Chemical Proteomics Strategies for Analyzing Protein Lipidation Reveal the Bacterial O-Mycoloylome.

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O-fatty acylation in Corynebacterium, termed protein O-mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms' outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O-mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O-mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O-acylated peptides. Using these tools in the model organism Corynebacterium glutamicum, we identified approximately 30 candidate O-mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O-mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O-mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae, Mycobacterium tuberculosis), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.

Chemical remodeling of the mycomembrane with chain-truncated lipids sensitizes mycobacteria to rifampicin.

The outer mycomembrane of Mycobacterium tuberculosis and related pathogens is a robust permeability barrier that protects against antibiotic treatment. Here, we demonstrate that synthetic analogues of the mycomembrane biosynthetic precursor trehalose monomycolate bearing truncated lipid chains increase permeability of Mycobacterium smegmatis cells and sensitize them to treatment with the first-line anti-tubercular drug rifampicin. The reported strategy may be useful for enhancing entry of drugs and other molecules to mycobacterial cells, and represents a new way to study mycomembrane structure and function.

A Far-Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug-Susceptibility Testing.

Increasing the speed, specificity, sensitivity, and accessibility of mycobacteria detection tools are important challenges for tuberculosis (TB) research and diagnosis. In this regard, previously reported fluorogenic trehalose analogues have shown potential, but their green-emitting dyes may limit sensitivity and applications in complex settings. Here, we describe a trehalose-based fluorogenic probe featuring a molecular rotor turn-on fluorophore with bright far-red emission (RMR-Tre). RMR-Tre, which exploits the unique biosynthetic enzymes and environment of the mycobacterial outer membrane to achieve fluorescence activation, enables fast, no-wash, low-background fluorescence detection of live mycobacteria. Aided by the red-shifted molecular rotor fluorophore, RMR-Tre exhibited up to a 100-fold enhancement in M. tuberculosis labeling compared to existing fluorogenic trehalose probes. We show that RMR-Tre reports on M. tuberculosis drug resistance in a facile assay, demonstrating its potential as a TB diagnostic tool.

An essential periplasmic protein coordinates lipid trafficking and is required for asymmetric polar growth in mycobacteria.

Mycobacteria, including the human pathogen Mycobacterium tuberculosis, grow by inserting new cell wall material at their poles. This process and that of division are asymmetric, producing a phenotypically heterogeneous population of cells that respond non-uniformly to stress (Aldridge et al., 2012; Rego et al., 2017). Surprisingly, deletion of a single gene - lamA - leads to more symmetry, and to a population of cells that is more uniformly killed by antibiotics (Rego et al., 2017). How does LamA create asymmetry? Here, using a combination of quantitative time-lapse imaging, bacterial genetics, and lipid profiling, we find that LamA recruits essential proteins involved in cell wall synthesis to one side of the cell - the old pole. One of these proteins, MSMEG_0317, here renamed PgfA, was of unknown function. We show that PgfA is a periplasmic protein that interacts with MmpL3, an essential transporter that flips mycolic acids in the form of trehalose monomycolate (TMM), across the plasma membrane. PgfA interacts with a TMM analog suggesting a direct role in TMM transport. Yet our data point to a broader function as well, as cells with altered PgfA levels have differences in the abundance of other lipids and are differentially reliant on those lipids for survival. Overexpression of PgfA, but not MmpL3, restores growth at the old poles in cells missing lamA. Together, our results suggest that PgfA is a key determinant of polar growth and cell envelope composition in mycobacteria, and that the LamA-mediated recruitment of this protein to one side of the cell is a required step in the establishment of cellular asymmetry.

Chemical probes for tagging mycobacterial lipids.

Mycobacteria, which cause tuberculosis and related diseases, possess a diverse set of complex envelope lipids that provide remarkable tolerance to antibiotics and are major virulence factors that drive pathogenesis. Recently, metabolic labeling and bio-orthogonal chemistry have been harnessed to develop chemical probes for tagging specific lipids in live mycobacteria, enabling a range of new basic and translational research avenues. A toolbox of probes has been developed for labeling mycolic acids and their derivatives, including trehalose-, arabinogalactan-, and protein-linked mycolates, as well as newer probes for labeling phthiocerol dimycocerosates (PDIMs) and potentially other envelope lipids. These lipid-centric tools have yielded fresh insights into mycobacterial growth and host interactions, provided new avenues for drug target discovery and characterization, and inspired innovative diagnostic and therapeutic strategies.

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

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.