A chemical reporter strategy for detecting and identifying O-mycoloylated proteins in Corynebacterium

Kavunja, Herbert W.; Piligian, Brent F.; Fiolek, Taylor J; Foley, Hannah N.; Nathan, Temitope O.; Swarts, Benjamin M.

doi:10.1039/c6cc07143k

Cited by 28 publications

(56 citation statements)

References 32 publications

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“…As noted above and depicted in Figure A, TMM reporters have the potential to label both major mycomembrane components, including inner‐leaflet AGM and outer‐leaflet TDM . We previously determined that O‐AlkTMM‐C7 mainly incorporated into the AGM‐containing cellular fraction from Msmeg and less so into the TDM‐containing extractable lipids fraction, which is consistent with AGM being the most abundant mycolate‐containing envelope component . Here, we performed the same cellular fractionation experiments following treatment of Msmeg with 4 – 6 .…”

Section: Resultssupporting

confidence: 63%

“…Cglut encodes six mycoloyltransferases, only one of which, CgMytC, transfers mycoloyl groups from TMM onto polypeptides to generate O‐mycoloylated proteins (Figure A) . Our group previously showed that O‐AlkTMM‐C7 could be used to metabolically label O‐mycoloylated proteins in Cglut with alkyne tags, enabling their subsequent visualization using CuAAC followed by SDS‐PAGE with in‐gel fluorescence analysis . We reasoned that using this experimental platform with a Cglut mutant lacking CgMytC would allow us to assess whether TMM reporters operate in a mycoloyltransferase‐dependent manner, because: 1) CgMytC is not essential; and 2) this system eliminates the issue of mycoloyltransferase redundancy‐again, available evidence suggests that only CgMytC adds O‐mycolates to proteins in Cglut .…”

Section: Resultsmentioning

confidence: 99%

“…O‐AlkTMM‐C7 was also used to assist in the functional characterization of a protein involved in mycomembrane construction, namely the mycobacterial lipid transporter, LprG . In another example, O‐AlkTMM‐C7 was shown to label mycomembrane‐resident O‐mycoloylated proteins in Cglut (via the pathway shown in Figure A), providing a chemical biology approach to the discovery and characterization of this distinctive class of proteins . In the context of studying this post‐translational modification, the ability of O‐AlkTMM‐C7 to exclusively label O‐mycoloylated proteins provides a substantial advantage over using alkynyl fatty acids, which label all types of lipidated proteins in bacteria and thus lack selectivity for O‐mycoloylation .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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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.

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Section: Resultssupporting

confidence: 63%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2019

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“…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) . In addition to hexose monosaccharide reporters, several azido‐pentose reporters have been developed to label specific glycans in the mycobacterial cell wall (Figure H) .…”

Section: Chemical Reporters For Microbiologymentioning

confidence: 99%

Chemical Reporters for Exploring Microbiology and Microbiota Mechanisms

et al. 2019

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The advances made in bioorthogonal chemistry and the development of chemical reporters have afforded new strategies to explore the targets and functions of specific metabolites in biology. These metabolite chemical reporters have been applied to diverse classes of bacteria including Gram‐negative, Gram‐positive, mycobacteria, and more complex microbiota communities. Herein we summarize the development and application of metabolite chemical reporters to study fundamental pathways in bacteria as well as microbiota mechanisms in health and disease.

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“…Recently, we also observed that O-AlkTMM can label and facilitate the identification of proteins modified by mycoloyl groups in C. glutamicum . 105 This form of protein post-translational modification (PTM)—termed protein O -mycoloylation—was recently discovered in Corynebacterineae 106,107 but its scope and functional consequences remain unknown. O-AlkTMM will be a useful tool for identifying and characterizing proteins that contain this PTM and for probing the link between protein lipidation and trehalose metabolism in C. glutamicum and related organisms.…”

Section: Applications Of Trehalose Analoguesmentioning

confidence: 99%

Tailoring trehalose for biomedical and biotechnological applications

O'Neill

Piligian

Olson

et al. 2017

Pure and Applied Chemistry

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Trehalose is a non-reducing sugar whose ability to stabilize biomolecules has brought about its widespread use in biological preservation applications. Trehalose is also an essential metabolite in a number of pathogens, most significantly the global pathogen Mycobacterium tuberculosis, though it is absent in humans and other mammals. Recently, there has been a surge of interest in modifying the structure of trehalose to generate analogues that have applications in biomedical research and biotechnology. Non-degradable trehalose analogues could have a number of advantages as bioprotectants and food additives. Trehalose-based imaging probes and inhibitors are already useful as research tools and may have future value in the diagnosis and treatment of tuberculosis, among other uses. Underlying the advancements made in these areas are novel synthetic methods that facilitate access to and evaluation of trehalose analogues. In this review, we focus on both aspects of the development of this class of molecules. First, we consider the chemical and chemoenzymatic methods that have been used to prepare trehalose analogues and discuss their prospects for synthesis on commercially relevant scales. Second, we describe ongoing efforts to develop and deploy detectable trehalose analogues, trehalose-based inhibitors, and non-digestible trehalose analogues. The current and potential future uses of these compounds are discussed, with an emphasis on their roles in understanding and combatting mycobacterial infection.

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A chemical reporter strategy for detecting and identifying O-mycoloylated proteins in Corynebacterium

Cited by 28 publications

References 32 publications

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

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

Chemical Reporters for Exploring Microbiology and Microbiota Mechanisms

Tailoring trehalose for biomedical and biotechnological applications

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