Isolation and characterization of lipid rafts in Emiliania huxleyi: a role for membrane microdomains in host–virus interactions

Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signa...

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Bibliographic Details
Published in:Environmental microbiology 2014-04, Vol.16 (4), p.1150-1166
Main Authors: Rose, Suzanne L, Fulton, James M, Brown, Christopher M, Natale, Frank, Van Mooy, Benjamin A. S, Bidle, Kay D
Format: Article
Language:English
Subjects:
antiserum
apoptosis
bioinformatics
biomarkers
capsid
Coccolithus huxleyi
death
Emiliania huxleyi
genes
glycosphingolipids
Glycosphingolipids - analysis
Haptophyta - physiology
Haptophyta - virology
Host-Pathogen Interactions
immunity
lipid content
Lipids
mass spectrometry
Membrane Microdomains - chemistry
Membrane Microdomains - physiology
Membrane Proteins - analysis
Phycodnaviridae - pathogenicity
polymerase chain reaction
protein content
Proteins
proteomics
Proteomics - methods
transmission electron microscopy
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Summary:Coccolithoviruses employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Lipid rafts, chemically distinct membrane lipid microdomains that are enriched in GSLs and are involved in sensing extracellular stimuli and activating signalling cascades through protein–protein interactions, likely play a fundamental role in host–virus interactions. Using combined lipidomics, proteomics and bioinformatics, we isolated and characterized the lipid and protein content of lipid rafts from control E. huxleyi cells and those infected with EhV86, the type strain for Coccolithovirus. Lipid raft‐enriched fractions were isolated and purified as buoyant, detergent‐resistant membranes (DRMs) in OptiPrep density gradients. Transmission electron microscopy of vesicle morphology, polymerase chain reaction amplification of the EhV major capsid protein gene and immunoreactivity to flotillin antisera served as respective physical, molecular and biochemical markers. Subsequent lipid characterization of DRMs via high performance liquid chromatography‐triple quadrapole mass spectrometry revealed four distinct GSL classes. Parallel proteomic analysis confirmed flotillin as a major lipid raft protein, along with a variety of proteins affiliated with host defence, programmed cell death and innate immunity pathways. The detection of an EhV86‐encoded C‐type lectin‐containing protein confirmed that infection occurs at the interface between lipid rafts and cellular stress/death pathways via specific GSLs and raft‐associated proteins.
ISSN:1462-2912
1462-2920
DOI:10.1111/1462-2920.12357