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

Abstract

Proximity labeling is a technology for tagging proteins and other biomolecules in living cells. These methods use enzymes that generate reactive species whose properties afford high spatial resolution for the localization of proteins to subcellular compartments and the identification of endogenous interaction partners. Here we present the adaptation of the engineered peroxidase APEX2 to proximity labeling in mycobacteria, including the human pathogen Mycobacterium tuberculosis. APEX2 is uniquely suited for general use in bacteria because unlike other proximity labeling enzymes, it does not depend on metabolites like ATP that are found in the cytoplasm, but are absent from the bacterial periplasm. Importantly, we found that in slow-growing mycobacteria like M. tuberculosis, codon usage optimization is required for APEX2 export into the periplasm via fusion to an N-terminal secretion signal. APEX2 expressed from codon-optimized genes affords robust, compartment-specific protein labeling in the cytoplasm and the periplasm of both fast- and slow-growing species. Here we detail these updated constructs and provide an optimized protocol for APEX2-mediated protein labeling in mycobacteria. We expect this approach to be broadly useful for determining the localization of specific proteins, cataloging subcellular proteomes, and identifying interaction partners of 'bait' proteins expressed as fusions to APEX2.

Keywords: APEX2; Mycobacteria; Periplasm; Peroxidase; Protein labeling; Proximity labeling; Tuberculosis.

Figure 1.. Compartment-specific biotinylation of proteins in M. smegmatis by the engineered peroxidase APEX2. M. smegmatis expressing APEX2, Sec-APEX2 or Tat-APEX2 was grown without or with theophylline and subjected to the labeling protocol with biotin-phenol.

(A) Streptavidin blot analysis of total lysates was used to detect protein biotinylation. Asterisks and arrowheads indicate examples of APEX2 expression-independent and -dependent bands; respectively. Data are representative of >3 independent experiments. (B) Biotinylated proteins were enriched by avidin affinity purification and analyzed by immunoblot with antibody against the M. tuberculosis antigen 85 complex, a group of three homologous proteins expressed in the cell wall. Purified M. tuberculosis Ag85A (34 kDa) was included as a positive control for the antibody. All data are from the same image; intervening lanes were removed for clarity. Data are representative of 2 independent experiments. (C) M. smegmatis expressing APEX2 or Sec-APEX2 from a multi-copy episomal plasmid and the cell wall protein-epitope fusion LprG-3XFLAG (27 kDa) or NA-LprG-3XFLAG (which lacks the N-terminal secretion signal (Drage et al., 2010) and thus accumulates in the cytosol; 24 kDa) from an integrated chromosomal copy were grown without or with theophylline and subjected to the labeling protocol with biotin-phenol. Biotinylated proteins were enriched by avidin affinity purification. Anti-FLAG immunoblot analysis with chemiluminescence detection was used to assess expression and enrichment of LprG-3XFLAG and NA-LprG-3XFLAG. Data are representative of 3 independent experiments. Lane labels I and O indicate input and output for avidin enrichment. Figure adapted and used with permission (Ganapathy et al., 2018).

Figure 2.. Compartment specific labeling of proteins in M. tuberculosis by APEX2 expressed from a gene optimized for M. tuberculosis H37Rv codon usage.

M. tuberculosis mc²6020 expressing APEX2 or Sec-APEX2 from the codon usage-optimized APEX2m gene were grown with or without theophylline and subjected to the labeling protocol for (A) biotin-phenol or (B) tyramide azide followed by CuAAC coupling to fluorescein-alkyne. Total lysates were analyzed by (A) streptavidin blot or (B) fluorescence imaging. In (B) cultures of two independent clones were analyzed. Asterisks indicate examples of APEX2 expression-independent bands. Data are represented of >3 independent experiments.

Figure 3.. Plasmid maps for pRibo-Sec-APEX2m and pRiboI-Sec-APEX2m.

The restriction sites most relevant to cloning are indicated. Plasmids and partial sequences are available from Addgene (ID: 176844, 176845). Full maps and sequences are available from the authors upon request. Figure created with SnapGene.

Figure 4.. Experimental flow for APEX2 expression and protein labeling in mycobacteria.

The procotol specifies labeling with tyramide azide followed by coupling to fluorescein (FAM-alkyne), but is compatible with other alkyne reagents for other modes of detection or downstream applications, including enrichment as shown for proteins labeled with biotin-phenol. Figure created with BioRender.com.

Figure 5.. A colorimetric assay enables rapid assessment of APEX2 activity

in whole cells. M. smegmatis was grown with or without theophylline. Guaiacol substrate and hydrogen peroxide were added to an aliquot of culture to initiate the peroxidase reaction.

Figure 6.. Detection of APEX2 by anti-APX2 antibody.

M. smegmatis expressing APEX2 or Sec-APEX2 was growth with or without theophylline. Total lysates were analyzed by immunoblot using an anti-APX2 antibody raised against ascorbate peroxidase from Arabidopsis thaliana. The doublet for Sec-APEX2 may indicate protein before (31 kDa predicted molecular weight) and after (28 kDa) cleavage of the N-terminal secretion signal by signal peptidase in the periplasm. Asterisks indicate non-specific bands. Data are representative of >3 independent experiments.