Chapter 18
Metabolic Labeling of Live Mycobacteria
with Trehalose-Based Probes
Nicholas Banahene and Benjamin M. Swarts
Abstract
The mycobacterial cell envelope includes a unique outer membrane, also known as the mycomembrane,
which is the major defense barrier that confers intrinsic drug tolerance to Mycobacterium tuberculosis (Mtb)
and related bacteria. The mycomembrane is typified by long-chain mycolic acids that are esterified to
various acceptors, including: (1) trehalose, forming trehalose mono- and di-mycolate; (2) arabinogalactan,
forming arabinogalactan-linked mycolates; and (3) in some species, protein serine residues, forming Omycoloylated proteins. Synthetic trehalose and trehalose monomycolate analogs have been shown to
specifically and metabolically incorporate into mycomembrane components, facilitating their analysis in
native contexts and opening new avenues for the specific detection and therapeutic targeting of mycobacterial pathogens in complex settings. This chapter highlights trehalose-based probes that have been
developed to date, briefly discusses their applications, and describes protocols for their use in mycobacteria
research.
Key words Trehalose, Mycomembrane, Glycolipids, O-mycoloylated proteins, Click chemistry, Tetrazine ligation, Fluorescence imaging
1 Introduction
Compared to canonical Gram-positive and -negative bacteria,
mycobacteria have a distinctive cell envelope highlighted by a
unique outer membrane called the mycomembrane. The dualleaflet mycomembrane consists mainly of long-chain (C 60 –C 90 ),
α-branched, β-hydroxyl fatty acids called mycolic acids. These
mycolic acids are esterified—via a process known as mycoloylation—to various acceptors, including trehalose, forming outerleaflet trehalose mono- and dimycolate (TMM and TDM), and
arabinogalactan, forming inner-leaflet arabinogalactan mycolate
(AGM) [1–4]. In closely related corynebacteria, this process is
also known to occur on protein serine residues, forming O-mycoloylated proteins [5, 6]. Collectively, these mycolate-containing
glycolipids and proteins form a hydrophobic defense barrier that
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_18, © Springer Science+Business Media, LLC, part of Springer Nature 2021
385
Metabolic Labeling of Live Mycobacteria
with Trehalose-Based Probes
Nicholas Banahene and Benjamin M. Swarts
Abstract
The mycobacterial cell envelope includes a unique outer membrane, also known as the mycomembrane,
which is the major defense barrier that confers intrinsic drug tolerance to Mycobacterium tuberculosis (Mtb)
and related bacteria. The mycomembrane is typified by long-chain mycolic acids that are esterified to
various acceptors, including: (1) trehalose, forming trehalose mono- and di-mycolate; (2) arabinogalactan,
forming arabinogalactan-linked mycolates; and (3) in some species, protein serine residues, forming Omycoloylated proteins. Synthetic trehalose and trehalose monomycolate analogs have been shown to
specifically and metabolically incorporate into mycomembrane components, facilitating their analysis in
native contexts and opening new avenues for the specific detection and therapeutic targeting of mycobacterial pathogens in complex settings. This chapter highlights trehalose-based probes that have been
developed to date, briefly discusses their applications, and describes protocols for their use in mycobacteria
research.
Key words Trehalose, Mycomembrane, Glycolipids, O-mycoloylated proteins, Click chemistry, Tetrazine ligation, Fluorescence imaging
1 Introduction
Compared to canonical Gram-positive and -negative bacteria,
mycobacteria have a distinctive cell envelope highlighted by a
unique outer membrane called the mycomembrane. The dualleaflet mycomembrane consists mainly of long-chain (C 60 –C 90 ),
α-branched, β-hydroxyl fatty acids called mycolic acids. These
mycolic acids are esterified—via a process known as mycoloylation—to various acceptors, including trehalose, forming outerleaflet trehalose mono- and dimycolate (TMM and TDM), and
arabinogalactan, forming inner-leaflet arabinogalactan mycolate
(AGM) [1–4]. In closely related corynebacteria, this process is
also known to occur on protein serine residues, forming O-mycoloylated proteins [5, 6]. Collectively, these mycolate-containing
glycolipids and proteins form a hydrophobic defense barrier that
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_18, © Springer Science+Business Media, LLC, part of Springer Nature 2021
385