Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues

• Published in: Chembiochem : a European journal of chemical biology 2019-05, Vol.20 (10), p.1282-1291
• Main Authors: Fiolek, Taylor J., Banahene, Nicholas, Kavunja, Herbert W., Holmes, Nathan J., Rylski, Adrian K., Pohane, Amol Arunrao, Siegrist, M. Sloan, Swarts, Benjamin M.
• Format: Article
• Language: English
• Subjects: Acyltransferases - metabolism; Alkynes; Alkynes - chemical synthesis; Alkynes - chemistry; Alkynes - metabolism; Antigens; Azides - chemical synthesis; Azides - chemistry; Azides - metabolism; Bacillus subtilis - chemistry; bioorthogonal chemistry; Biosynthesis; Carbohydrates; Cell Engineering - methods; Cell Membrane - chemistry; Cell Membrane - metabolism; Cell surface; chemical reporters; Chemical synthesis; Click Chemistry; Cord Factors - chemical synthesis; Cord Factors - chemistry; Cord Factors - metabolism; Corynebacterium - chemistry; Dependence; Drug development; Engineering; Escherichia coli - chemistry; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Fluorescent Dyes - metabolism; imaging; Immunological tolerance; Labeling; Metabolism; Molecular Structure; mycobacteria; Mycobacterium smegmatis - chemistry; Mycobacterium tuberculosis - chemistry; Mycolic acids; Organic chemistry; Permeability; Substrates; Trehalose; Tuberculosis
• ISSN: 1439-4227; 1439-7633; 1439-7633
• DOI: 10.1002/cbic.201800687

Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)‐catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne‐modified TMM analogue (O‐AlkTMM‐C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM‐based reporters bearing alkyne, azide, trans‐cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one‐ or two‐step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell‐surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole‐cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies. Modifying mycobacteria by mycoloylation: A suite of trehalose monomycolate (TMM)‐based metabolic reporters provides versatility and specificity for analyzing and engineering the outer membrane of living mycobacteria. These compounds gave insight into the substrate tolerance of mycoloyltransferases and allow one‐ or two‐step cell labeling, live cell labeling, and rapid cell labeling through tetrazine ligation.