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Bacteria CLICK-Detection

Metabolic labeling with CLICKable Azide saccharides
   



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Glycan conjugates are an essential part of cell membranes[1]. Each organism possesses a unique glycan conjugate signature that is thus an ideal target for the development of organism specific detection tools.

A number of Clickable Azide-functionalized saccharides such as Kdo-Azide[2], 6-Azido-Trehalose[3] pLeg-Azide[4] are now available for non-radioactive metabolic labeling of bacterial cell membrane (Fig. 1, Tab. 1). They are cell-permeable and biosynthetically processed instead of their natural counterparts.

The resulting Azide-functionalized bacterial cell membrane can subsequently be visualized via Cu(I)-catalyzed Alkyne-Azide cycloaddition (CuAAC) or Cu(I)-free strain-promoted Alkyne-Azide cycloaddition (SPAAC) that allows to introduce

  • a (Desthio)Biotin group for subsequent purification (via Alkynes of (Desthio)Biotin or DBCO-containing (Desthio)Biotin) or
  • a fluorescent group for subsequent microscopic imaging (via fluorescent Alkynes or DBCO-containing fluorescent dyes)

Figure 1: CLICK-labeling of bacterial cell membrane is performed by a two-step procedure. 1) Azide-functionalization of bacterial cell membrane by metabolic incorporation of correspondingly-functionalized saccharides and 2) CLICK-labeling via Alkyne- or DBCO-functionalized detection molecules by CuAAC or SPAAC, respectively.


Table 1: Available Azide-functionalized saccharides for bacterial cell membrane labeling.


Detection of... Metabolic labeling
compound
Tested bacterial strains Reference
...Gram-negative bacteria Kdo-Azide Legionella pneumophila
Salmonella typhimurium
Escherichia coli O86
Escherichia coli K12
[2]
...Mycobacteria 6-Azido-Trehalose Mycobacterium smegmatis mc2155
Mycobacterium tuberculosis H37Rv
Mycobacterium bovis BCG
[3]
...Legionella pneumophila pLEG-Azide Legionella pneumophila SG1 (Paris), SG1 (Lens), SG1 (Philadelphia), SG3, SG4, SG5, SG6 [4]


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[1] Singer et al. (1972) The fluid mosaic model of the structure of cell membranes. Science 175:720.
[2] Dumont et al. (2012) Click-Mediated Labeling of Bacterial Membranes through Metabolic Modification of the Lipopolysaccharide Inner Core. Angew. Chem. Int. Ed. 51:3143.
[3] Swarts et al. (2012) Probing the Myobacterial Trehalome with Bioorthogonal Chemistry. J. Am. Chem. Soc. 134 (39):16123.
[4] Mas Pons et al. (2014) Identification of Living Legionella pneumophila using species-specific metabolic lipopolysaccharide Labeling. Angew. Chem. Int. Ed. 53:1275.

   
   
   
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