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.

The genetic proteome: Using genetics to inform the proteome of mycobacterial pathogens

“…Likewise, chemical probes for detecting direct interactions between proteins in the mycobacterial cell envelope have been coupled to quantitative proteomics. These approaches have revealed more than 100 envelope proteins and their binding partners in M. smegmatis [134]. Another interesting example of the genetic proteome is a recent study in which genetics were used to alter the proteome of microvesicles produced by M. tuberculosis.…”

Chemical Biology Tools for Examining the Bacterial Cell Wall

“…However, its protein composition has eluded classic biochemical techniques for a long time, in part because of the difficulty of cleanly separating the covalently bound mycomembrane from other layers of the complex mycobacterial envelope. Kavunja et al recently developed the first photocrosslinking probes for the mycomembrane to analyze mycolate-protein interactions in vivo (Figure 2E) (Kavunja et al, 2020). They synthesized a TMM analog that specifically incorporates into the TDM portion of the mycomembrane via previously reported conserved, substrate-promiscuous Ag85 mycoloyltransferases (Fiolek et al, 2019).…”

“…Previously, Sarkar et al exploited the MurF ligation process to insert a D-Ala-that was biofunctionalized with an alkyne and photocrosslinking handles into Lipid II to mine the protein-interacting partners (Sarkar et al, 2016). Targeted acquisition of cell envelope interactomes may reveal new potential targets for antibiotic therapies (Kavunja et al, 2020).…”

Multi-Omics Technologies Applied to Tuberculosis Drug Discovery

“…Glycans are vital in a broad range of processes, their direct recognition by glycan-binding proteins is important for many processes that may be essential and druggable. Recently, Kavunja et al used a glycomics approach to identify mycolate-interacting proteins associated with synthesis and remodeling of the membrane in M. smegmatis that could lead to the validation of novel therapeutic targets [111]. Such techniques offer unique opportunities for biological discovery and new target identification that will expand as methodologies develop to increase sensitivity and reduce complexity.…”