Correlation of Phenotypic Profiles Using Targeted Proteomics Identifies Mycobacterial Esx‑1 Substrates

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.