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Membrane Insertion for the Detection of Lipopolysaccharides: Exploring the
Dynamics of Amphiphile-in-Lipid Assays
#MMPMID27227979
Stromberg LR
; Hengartner NW
; Swingle KL
; Moxley RA
; Graves SW
; Montaņo GA
; Mukundan H
PLoS One
2016[]; 11
(5
): e0156295
PMID27227979
show ga
Shiga toxin-producing Escherichia coli is an important cause of foodborne
illness, with cases attributable to beef, fresh produce and other sources. Many
serotypes of the pathogen cause disease, and differentiating one serotype from
another requires specific identification of the O antigen located on the
lipopolysaccharide (LPS) molecule. The amphiphilic structure of LPS poses a
challenge when using classical detection methods, which do not take into account
its lipoglycan biochemistry. Typically, detection of LPS requires heat or
chemical treatment of samples and relies on bioactivity assays for the conserved
lipid A portion of the molecule. Our goal was to develop assays to facilitate the
direct and discriminative detection of the entire LPS molecule and its O antigen
in complex matrices using minimal sample processing. To perform serogroup
identification of LPS, we used a method called membrane insertion on a waveguide
biosensor, and tested three serogroups of LPS. The membrane insertion technique
allows for the hydrophobic association of LPS with a lipid bilayer, where the
exposed O antigen can be targeted for specific detection. Samples of beef lysate
were spiked with LPS to perform O antigen specific detection of LPS from E. coli
O157. To validate assay performance, we evaluated the biophysical interactions of
LPS with lipid bilayers both in- and outside of a flow cell using fluorescence
microscopy and fluorescently doped lipids. Our results indicate that membrane
insertion allows for the qualitative and reliable identification of amphiphilic
LPS in complex samples like beef homogenates. We also demonstrated that
LPS-induced hole formation does not occur under the conditions of the membrane
insertion assays. Together, these findings describe for the first time the
serogroup-specific detection of amphiphilic LPS in complex samples using a
membrane insertion assay, and highlight the importance of LPS molecular
conformations in detection architectures.
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