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In this blog, we introduce our new set of click chemistry reagents for the specific visualisation of Legionella and describe their applications for basic research.

Legionella

Legionella are a genus of pathogenic gram-negative bacteria found in freshwater and aerated soil. Legionella are known for causing respiratory disease in humans via the inhalation of contaminated water droplets or consumption of contaminated drinking water.

The Legionella genus got its name after an outbreak resulted in 221 cases and 34 deaths among attendees of an American Legion convention in a Philadelphia hotel, July 1976. In January of the following year, the Legionella bacterium was isolated and identified as the culprit, giving rise to the name Legionnaires’ disease [1]. Legionella were found to be growing in the cooling tower of the hotel’s air conditioning system and had been inhaled by many of those attending. Since its discovery, Legionella have been known to cause pneumonia outbreaks in community and hospital settings from contaminated water supplies. The largest and most common source of Legionnaires’ disease outbreaks are cooling towers, due to their widespread dissemination of water.

Upon inhalation, Legionella enter the lungs where they are phagocytosed by alveolar macrophages as part of the innate immune response. Once internalised, however, Legionella surround themselves in a membrane-bound vacuole that does not fuse with lysosomes or become highly acidic [2]. In this protected compartment, the bacteria are able to proliferate and exert their pathogenic effects, resulting in Legionnaires’ disease and a similar illness called Pontiac fever [3].

Right: Acanthamoeba polyphaga infected with L. pneumophila [4].

Legionella Click Chemistry

LegioTag is an azide-functionalised Legionaminic acid (LEG) for metabolic lipopolysaccharide (LPS) labelling of Legionella pneumophila. LEG is an essential lipopolysaccharide which is incorporated by L. pneumophila into its cell wall during active glycan biosynthesis. When cell-permeable LegioTag is added to an actively growing culture containing L. pneumophila it is intracellularly processed and utilized instead of its natural LEG counterpart.

This azide-modified glycan can then be labelled with either a fluorescent alkyne for imaging or a biotin alkyne for purification. The advantage of using azide and alkyne groups, are that they conjugate to one another with high efficiency but do not react or interfere with other functional groups found in biological samples. LegioTag can be added directly to a Gram-negative bacteria culture in log growth phase, following dilution in fresh media. The culture is then incubated overnight at the appropriate temperature. Following incubation, the culture is washed with buffer prior to ‘click’chemistry labelling using a commercially available Cell Reaction Buffer Kit and Alkyne labelled probe. A variety of different probes are available which can be used for fluorescence detection by microscopy or for bead-based purification, amongst others.

 

Reagents from The Native Antigen Company

The Native Antigen Company offers a range of reagents for Legionella research, immunoassay and vaccine development:

 

LegioTag is an L. pneumophila-specific label comprised mannose analogue that contains an azido moiety for species-specific detection and labeling of L. pneumophila. This allows specific detection and enrichment of culturable L. pneumophila using click chemistry and the use of fluorescent reporters, purification tags or magnetic beads.

 

BacTag is an azide-functionalised 3-Deoxy-D-manno-octulosonic acid (KDO) molecule for metabolic lipopolysaccharide (LPS) labelling of Gram-negative bacteria. This allows specific detection and enrichment of culturable Gram-negative bacteria, using click chemistry and the use of fluorescent reporters, purification tags or magnetic beads.

 

Our Legionella IgG ELISA is for the qualitative determination of IgG class antibodies against Legionella pneumophila in human serum or plasma. This assay shows high sensitivity and specificity with a total assay time of 2-3 hours.

 

Our Legionella IgM ELISA is for the qualitative determination of IgM class antibodies against Legionella pneumophila in human serum or plasma. This assay shows high sensitivity and specificity with a total assay time of 2-3 hours.

 

Our L. pneumophila antigen is a native extract harvested from cell culture and inactivated by detergent.

 

We also provide a range of mouse monoclonal antibodies, specific to Legionella and LPS that are suitable for use in immunoassay development.

 

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