Metabolic Labeling of Bacterial Membranes

Title:  Click-Mediated Labeling of Bacterial Membranes through Metabolic Modification of the Lipopolysaccharide Inner Core

Authors: Dr. Audrey Dumont1, Dr. Annie Malleron2,3, Monzer Awwad2,3,  Dr. Sam Dukan1,*,  Dr. Boris Vauzeilles2,3

Journal: Angewandte Chemie International Edition

Affiliation: 1Aix Marseille Université,Laboratoire de Chimie Bactérienne, 2CNRS and Université Paris-Sud Glycochimie Moléculaire et Macromoléculaire, 3Laboratory of Excellence in Research on Medication and Innovative Therapeutics

Recently the labeling of cell-surface glycans by addition of a synthetically modified monosaccharide that acts as a reporter has become a very popular way to study many multi-cellular organisms.  However the authors mention that there have been very few studies using this method in bacterial systems.  Gram-positive bacteria are encased in a cell wall made of peptidoglycans but Gram-negative bacteria are coated in lipopolysaccharides that are interesting target for such experiments.   Previous studies used a very limited modification that would only be applicable to a few Gram-negative bacteria and only with the use of an alternative pathway enabled through genetic engineering.  The authors of this paper wanted to label the lipopolysaccharides of Gram-negative bacteria without using genetic engineering.

The authors selected 3-deoxy-D-manno-octulosonic acid (KDO) as their target, because it is a necessary part of the inner core of lipopolysaccharides and is present in almost all Gram-negative bacteria.  They created a modified KDO analog – 8-azido-8-deoxy-KDO.  This analog could be incorporated through the KDO pathway and once incorporated could be detected by the click reaction between an azide and alkyne.

The researchers cultured E. coli K12 overnight in the presence of their KDO analog.  They then treated the culture with fluorophore containing a terminal alkyne.  After five minutes of incubation, they could easily detect the bacteria.  Additionally, the bulk of the fluorescence occurred at the edge of the cell indicating that membranes were labeled preferentially.  The authors tested three other Gram-negative bacteria and three negative controls: a bacteria that has been shown to use 8-amino-8-deoxy-KDO instead of KDO and two Gram-positive bacteria.  The Gram-negative bacteria showed labeling, while the negative controls showed none.

This article demonstrates the metabolic labeling of bacteria without genetic engineering.  This technique could be applied to bacterial cell imaging, drug delivery, and the extraction and characterization of lipopolysaccharides.

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