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

Abstract

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.

Metabolic labeling probes for interrogation of the host–pathogen interaction

“…The same group recently expanded the bioorthogonal TMM library to include trans-cyclooctene TMM analogues reactive in an inverse electron-demand Diels-Alder ligation reaction. 106 Recently, various trehalose analogues have emerged as novel potential point-of-care diagnostic tools for Mtb. This is a large unmet clinical need, as the current diagnostic methods are notoriously unreliable.…”

Metabolic Labeling of Bacterial Glycans

“…103 Additional probes, mimicking the mycoloyl donor TMM, were developed by the group of Swarts in order to target mycoloyltransferases in mycobacteria. [104][105][106] In these probes, the complex structure of mycolic acids was replaced by a short linear alkyl chain bearing a bioorthogonal group (terminal alkyne, azide or TCO) or fluorescein at the end of the alkyl chain. This has considerably simplified access to new mycomembrane probes since these probes were easier to synthesize than compounds modified by a bioorthogonal function or fluorophore on trehalose core.…”

Chemical Reporters for Exploring Microbiology and Microbiota Mechanisms

“…For example, trehalose analogues have been developed for visualization of mycobacteria cell division (Figure E), visualization of antibiotic activity on mycobacterial cell wall (Figure C), and rapid detection of live Mycobacterium tuberculosis (Mtb) in sputum using solvatochromic dyes for clinical TB diagnosis (Figure D) . Azide and alkyne reporters of trehalose have also been developed for two‐step labeling of mycobacteria (Figure F) . Moreover, a TMM chemical reporter has been developed for detection and identification of protein O‐mycoloylation in Corynebacterium (Figure G) .…”

“…[59] Azide anda lkyne reporters of trehalose have also been developed for two-step labeling of mycobacteria ( Figure 8F). [60] Moreover,aTMM chemical reporter hasb een developed for detection and identification of protein O-mycoloylation in Corynebacterium ( Figure 8G). [61] In addition to hexosem onosaccharide reporters, severala zido-pentose reporters have been developed to labels pecific glycans in the mycobacterial cell wall ( Figure 8H).…”