Photoactivatable Glycolipid Probes for Identifying Mycolate-Protein Interactions in Live Mycobacteria

Abstract

Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling in vivo photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this strategy enriched over 100 proteins, including the mycomembrane porin (MspA), several proteins with known mycomembrane synthesis or remodeling functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting proteins. Our approach is highly versatile, as it (i) enlists click chemistry for flexible protein functionalization; (ii) in principle can be applied to any mycobacterial species to identify endogenous bacterial proteins or host proteins that interact with mycolates; and (iii) can potentially be expanded to investigate protein interactions with other mycobacterial lipids. This tool is expected to help elucidate fundamental physiological and pathological processes related to the mycomembrane and may reveal novel diagnostic and therapeutic targets.

Figure 1.

(A) Metabolism and host interactions of mycolate glycolipids. (B) Strategy for in vivo capture and analysis of mycolate-interacting proteins using photoactivatable probes (see Scheme S1 and Supporting Information Discussion).

Figure 2.

(A) Syntheses of N- and O-x-AlkTMM-C15. (B) UV-dependent photo-cross-linking of BSA with probes followed by CuAAC-mediated product detection.

Figure 3.

Mycomembrane labeling with N- and O-x-AlkTMM-C15. (A) Bacteria were cultured in probe (25 μM), reacted with azido-488 by CuAAC, and analyzed by microscopy (Figure S4, flow cytometry). (B) Probe-treated Msmeg was reacted with azido-488 by CuAAC and fractionated into PG-AGM- and TDM-containing fractions, and fluorescence was measured. Error bars denote the standard deviation of three replicates. MFI, mean fluorescence intensity in arbitrary units.

Figure 4.

N-x-AlkTMM-C15-mediated affinity enrichment of mycolate-interacting proteins. Msmeg was cultured in N-x-AlkTMM-C15 (100 μM), UV-irradiated, and lysed. Lysates were reacted with AzTB by CuAAC and then analyzed using the indicated method before (input) and after (output) incubation with avidin beads to evaluate enrichment of (A) proteins in general and (B) MspA and Ag85. Data are representative of three independent experiments.

Figure 5.

Volcano plots showing proteins in red that were significantly enriched in N-x-AlkTMM-C15-treated, UV-exposed (+probe+UV) versus nonirradiated (+probe−UV) Msmeg grown to OD₆₀₀ (A) ~1.2 or (B) ~4 using click-mediated protein affinity enrichment, tryptic digestion, and LC−MS/MS analysis. Selected proteins of interest are indicated. (C) Venn diagram of proteins enriched in (A) and (B).