.lastname: Williams
authors.forename: Richard S
authors.initials: RS
authors.lastname: Pinapati
authors.forename: Patricia A DiGiuseppe
authors.initials: PA
authors.lastname: Champion
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D019910:Mycobacterium marinum / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D010641:Phenotype; D025941:Protein Interaction Mapping; D021381:Protein Transport; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C093276:ESAT-6 protein, Mycobacterium tuberculosis
keywords: Esx-1; EsxA; MRM/SRM; Mycobacterium marinum; RD1; secretion; substrate identification; targeted proteomics
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