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Review
. 2022 Feb 9;122(3):3336-3413.
doi: 10.1021/acs.chemrev.1c00729. Epub 2021 Dec 14.

Chemical Reporters for Bacterial Glycans: Development and Applications

Nicholas Banahene  1   2 , Herbert W Kavunja  1   2 , Benjamin M Swarts  1   2
Affiliations
Review

Chemical Reporters for Bacterial Glycans: Development and Applications

Nicholas Banahene et al. Chem Rev. .

Abstract

Bacteria possess an extraordinary repertoire of cell envelope glycans that have critical physiological functions. Pathogenic bacteria have glycans that are essential for growth and virulence but are absent from humans, making them high-priority targets for antibiotic, vaccine, and diagnostic development. The advent of metabolic labeling with bioorthogonal chemical reporters and small-molecule fluorescent reporters has enabled the investigation and targeting of specific bacterial glycans in their native environments. These tools have opened the door to imaging glycan dynamics, assaying and inhibiting glycan biosynthesis, profiling glycoproteins and glycan-binding proteins, and targeting pathogens with diagnostic and therapeutic payload. These capabilities have been wielded in diverse commensal and pathogenic Gram-positive, Gram-negative, and mycobacterial species─including within live host organisms. Here, we review the development and applications of chemical reporters for bacterial glycans, including peptidoglycan, lipopolysaccharide, glycoproteins, teichoic acids, and capsular polysaccharides, as well as mycobacterial glycans, including trehalose glycolipids and arabinan-containing glycoconjugates. We cover in detail how bacteria-targeting chemical reporters are designed, synthesized, and evaluated, how they operate from a mechanistic standpoint, and how this information informs their judicious and innovative application. We also provide a perspective on the current state and future directions of the field, underscoring the need for interdisciplinary teams to create novel tools and extend existing tools to support fundamental and translational research on bacterial glycans.

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Figures

Figure 1.
Figure 1.
Cell envelope architectures and major glycans of common types of bacteria: (A) Gram-positive bacteria; (B) Gram-negative bacteria; (C) mycobacteria. AG, arabinogalactan; CL, capsular layer; LAM, lipoarabinomannan; LM, lipomannan; LPS, lipopolysaccharide; MM, mycomembrane; OM, outer membrane; PG, peptidoglycan; PIM, phosphatidylinositol; PM, plasma membrane.
Figure 2.
Figure 2.
Overview of the bioorthogonal chemical reporter strategy as applied to studying bacterial glycans and workflow for the development of chemical reporters. The chemical reporter (shown as a hexose ring with a bioorthogonal functional group X) is fed to a living bacterial cell and metabolically incorporated into a glycan of interest. Next, the X-labeled glycan is reacted with an exogenously delivered reagent (e.g., a fluorophore) containing a complementary bioorthogonal functional group Y. A highly selective bioorthogonal reaction takes place on the cell surface between X and Y, leading to covalent ligation of the delivered cargo to the glycan of interest, enabling its analysis or modulation. See Figure 3 for common bioorthogonal reactions. In the one-step metabolic incorporation approach, X is typically a fluorophore and does not require a subsequent bioorthogonal reaction. The development of chemical reporters involves three stages (highlighted in blue text): (1) the reporter molecule must be designed and synthesized after selection of the target glycan and analysis of its structure and biosynthesis; (2) next, the reporter must be tested in bacterial cells to determine whether it successfully labels the glycan of interest and to elucidate the pathway of incorporation; (3) once the reporter’s behavior in cells is established, it can potentially be used for a variety of applications, ranging from live-cell imaging of glycan biosynthesis and dynamics to targeting the bacterium with therapeutic or diagnostic cargo within a host.
Figure 3.
Figure 3.
Commonly used bioorthogonal reactions.
Figure 4.
Figure 4.
Representative PG structure from Gram-negative bacteria. PG is also present in Gram-positive bacteria and mycobacteria.
Figure 5.
Figure 5.
Representative biosynthesis of PG in Gram-negative bacteria.
Figure 6.
Figure 6.
PG precursor derivatives designed to label the PG stem peptide based on (A) Park’s nucleotide and (B) Lipid I and II. (C) Convergent chemoenzymatic synthesis of PG precursor derivatives 1 and 2. AAs, amino acid building blocks; DIEA, N,N-diisopropylethylamine; DMF, N,N-dimethylformamide; HBTU, (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate; NHS, N-hydroxysuccinimidyl; NADPH, nicotinamide adenine dinucleotide phosphate hydride; PEP, phosphoenolpyruvate; UDP, uridine diphosphate.
Figure 7.
Figure 7.
D-amino acid derivatives targeting the PG stem peptide. (A) Metabolic incorporation of unnatural D-amino acid derivatives (modified with an unnatural R group) is proposed to predominantly occur through periplasmic TPase-mediated PG remodeling. Incorporation by PBP D,D-TPases to install the probe at the 5-position of the peptide is shown; incorporation by L,D-TPases to install the probe at the 4-position can also occur. Smaller D-amino acid derivatives may additionally incorporate into PG via an intracellular route. (B and C) Structures of D-amino acid derivatives bearing selectively reactive functional groups (B) and fluorophores (C).
Figure 8.
Figure 8.
Two-step chemical synthesis of D-amino acid derivatives. Boc, tert-butoxycarbonyl; LG, leaving group; TFA, trifluoroacetic acid.
Figure 9.
Figure 9.
Examples of in vitro and in vivo D-amino acid reporter labeling of PG. (A) Metabolic incorporation of HADA (17) into E. coli, B. subtilis, and A. tumefaciens. Bacteria were incubated in HADA, fixed, and imaged directly (top panel) or subjected to sacculus isolation (i.e., intact PG isolation) and then imaged (bottom panel). WGA595 is a red-fluorescent lectin that binds to PG glycan strands. Reproduced with permission from ref . Copyright 2012 Wiley-VCH. (B) Metabolic incorporation of AlkDA (10) into E. coli (Ec), L. monocytogenes (Lm), C. glutamicum (Cg), and M. tuberculosis (Mt). Bacteria were incubated in either D-Ala (control, top panel) or AlkDA (bottom panel), fixed, subjected to CuAAC with azido-488, and imaged. (C) Imaging of L. monocytogenes-infected macrophages with AlkDA (10). Macrophages were infected with red fluorescent protein (RFP)-expressing L. monocytogenes and extracellular bacteria were removed, then incubated in AlkDA (10), fixed, subjected to CuAAC with azido-488, and imaged. (B) and (C) were reproduced with permission from ref (https://pubs.acs.org/doi/full/10.1021/ja505668f). Copyright 2013 American Chemical Society. Further permission related to the material excerpted should be directed to the American Chemical Society. (D) Sequential labeling of S. venezuelae with variable-color fluorescent D-amino acid derivatives. Bacteria were sequentially incubated for short pulses with different dye-conjugated D-amino acids possessing orthogonal excitation/emission wavelengths (structures not shown in Figure 7), fixed, and imaged. Arrows indicate new branching sites of the cell wall. Reproduced from ref with permission from the Royal Society of Chemistry. (E) Ex vivo and in vivo labeling of S. aureus PG in host C. elegans using the tetrazine reagent TetDAC (25, Figure 10A). C. elegans was infected with green fluorescent protein (GFP)-expressing S. aureus that had been (i) pre-treated with TetDAC (25) for ex vivo labeling or (ii) not pre-treated for in vivo labeling. C. elegans was washed to remove uncolonized bacteria, then (i) reacted with TCO-Cy5 fluorophore and imaged for ex vivo labeling or (ii) treated with TetDAC (25), reacted with TCO-Cy5 fluorophore, and imaged for in vivo labeling. Reproduced with permission from ref . Copyright 2017 American Chemical Society.
Figure 10.
Figure 10.
D-amino carboxamide derivatives for PG labeling. (A) Tetrazine ligation-capable reporters bearing strained norbornene (NBDAC, 24) and tetrazine (TetDAC, 25) groups on the D-amino carboxamide scaffold. (B) Fluorescent D-amino carboxamide FDL-NH2 (27) labels B. subtilis more efficiently than D-amino acid FDL (26). B. subtilis was cultured in the probes and imaged. Reproduced with permission from ref (https://pubs.acs.org/doi/full/10.1021/ja505668f). Copyright 2014 American Chemical Society. Further permission related to the material excerpted should be directed to the American Chemical Society.
Figure 11.
Figure 11.
D-Ala-D-Ala dipeptide and L-Ala-D-iGlu-L-Lys tripeptide derivatives target the stem peptide of nascent PG via cytoplasmic routes of incorporation. (A) Metabolic incorporation of exogenous D-amino acid dipeptides (modified with an unnatural R group) is proposed to be initiated by MurF in the cytoplasm. Metabolic incorporation of exogenous tripeptide derivatives (modified with an NBD fluorophore) is proposed to occur through a recycling pathway in which tripeptides are taken up and ligated to UDP-MurNAc by Mpl, followed by MurF-catalyzed coupling with D-Ala-D-Ala and downstream incorporation into PG as shown in Figure 5. (B) Structures of D-amino acid dipeptide derivatives bearing bioorthogonal and photo-cross-linking functional groups.
Figure 12.
Figure 12.
Imaging and proteomics applications of D-amino acid dipeptide reporters for PG. (A) First direct detection of PG in Chlamydia. Mouse fibroblasts were infected with C. trachomatis in the presence of AlkDADA (28), fixed, subjected to CuAAC with azido-488, and imaged. Blue, DAPI nuclear staining of host cells; green, C. trachomatis PG labeling; red, C. trachomatis major outer membrane protein (MOMP) staining. Reproduced with permission from ref . Copyright 2014 Springer Nature. (B) Mycobacterial sidewall synthesis of PG in response to cell wall damage. M. smegmatis was incubated in the absence (−) or presence (+) of PG-damaging enzymes lysozyme (lys) and mutanolysin (mut), then treated with AlkDADA (28), fixed, subjected to CuAAC with azido-488, and imaged. While polar labeling is predominant in the -lys/mut condition, enhanced peripheral labeling is visually evident in the +lys/mut condition, and was quantified in ref . Reproduced from ref . (C) Identification of Lipid II-interacting proteins in B. subtilis. Bacteria were incubated in photo-cross-linking probe x-DADA-Alk (32), exposed to UV irradiation, subjected to CuAAC with azido-biotin, and then biotinylated proteins were either detected by Western blot (left) or avidin-enriched, trypsinized, and analyzed by LC-MS/MS (right). Several PBPs and other PG-related proteins were identified. Reproduced with permission from ref . Copyright 2016 Wiley-VCH.
Figure 13.
Figure 13.
Fluorescent reporter probes for TPase-mediated PG cross-linking. (A) Fluorescent stem pentapeptide mimics resembling the donor strand (FSPPM) are proposed to incorporate via D,D-TPase activity as shown for S. aureus PG; FSPPMs could theoretically also incorporate via L,D-TPase activity if the terminal D-Ala were cleaved first by a CPase. (B) Fluorescent stem tetrapeptide mimics resembling the donor strand (FSTPM), which lack the terminal D-Ala of FSPPMs, are proposed to incorporate exclusively via L,D-TPase activity.
Figure 14.
Figure 14.
(A) Biosynthesis of UDP-GlcNAc from fructose-6-phosphate proceeds via a GlcNAc-1-phosphate intermediate in the cytoplasm (note: GlmU is bifunctional). Subsequent incorporation of UDP-GlcNAc into PG is shown in Figure 5. (B) GlcNAc-1-P analogues as potential chemical reporters for the PG glycan core.
Figure 15.
Figure 15.
(A) Cytoplasmic recycling pathway for MurNAc in P. putida. Since this pathway is not present in many bacteria, it must be introduced via genetic engineering in those cases. (B) Representative MurNAc derivatives for labeling the PG glycan core, modified at the N-acyl group (B) and the D-Lac group (C). (D) Chemical synthesis of N-acyl-modified MurNAc derivatives 41–46 and chemoenzymatic synthesis of their corresponding sugar phosphate and UDP sugar derivatives (box).
Figure 16.
Figure 16.
Imaging the PG glycan backbone using MurNAc reporters. (A) E. coli ΔMurQ-KU was incubated in the presence of alkynyl MurNAc 42 and fosfomycin, fixed, subjected to CuAAC with azido-Cy5, and imaged by structured illuminated microscopy (SIM). Left, differential interference contrast (DIC); middle, SIM image; right, boxed area from middle image. (B) E. coli ΔMurQ-KU treated as in (A) and imaged by super-resolution 3D stochastic optical reconstruction microscopy (STORM). Red triangle indicates septal division plane. (C) E. coli ΔMurQ-KU were pre-treated with 42 and fosfomycin, then used to invade J774 macrophages. After fixation, CuAAC with azido-488, and nuclear staining with DAPI, invaded macrophages were imaged. White triangles indicate fluorescent fragments released from intracellular bacteria. Images in (A–C) are reproduced from ref . (D) H. pylori HJH1 was incubated in the presence of alkynyl MurNAc 42 for a short pulse, fixed, subjected to CuAAC with azido-Alexa Fluor 555, counterstained with the fluorescent lectin WGA-Alexa Fluor 488, and imaged by 3D SIM. Image in (D) reproduced from ref .
Figure 17.
Figure 17.
Representative structure of LPS from Gram-negative E. coli. Hep, heptose; Hex, hexose; KDO, keto-deoxyoctulosonate.
Figure 18.
Figure 18.
Representative biosynthesis of LPS in Gram-negative E. coli.
Figure 19.
Figure 19.
KDO-based chemical reporters. (A) Chemical synthesis and structure of KDO-N3 (55). Ts, tosyl. (B) Structures of alkyne- and endocyclic nitrone-modified KDO analogues (56 and 57).
Figure 20.
Figure 20.
Imaging the LPS core region using KDO reporters. (A) E. coli K12 was incubated in the presence of KDO-N3 (55), fixed, subjected to CuAAC with azido-488, and imaged by fluorescence microscopy. Right, expanded deconvoluted image of labeled E. coli cell. Scale bars, 1 μm. Reproduced with permission from ref . Copyright 2012 Wiley-VCH. (B) E. coli was incubated in the presence of KDO-N3 (55) and subjected to SPAAC with a cyclooctyne-488 reagent. LPS was extracted and separated by SDS-PAGE and visualized by fluorescence imaging (top) or by LPS staining with Pro-Q Emerald 300 LPS staining kit (bottom). Reproduced with permission from ref . Copyright 2017 American Society for Biochemistry and Molecular Biology. (C) Mouse gut microbiotas were labeled with KDO-N3 (55, shown in red text as 8AzKdo), subjected to CuAAC with alkyne-TAMRA, labeled with fluorophore-conjugated vancomycin (shown in green text as Vanco), and imaged by confocal fluorescence microscopy. Fluorescence, DIC, and merged images are shown. Scale bar, 20 μm. Reprinted with permission from ref . Copyright 2017 American Chemical Society.
Figure 21.
Figure 21.
Fucose-based chemical reporters for metabolic labeling of E. coli O86 LPS O-antigen. (A) Structure of the E. coli O86 LPS O-antigen. (B) Fucose salvage pathway from Bacteroides engineered into E. coli O86. The bifunctional enzyme Fkp possesses both fucose kinase and GDP fucose pyrophosphorylase activity. Subsequent polymerization of the O-antigen subunit and assembly into mature LPS is shown in Figure 18. (B) Representative fucose analogues as chemical reporters for the E. coli O86 LPS O-antigen.
Figure 22.
Figure 22.
MandiNAc-based chemical reporters for labeling L. pneumophila LPS O-antigen. (A) Structure of the L. pneumophila LPS O-antigen. (B) Biosynthesis of O-antigen precursor CMP-Leg5Ac7Ac, which arises from a MandiNAc intermediate. Subsequent polymerization of the O-antigen subunit and assembly into mature LPS is shown in Figure 18. (C) Structures and (D) chemical synthesis of azide-modified MandiNAc analogues as chemical reporters for the L. pneumophila LPS O-antigen. Bz, benzoyl; CAN, ceric ammonium nitrate; Ms, mesyl; PMP, para-methoxyphenyl.
Figure 23.
Figure 23.
AltdiNAc-based chemical reporters for labeling pseudaminic acid residues of LPS O-antigen. (A) Structure of a pseudaminic acid-containing LPS O-antigen from P. aeruginosa. FucNAc, N-acetylfucosamine; Pse5Ac7Fo, pseudaminic acid derivative with 5-acetamido and 7-formamido groups; Xyl, xylose. (B) Biosynthesis of O-antigen precursor CMP-Pse, which arises from an AltdiNAc intermediate. Subsequent polymerization of the O-antigen subunit and assembly into mature LPS is shown in Figure 18. (C) Structures of azide-modified AltdiNAc chemical reporters.
Figure 24.
Figure 24.
Sialic acid-based chemical reporters for labeling LOS in H. influenzae.
Figure 25.
Figure 25.
Representative structures of bacterial glycoproteins. A Gram-negative cell envelope is shown, although glycoproteins exist in Gram-positive bacteria and mycobacteria as well.
Figure 26.
Figure 26.
General scheme for the biosynthesis of N- and O-linked bacterial glycoproteins via OTase-dependent or independent pathways.
Figure 27.
Figure 27.
(A) Chemoenzymatic synthesis of AltNAc4NAz (75), a chemical reporter for pseudaminic acid-containing flagellin glycoprotein in C. jejuni. (B) Analysis of AltNAc4NAz-labeled flagellin from C. jejuni by Western blot. Wild-type or PseG-deficient C. jejuni were grown on agar containing 75, reacted with phosphine-biotin, lysed, and probed by streptavidin-horseradish peroxidase (HRP) or anti-flagellin Western blot. For streptavidin-HRP blot: lane 1, C. jejuni ΔpseG + 75; lane 2, C. jejuni ΔpseG no probe; lane 3, C. jejuni wild type no probe; lane 4, C. jejuni wild type + 75. For anti-flagellin blot: lane 1, C. jejuni ΔpseG + 75; lane 2, C. jejuni ΔpseG no probe; lane 3, C. jejuni wild type no probe. Reprinted with permission from ref . Copyright 2009 Wiley-VCH.
Figure 28.
Figure 28.
(A) MandiNAc-based chemical reporters for labeling legionaminic acid residues of C. jejuni flagellin glycoproteins. See Figure 22B for a relevant biosynthetic pathway and the structure of MandiNAc. (B) Imaging of C. jejuni flagellin glycans using compound 83. C. jejuni was incubated in the presence of 83, fixed, subjected to SPAAC with cyclooctyne-PEG-biotin, stained with streptavidin-488, and imaged by fluorescence microscopy. A secondary dye (red) stained the body of the bacterium. Scale bar, 1 μm. Reproduced from ref .
Figure 29.
Figure 29.
Chemical reporters for labeling bacterial glycoproteins. (A) GlcNAc-based reporters developed for labeling glycoproteins in H. pylori. (B) Rare bacterial sugar-based reporters developed for labeling glycoproteins in various species.
Figure 30.
Figure 30.
Representative structures of wall teichoic acids and lipoteichoic acids in Gram-positive bacteria.
Figure 31.
Figure 31.
Common pathway of phosphocholine tailoring of wall teichoic acids and lipoteichoic acids in S. pneumoniae. See Figure 30 for the structure of the teichoic acid repeating unit (biosynthesis of the repeating unit is not shown here).
Figure 32.
Figure 32.
(A) Choline derivatives for labeling phosphocholine-modified teichoic acids in S. pneumoniae. (B) Imaging of S. pneumoniae teichoic acids with propargyl-Cho (90). S. pneumoniae at different growth phases (i–iii represent early to late growth stages) was incubated in the presence of propargyl-Cho (90) for a 30 min pulse, subjected to CuAAC with azido-coumarin, and imaged by fluorescence microscopy. Left, phase contrast; right, fluorescence. White triangles mark the septal plane. Scale bars, 1 μm. Reproduced with permission from ref . Copyright 2017 Royal Society of Chemistry. (C) Simultaneous imaging of S. pneumoniae teichoic acids and PG with propargyl-Cho (90) and HADA (17), respectively. S. pneumoniae was incubated in the presence of propargyl-Cho (80), cyclooctyne-594, and HADA (17) for a 5 min pulse, washed, and imaged by fluorescence microscopy. Top (red), teichoic acids; middle, phase contrast; bottom (blue), PG. E, elongation site; D, division site; N, new pole; O, old pole. Right-hand boxes show fluorescence intensity as a function of cell length. Reproduced with permission from ref . Copyright 2018 American Chemical Society.
Figure 33.
Figure 33.
Representative structures of the mycolate-containing glycolipids AGM, TMM, and TDM present in the outer mycomembrane of mycobacteria. AGM, arabinogalactan-linked mycolates; TDM, trehalose dimycolate; TMM, trehalose monomycolate.
Figure 34.
Figure 34.
Trehalose-mediated biosynthesis of mycolate-containing glycolipids in mycobacteria. Figure adapted from ref with permission from the Royal Society of Chemistry.
Figure 35.
Figure 35.
Trehalose derivatives for labeling trehalose mycolate glycolipids in mycobacteria. (A) Metabolic incorporation of unnatural trehalose derivatives (modified with an unnatural R group) is proposed to occur through mycoloyltransferase-catalyzed mycoloyl group transfer. Periplasmic Ag85-catalyzed transfer of mycoloyl group from native TMM to exogenous trehalose derivative is shown. A similar mechanism would occur for cytoplasmic Pks13-mediated mycoloyl group transfer onto intracellular trehalose derivatives. Depending on the position of the unnatural modification, mycoloyl groups could add to one 6-position to give a labeled TMM or both 6-positions to give a labeled TDM. Enz, enzyme with mycoloyltransferase activity (e.g., Ag85). (B–D) Structures of some published trehalose derivatives bearing fluorophores (B), bioorthogonal functional groups (C), and fluorine modifications (D).
Figure 36.
Figure 36.
Labeling and imaging trehalose mycolates in mycobacteria using fluorescent trehalose derivatives. (A) Synthesis of FITC-Tre (93). Cbz, carbobenzyloxy; TMSOTf, trimethylsilyl triflate. (B) M. tuberculosis expressing red fluorescent protein (RFP) was incubated in the presence of 100 μM FITC-Tre (93), fixed, and imaged by fluorescence microscopy. Left, FITC channel; right, merge of FITC and RFP channels. (C) J774 macrophages were infected with M. tuberculosis, treated with 200 μM FITC-Tre (93), fixed, and imaged by fluorescence microscopy. Scale bars, 5 μm. Images in (B) and (C) were reproduced with permission from ref . Copyright 2011 Springer Nature. (D) M. smegmatis was incubated for varying durations with 100 μM 6-TMR-Tre, fixed, and imaged by structured illumination microscopy. Scale bars, 2 μm. Reproduced with permission from ref . Copyright 2018 Wiley-VCH. (E) M. smegmatis (Ms) and C. glutamicum (Cg) were incubated with 100 μM fluorogenic DMN-Tre (98) or 100 μM non-fluorogenic 6-FlTre (97) and directly imaged by fluorescence microscopy without washing. Scale bars, 5 μm. Reproduced with permission from ref . Copyright 2018 The American Association for the Advancement of Science.
Figure 37.
Figure 37.
Labeling trehalose mycolates in mycobacteria using azido trehalose (TreAz) derivatives. (A) M. smegmatis (Msmeg) wild type, ΔsugC mutant lacking the trehalose transporter, or ΔsugC::sugC complement with transporter restored were incubated in the presence of 25 μM 6-TreAz (102), fixed, reacted with alkyne-488 via CuAAC, and imaged by fluorescence microscopy. Top, 488 channel; bottom, brightfield channel. Scale bars, 5 μm. Reproduced with permission from ref . Copyright 2014 Wiley-VCH. (B) M. smegmatis spheroplasts were incubated with 100 μM 6-TreAz (102) in the absence (top) or presence (bottom) of MmpL3 inhibitor BM212, reacted with cyclooctyne-biotin via SPAAC, stained with 488-streptavidin, and imaged by fluorescence microscopy. Scale bars, 3 μm. Reproduced from ref . (C) TreT-catalyzed one-step chemoenzymatic synthesis and all-aqueous purification of trehalose derivatives. Various unnatural acceptor substrates can be converted into products, with Y representing azido-, deoxy-, fluoro-, or stereochemical modifications, and X representing oxygen or sulfur atoms. Figure adapted from ref with permission from the Royal Society of Chemistry.
Figure 38.
Figure 38.
O- and N-linked TMM derivatives for selective labeling of mycolate-containing components of the mycomembrane. (A) O-linked TMM derivatives (modified with an unnatural R group) mimic the mycoloyl donor function of TMM and are proposed to undergo periplasmic mycoloyltransferase-catalyzed transfer of their unnatural acyl chain onto mycoloyl acceptors, producing labeled TDM, AGM, proteins, and potentially other mycolate-containing products. (B) N-linked TMM derivatives (modified with an unnatural R group) mimic the mycoloyl acceptor function of TMM and are proposed to undergo periplasmic mycoloyltransferase-catalyzed mycoloylation of their free 6-position hydroxyl group, producing only a labeled version of TDM. Enz, enzyme with mycoloyltransferase activity (e.g., Ag85). (C and D) Structures of published O-linked (C) and N-linked (D) TMM derivatives bearing bioorthogonal functional groups, fluorophores, and photo-cross-linking tags.
Figure 39.
Figure 39.
Labeling and imaging mycolates in mycobacteria using TMM derivatives. (A) General scheme for the synthesis of O-linked TMM derivatives. DCC, N,N’-dicyclohexylcarbodiimide; DMAP, 4-dimethylaminopyridine; TMS, trimethylsilyl. (B) Different species of bacteria were incubated in the presence of 50 μM O-AlkTMM-C7 (113) or left untreated, fixed, reacted with azido-488 via CuAAC, and imaged by fluorescence microscopy. For each condition, left is the transmitted light channel and right is the 488 channel. Scale bars, 5 μm. (C) M. smegmatis was incubated in the presence of 20 μM O-TCO-TMM (117) or left untreated, fixed, reacted with tetrazine-Cy3 for 1 min and imaged by fluorescence microscopy. Left, transmitted light channel; right, Cy3 channel. Scale bars, 5 μm. Reproduced with permission from ref . Copyright 2019 Wiley-VCH. (D) M. smegmatis was incubated in the presence of 50 μM O-AlkTMM-C7 (113) for varying durations, fixed, reacted with azido-488 via CuAAC, and imaged by fluorescence microscopy. Arrows indicate sites of septal mycomembrane synthesis in dividing cells. Scale bars, 5 μm. (B) and (D) reproduced with permission from ref . Copyright 2016 Wiley-VCH. (E) M. smegmatis was incubated in the presence of 50 μM O-AlkTMM-C7 (113) in 2% (carbon-rich) or 0.02% (carbon-depleted) glucose-supplemented medium, fixed, reacted with azido-488 via CuAAC, and imaged by fluorescence microscopy. Scale bars, 5 μm. Reproduced with permission from ref . Copyright 2021 American Society for Microbiology. (F) C. glutamicum was incubated in the presence of 50 μM O-DBF-TMM (120), fixed, photo-oxidized in the presence of diaminobenzidine, stained with osmium tetroxide, and imaged by transmission electron microscopy. The stained mycomembrane (MM) and peptidoglycan layer (PGL) are indicated. Reproduced with permission from ref . Copyright 2019 Springer Nature.
Figure 40.
Figure 40.
Imaging of mycoloyltransferase hydrolytic activity in mycobacteria using FRET-based fluorogenic QTF. (A) Upon hydrolysis by mycoloyltransferases (e.g., Ag85), the ester bond of QTF (125) is cleaved, releasing products 126 and 127, the former of which is fluorescent. (B) A mixed culture of mCherry-expressing C. glutamicum (red), B. subtilis, and E. coli was incubated in the presence of 1 μM QTF, fixed, and imaged by confocal fluorescence microscopy. An overlay of all channels is shown. Scale bar, 5 μm. Reproduced from ref .
Figure 41.
Figure 41.
TMM derivatives enable labeling of proteins that (A) are covalently modified by or (B) non-covalently interact with mycolates. (A) Labeling of O-mycoloylated proteins in C. glutamicum with O-AlkTMM-C7. C. glutamicum was incubated in 100 μM O-AlkTMM-C7 (113), then chloroform-methanol protein extracts were obtained, subjected to CuAAC with azido-488, subjected to NaOH treatment to cleave ester linkages, and analyzed by SDS-PAGE. Bands A–F represent proteins identified by Coomassie staining; all but band B were labeled by O-AlkTMM-C7 and bands A, C, D, and E were confirmed by MALDI-MS to be O-mycoloylated. The asterisk (*) marks background fluorescence signal. Reproduced from ref. with permission from the Royal Society of Chemistry. (B) Enrichment of mycomembrane proteins in M. smegmatis using photo-activatable N-x-AlkTMM-C15. M. smegmatis was incubated in 100 μM N-x-AlkTMM-C15 (122) and exposed to UV irradiation, then lysates were collected, subjected to CuAAC with azido-TAMRA-biotin, incubated with avidin beads, trypsinized, and analyzed by label-free quantitative LC-MS/MS. Volcano plot shows proteins in red that were significantly ≥4-fold enriched in 122-treated, UV-exposed versus non-UV-exposed bacteria. Selected proteins of interest are indicated. Reproduced with permission from ref. . Copyright 2020 American Chemical Society.
Figure 42.
Figure 42.
Analysis of Tdmh activity in mycobacteria using fluorogenic FRET-TDM. (A) Upon hydrolysis by Tdmh, an ester bond of FRET-TDM (128) is cleaved. Either ester bond of FRET-TDM can be cleaved by Tdmh; the possibility in which products 119 and 129 are generated is shown. (B) Recombinant purified wild-type Tdmh or its active site mutant S124A were resolved by native PAGE on two separate gels, one of which was Coomassie-stained (left) and the other was treated with 10 μM FRET-TDM (128) and scanned for 488 fluorescence (right). Reproduced with permission from ref. (https://pubs.acs.org/doi/10.1021/acsomega.9b00130). Copyright 2019 American Chemical Society. Further permission related to the material excerpted should be directed to the American Chemical Society.
Figure 43.
Figure 43.
Simplified structure of mycobacterial lipoarabinomannan (LAM). LAM is an arabinan-containing extension of lipomannan (LM), which itself is a heavily mannosylated extension of a phosphatidylinositolmannoside (PIM) core.
Figure 44.
Figure 44.
Biosynthesis of arabinan domains of AG, LAM, and AM in mycobacteria. AG, arabinogalactan; AraTs, arabinosyltransferases; AM, arabinomannan; DPA, decaprenyl-phospho-D-arabinofuranose; DPPR, decaprenylphosphoryl-5-phosphoribose; G6P, glucose-6-phosphate; LAM, lipoarabinomannan; PRPP, phosphoribosyl diphosphate.
Figure 45.
Figure 45.
(A) Structures of azido pentose derivatives for labeling M. tuberculosis. (B) M. tuberculosis expressing mCherry was incubated in the presence of 5 mM 5-AraAz (132), reacted with cyclooctyne-488 via SPAAC, fixed, and imaged by fluorescence microscopy. The control was not treated with 5-AraAz or cyclooctyne-488. Scale bars, 5 μm. Reproduced with permission from ref . Copyright 2017 Wiley-VCH.
Figure 46.
Figure 46.
Labeling and imaging arabinans in mycobacteria and corynebacteria using DPA derivatives. (A) Structure of FPA, a derivative of DPA with a truncated lipid. (B) Restoration of AG synthesis in a DPA-deficient C. glutamicum mutant using FPA. C. glutamicum ΔubiA was incubated in 500 μM FPA (133) or left untreated and visualized using transmission electron microscopy. Left, untreated; right, FPA-treated. CM, cytoplasmic membrane; PS, polysaccharide layer. Scale bars, 100 nm. Reproduced with permission from ref . Copyright 2019 American Chemical Society. (C) Structures of azide-modified DPA derivatives. (D) Imaging of arabinans using 5-AzFPA. C. glutamicum was incubated in 250 μM 5-AzFPA (136) and 500 μM HADA (17) for 2 h, fixed, and analyzed by fluorescence microscopy. Scale bars, 3 μm. Reprinted from ref with permission. Copyright 2021 American Chemical Society. (E) Representative synthesis of an AzFPA derivative. The late stage of the synthesis of 5-AzFPA (136) is shown. All, allyl; TBDPS, tert-butyldiphenylsilyl.
Figure 47.
Figure 47.
Representative structures of bacterial capsular polysaccharides and exopolysaccharides. Gram-negative cell envelope schematic is shown, although capsular polysaccharides and exopolysaccharides exist in Gram-positive bacteria and mycobacteria as well.
Figure 48.
Figure 48.
Labeling and imaging capsular polysaccharides in gut commensal bacteria using GalNAc derivatives. (A) Structures of monosaccharide reporters Ac4GalNAz (139), Ac4ManNAz (140), and GalNAc-CCP (141). (B) B. fragilis was incubated in Ac4GalNAz (139), subjected to SPAAC with cyclooctyne-488, and imaged by confocal microscopy. Left, confocal image of labeled bacteria. Scale bar, 5 μm. Right, 3D-rendered confocal image of Ac4GalNAz-labeled bacteria counterstained with propidium iodide. Scale bar, 1 μm. (C) B. fragilis wild type strain (WT), PS A1 constitutive expression strain (ΔmpiM44), and PS A1-deficient strain (ΔPSA) were incubated in Ac4GalNAz (139), subjected to SPAAC with cyclooctyne-647, inactivated, and analysed by SDS-PAGE with fluorescence scanning or blotting with anti-PS A1 antibody. ctrl, control; F, fluorescent; MW, molecular weight; NF, non-fluorescent. (D) B. fragilis pre-labeled with Ac4GalNAz (139) and cyclooctyne-Cy7 was introduced to mice and whole-body longitudinal imaging (top) and imaging of dissected intestines (bottom) were performed. ROI, region of interest. (B–D) were reproduced with permission from ref . Copyright 2015 Springer Nature. (E) B. vulgaris was labeled with coumarin probe HADA (17), KDO-N3 (55), and GalNAc-CCP (141), then reacted with cyclooctyne-rhodamine (to label azides) and tetrazine-TAMRA (to label cyclopropenes). Triple fluorescent-labeled bacteria were directly introduced into surgically exposed intestines of live mice, then imaging was performed by intravital two-photon microscopy. WGA-633 is a lectin used to stain the epithelium. Scale bars, 10 μm. Reproduced with permission from ref . Copyright 2015 Springer Nature.
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