A Screen for Protein–Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A

Touchette, Megan H.; Vlack, Erik R. Van; Bai, Lu; Kim, Jia; Cognetta, Armand B.; Previti, Mary Lou; Backus, Keriann M.; Martin, Dwight W.; Cravatt, Benjamin F.; Seeliger, Jessica C.

doi:10.1021/acsinfecdis.6b00179

Cited by 25 publications

(27 citation statements)

References 51 publications

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“…Compounds that inhibit proper mycolyltransferase localization could be efficacious, as deletion of a single mycolyltransferase can profoundly affect cell viability (44). Moreover, mycolyltransferases can engage in protein-protein interactions, as Msmeg_6398 has been found to interact with LprG, a lipid transport protein; this interaction could confine the enzyme within the cell envelope (45). Alternatively, since mycolyltransferases are secreted, their mechanism of export could influence their localization.…”

Section: Discussionmentioning

confidence: 99%

Imaging mycobacterial growth and division with a fluorogenic probe

Hodges

Brown

Crooks

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

105

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Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.

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Section: Discussionmentioning

confidence: 99%

Imaging mycobacterial growth and division with a fluorogenic probe

Hodges

Brown

Crooks

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

105

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“…For instance, O‐AlkTMM‐C7 allowed the first direct visualization of AGM biosynthesis during cell growth and division in M. smegmatis ( Msmeg ) . 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 .…”

Section: Introductionmentioning

confidence: 99%

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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“…This pointed at a metabolic defect in the deletion mutant being responsible for decreased virulence in mice and further lent support to the operon’s role in the transport of triacylated lipids. In a very recent study, LprG was found to interact with various periplasmic proteins, including mycolyltransferase Ag85A, which catalyzes the formation of TDM in the cell wall (Touchette et al , ).…”

Section: Introductionmentioning

confidence: 99%

Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG

Höhl

Remm

Eskandarian

et al. 2019

Molecular Microbiology

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Summary The major facilitator superfamily transporter Rv1410 and the lipoprotein LprG (Rv1411) are encoded by a conserved two‐gene operon and contribute to virulence in Mycobacterium tuberculosis. Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of Mycobacterium smegmatis lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in Lactococcus lactis did not confer drug resistance, deletion of the operon in Mycobacterium abscessus and M. smegmatis resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon’s function. Interestingly, influx of the fluorescent dyes BCECF‐AM and calcein‐AM in de‐energized M. smegmatis cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.

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A Screen for Protein–Protein Interactions in Live Mycobacteria Reveals a Functional Link between the Virulence-Associated Lipid Transporter LprG and the Mycolyltransferase Antigen 85A

Cited by 25 publications

References 51 publications

Imaging mycobacterial growth and division with a fluorogenic probe

Imaging mycobacterial growth and division with a fluorogenic probe

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

Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG

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