Optimized APEX2 peroxidase-mediated proximity labeling in fast- and slow-growing mycobacteria

Cell wall proteomics in liveMycobacterium tuberculosisuncovers exposure of ESX substrates to the periplasm

“…Optimization and validation of subcellular compartment-specific labeling by APEX2 in Mycobacterium tuberculosis. We have previously reported optimization of APEX2 for compartment-specific labeling in both M. smegmatis and Mtb 23 . Briefly, we found that in Mtb codon optimization of the APEX2 gene was necessary for export of APEX2 into the cell wall via fusion to the N-terminal signal peptide from the Mtb gene mpt63 (hereafter referred to as Sec-APEX2): Only codon-optimized Sec-APEX2 was enzymatically active in whole cells with the colorimetric substrate guaiacol and yielded a distinct pattern of labeling by the substrate biotin-phenol compared to APEX2 expressed in the cytoplasm (Cyt-APEX2) ( Figure 1A; Figure S1A ).…”

“…APEX2-mediated labeling and enrichment of biotinylated proteins. Growth, APEX2mediated labeling, and enrichment of biotinylated proteins was performed as reported 23 . Briefly, Mtb/APEX2m and Mtb/Sec-APEX2m were cultured in liquid medium to an optical density at 600 nm (OD600) of 0.8-1.…”

Protease shaving ofMycobacterium tuberculosisfacilitates vaccine antigen discovery and delivery of novel cargoes to the Mtb surface

“…Our ability to fuse five different epitope tags to PPE18 is in agreement with previous studies that have used similarly sized tags to detect the expression of recombinant proteins in mycobacteria 53 . For larger proteins, previous strategies were devised that fuse signal peptides from validated substrates of the Sec protein secretion system in order to dictate localization of an enzyme to a compartment of interest, like the periplasm 27 . This strategy is less optimal for purposes studying the Mtb surface interface as many of the surface accessible, outer membrane proteins we identified were associated with the ESX systems of the Type VII secretion system, where a verified signal peptide is not known.…”

“…Several biotechnologies desired in the field would be enabled with an Mtb strain that can be functionalized with heterologous proteins including surface display of heterologous enzymes to monitor bacterial infection, phagocyte interaction, and characterization of pathogen-specific compartments. Previous studies have shown that APEX2, an enzyme that facilitates proximity labeling, could be targeted to the periplasm of Mtb and Mycobacterium smegmatis 26,27 . We thus sought to determine if fusion of APEX2 containing a C-terminal HA tag to PPE18 (PPE18-APEX2-HA) would facilitate export of APEX2 to the Mtb surface (Fig.…”