Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.

The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope. [Display omitted] • APEX2 proximity labeling accurately identifies mycobacterial cell wall proteins • "Impermeable" N-hydroxysuccinimide reagents do not selectively tag surface proteins • Cytoplasmic or cell wall tagging of membrane proteins informs their topology • Cell wall tagging of Type VII secretion substrates supports role in export mechanism The cell wall of the bacterial pathogen Mycobacterium tuberculosis is a protective barrier, but also an obstacle to the flow of nutrients and other biomolecules. Jaisinghani et al. use APEX2-mediated proximity labeling in live cells to identify cell wall proteins and thereby inform the mechanism of poorly understood transport processes. [ABSTRACT FROM AUTHOR]