| Coccolithoviruses (EhVs) employ a suite of glycosphingolipids (GSLs) to successfully infect the globally important coccolithophore Emiliania huxleyi. Given their enrichment in glycosphingolipids and their involvement in sensing extracellular stimuli and activating signaling cascades through protein-protein interactions, lipid rafts and associated microdomains likely play a heretofore unappreciated role in viral infectivity and resistance mechanisms. Indeed, the lipid raft protein flotillin carries an evolutionary conserved prohibitin homology (PHB) domain, which has been shown to influence pathogen resistance by inducing hypersensitive response and cell death. Little to nothing is known about lipid rafts in unicellular photoautotrophic organisms, especially those from the marine environment. We combined bioinformatics, detergent treatment, density gradient purification, western blotting, lipidomics and genome-enabled proteomics to isolate and characterize the lipid and protein content of lipid rafts from control and EhV86-infected E. huxleyi strain CCMP1516.;Using bioinformatic analysis, we identified a flotillin-like protein (Protein ID 363433) in the E. huxleyi CCMP1516 genome, which contained several evolutionary-conserved features of known flotillin proteins. Western blot analysis further revealed immunoreactivity of a 67 kDa protein in E. huxleyi cell extracts to a human anti-flotillin antibody, consistent with the dimerized form of this protein and thereby providing a biochemical marker for lipid raft isolation. Lipid raft-enriched fractions were isolated and purified as detergent (Brij96) resistant membranes (DRMs) in Optiprep density gradients and their lipid and protein composition were subsequently characterized. TSQ mass spectrometry of the lipids in DRM fractions showed novel host-derived GSLs which co-purified with flotillin immunoreactive fractions. These fractions also contained notable vesicle morphology as confirmed by TEM.;Subsequent analysis of lipid raft proteome in both uninfected and EhV86-infected E. huxleyi cells collected at 2 h confirmed flotillin as one of the major proteins and more broadly provided key insight into host defense and virus infection strategies at the interface of lipid rafts and stress, programmed cell death, and innate immunity pathways. In particular, the detection of an EhV86-encoded C-type lectin (CTL)-containing protein (ehv149), with putative roles in cell adhesion and pathogen recognition receptors, suggests that EhV86 infection of E. huxleyi occurs by way of the recognition of specific glycosphingolipids and raft-associated proteins. |
