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Environmental Microbiology (2014)
doi:10.1111/1462-2920.12357
Isolation and characterization of lipid rafts in Emiliania
huxleyi: a role for membrane microdomains in
host–virus interactions
Suzanne L. Rose,1† James M. Fulton,2
Christopher M. Brown,1 Frank Natale,1
Benjamin A. S. Van Mooy2 and Kay D. Bidle1*
1
Institute of Marine and Coastal Sciences, Rutgers
University, New Brunswick, NJ, USA.
2
Department of Marine Chemistry and Geochemistry,
Woods Hole Oceanographic Institution, Woods Hole,
MA, USA.
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.
Received 20 June, 2013; accepted 5 December, 2013. *For
correspondence. E-mail [email protected]; Tel. (+1) 848
932 3467; Fax (+1) 732 932 4083. †Present address: University of
Nebraska – Lincoln, Department of Biochemistry, Lincoln, NE,
USA.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd
The detection of an EhV86-encoded C-type lectincontaining protein confirmed that infection occurs at
the interface between lipid rafts and cellular stress/
death pathways via specific GSLs and raft-associated
proteins.
Introduction
The coccolithophore Emiliania huxleyi (Lohmann) Hay &
Mohler (Prymnesiophyceae, Haptophyta) forms massive
spring blooms in the North Atlantic that are routinely
infected and terminated by lytic, double-stranded DNAcontaining viruses (EhVs) (Bratbak et al., 1993; Jordan
and Chamberlain, 1997; Tyrell and Merico, 2004; Allen
et al., 2006a). As members of the Phycodnaviridae, EhVs
are giant microalgal viruses (diameter ∼180 nm) with
an extensive genetic capability (∼407 kb genomes) to
manipulate host metabolic pathways for their replication
(Castberg et al., 2002; Schroeder et al., 2002; Van Etten
et al., 2002; Wilson et al., 2005). Given the array of sensitive and resistant host strains in culture along with a
number of genetically diverse EhVs, the E. huxleyi–EhV
host–virus system has emerged as one of the best characterized model systems to investigate marine algal host–
virus interactions and the cellular processes mediating
infection dynamics (Mackinder et al., 2009; Bidle and
Vardi, 2011).
The determinants of host susceptibility or resistance
to viral infection, especially the factors involved in the
initial cellular response, are of fundamental interest
in host–virus interactions. Coccolithoviruses employ a
sophisticated, co-evolutionary biochemical ‘arms race’ to
manipulate host lipid metabolism, alter glycosphingolipid
(GSL) production and ultimately regulate cell fate via
induction of a programmed cell death (PCD) pathway [via
reactive oxygen species, metacaspase expression and
caspase activity (Bidle et al., 2007; Vardi et al., 2009;
2012; Bidle and Vardi, 2011)] in a manner reminiscent of
the ‘Red Queen’ dynamic (VanValen, 1973) driving plantand animal-pathogen interactions (Staskawicz et al.,
2001). Subtle differences in the physiological regulation of
the PCD machinery appear to be critical in host susceptibility (Bidle and Kwityn, 2012), perhaps as part of an
innate immune response to determine cell fate. Yet, little is
2
S. L. Rose et al.
known about the cellular determinants of infectivity as
they relate to lipid dynamics within host cell membranes.
The cell membrane fluid mosaic model originally proposed in 1972 (Singer and Nicolson, 1972) has evolved
over the past decade leading to the contemporary characterization of biological membranes as having discrete
organizational regions, or microdomains (Brown and
London, 2000; Edidin, 2003; Lingwood and Simons,
2010). Coined as ‘lipid rafts’, these microdomains are
enriched in sphingolipids, GSLs and sterols forming a
liquid-ordered phase distinct from the phospholipid containing liquid-disordered phase of the surrounding membrane (Van Meer and Simons, 1988; Rajendran and
Simons, 2005; Hancock, 2006; Hakomori, 2008). The
distinct biochemical nature of lipid rafts allows for their
isolation due to their insolubility in cold non-ionic
detergents (e.g. Brij-96 and Triton-X) and ability to
form buoyant ‘detergent-resistant membranes’ (DRMs)
(Radeva and Sharom, 2004; Borner et al., 2005) with
lipid raft domain sizes ranging from ∼150 to 250 nm in
diameter (Pike, 2003), perhaps deriving from smaller,
∼50 nm diameter assemblies that coalesce in response
to different cellular or environmental conditions (Simons
and Ehehalt, 2002).
The ability of lipid rafts to incorporate or exclude
specific proteins and facilitate selective protein–protein
interactions provides an important platform for the regulation of a wide range of cellular processes, including
cytoskeleton organization, cell adhesion, signal transduction pathways and apoptosis (Brown and London,
2000; Munro, 2003; Morrow and Parton, 2005; Hancock,
2006; Lingwood and Simons, 2010). Small individual lipid
raft size (∼10–50 nm) suggests their role in the regulation
of raft-associated proteins, whereby signalling proteins
are physically separated and thereby held in an inactivated state (Simons and Ehehalt, 2002). Subsequent
clustering of lipid rafts into a larger morphological platform
(∼200 nm) in turn facilitates protein–protein interactions
that are essential to trigger pathways involved in cell
growth, stress response, death and survival (Simons
and Ehehalt, 2002). Lipid rafts have been found to be
enriched in glycosylphosphatidylinositol (GPI)-anchored
proteins, Src family kinases, heterotrimeric G proteins and
stomatin, prohibitin, flotillin and HflC/K (SPFH) superfamily proteins, all of which are targeted to lipid rafts via
essential lipid modifications (Morrow and Parton, 2005;
Rajendran and Simons, 2005; Rivera-Milla et al., 2006).
Flotillin, an evolutionary conserved SPFH domaincontaining protein, has been proposed to function as a
lipid raft organizer, acting as a scaffold and linking the
aforementioned lipid raft domains and raft-dependent cellular process (Simons and Toomre, 2000; Langhorst et al.,
2005; Morrow and Parton, 2005). Flotillin-enriched lipid
rafts appear to be involved in host invasion indicating
a possibly important role in host–pathogen sensitivity
or resistance (Rivera-Milla et al., 2006; Babuke and
Tikkanen, 2007; del Cacho et al., 2007).
Lipid rafts are targeted and usurped by a vast number
of pathogens as sites of host attack in higher eukaryotes
(van der Goot and Harder, 2001). In the E. huxleyi–EhV
system, they are hypothesized to function as specific
points of viral attachment and entry, as well as in virus
assembly and budding (Mackinder et al., 2009; Bidle and
Vardi, 2011). EhVs induce and employ a unique suite of
virus-encoded GSLs (vGSLs) to successfully infect
E. huxleyi (Vardi et al., 2009; 2012; Bidle and Vardi,
2011). Furthermore, EhV86 is an icosahedral virus that is
enveloped with a lipid membrane composed of various
GSLs (Vardi et al., 2009; Fulton et al., 2014) and contains
a majority (23 of 28) of putative membrane proteins
with transmembrane domains (Allen et al., 2008). Thus,
lipids and lipid-associated proteins appear to play a critical role in EhVs and their ‘animal-like’, envelope fusionbased infection strategy for entering and exiting infected
host cells (Mackinder et al., 2009). To date, very little
is known about lipid raft characteristics in unicellular
marine phytoplankton, much less this globally important
coccolithophore.
In this study, we investigated the lipid and protein composition of lipid rafts in E. huxleyi, and their potential
involvement in E. huxleyi–EhV host–virus interactions.
Building off of previously published methods in higher
eukaryotes (Radeva and Sharom, 2004; Macdonald and
Pike, 2005) and utilizing a flotillin-like protein in E. huxleyi
as a biochemical marker, we developed a protocol for the
isolation of lipid rafts and associated proteins from both
uninfected and infected cell populations and characterized their dynamics and composition through genomeenabled proteomics and lipidomics. Our results provide
insight into the initial biochemical and biophysical basis of
viral–host interactions and provide a better mechanistic
understanding of the cell surface properties that serve to
regulate host response to viral infection.
Results and discussion
A primary goal of this study was to isolate and characterize lipid rafts, here defined as insoluble DRMs, particularly
their associated proteomes and lipidomes, in both
uninfected and EhV86-infected cell populations. Our
approach was to use buoyant densities and vesicle morphology, along with flotillin and GSL species as respective
protein and lipid biochemical markers, to inform our targeted isolation of lipid rafts for more detailed characterization. Flotillins are expressed in a wide variety of
eukaryotes and prokaryotes and play a central role in lipid
raft generation (protein–lipid binding domain), as well as
in the stabilization of raft-associated proteins (protein–
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Lipid rafts in Emiliania huxleyi
3
A
MEACGCVITSQDERKVVERCGKFEDVLDAGFSCVLPCLCQFVKGSISTRIQMLEINADTKTKDNVFVGIK70
VAVQYQVNGDSQSIQDAMYKLTNPRAQIESYVLDVVRSSVPKIDLDNVFLEKEEIAASIKEMLGETMGRF140
GYSILATPVTDIEPNLEVKRAMNEINKAKRLRQAAVDEGEAIKIRSIKEAEAEAARTEIQAKADAEAKFM210
QGQGIARQRQAIVSGLRDSVNCFKADVAGVDSKQVMDLLLVTQYFDMMKDVGGNSRSNAVFMNHSPGALQ280
DLTQAIQGGFMSSLPAAPSFAQAQRH306
hydrophobic Sphingolipid Binding Domains (13-41 aa; 133-151 aa)
conserved palmitoylation site
predicted α-helix which can dimerize flotillin monomers via a triple coil
MW
B
C
hc
Ehux
kDa
50
37
*
Fig. 1. Analysis of a flotillin-like protein in Emiliania huxleyi CCMP1516.
A. The 306 amino acid, 33.5 kDa putative flotillin-like protein (Protein ID 363433) contains notable lipid raft-associated domain characteristics.
Predicted secondary structural elements consist of the evolutionary conserved prohibitin homology (PHB)/stomatin prohibitin flotillin and HflC/K
(SPFH) domain residues (aa 4–166; underlined) and the hydrophobic sphingolipid binding domain (SBD) regions (aa 13–41; aa 133–151),
along with the conserved palmitoylation site, which contributes to membrane association and where α-helix coils are predicted to facilitate
dimerization of flotillin proteins.
B. Tertiary structure prediction (Phyre2 PDB SCOP code c3bk6C membrane protein; viewed with PyMOL software), with visualization of a
α-helix coil tail (orange-red).
C. Western blot analysis of E. huxleyi cell extracts challenged with a human anti-flotillin antibody displayed strong immunohybridization with a
67 kDa protein consistent with the predicted size of the dimerized protein (Protein ID 363433). Immunoblot control consisted of the predicted
42 kDa human flotillin (asterisk in lane ‘hc’; NCBI Accession #AAF17215; 253 aa) from human A-375 whole cell lysate of malignant melanoma
cells. Molecular weight markers (MW; kDa) are indicated.
protein binding domain), both of which are fundamental
in the regulation of diverse processes (i.e. signal
transduction, membrane trafficking and pathogen entry)
(Borner et al., 2005; Morrow and Parton, 2005).
Bioinformatic analysis of the E. huxleyi CCMP1516
annotated proteome (http://genome.jgi-psf.org/Emihu1/
Emihu1.home.html) revealed a 306 amino acid, 33.5 kDa
‘flotillin-like’, SPFH domain-containing protein (Protein ID
363433; Accession# EOD34547) that was also characterized by ‘Band_7’ protein domains (IPR001107 and
cd03407; e-value = 1.42e-93) diagnostic of other flotillin
(reggie)-like proteins. This particular Band_7 subgroup
(cd03407) contains proteins similar to SPFH and podicin,
many of which are lipid raft-associated. The putative
E. huxleyi flotillin-like protein also showed high sequence
identity to a ‘hypersensitive-induced response protein’ in
Arabidopsis thaliania (NP_177142; e-value of 6e-88)
(Theologis et al., 2000) that is linked to PCD pathway in
higher plants (Lam et al., 2001).
Secondary structure analysis of the E. huxleyi flotillinlike protein revealed the evolutionary conserved SPFH
domain (aa residues 4–166; Fig. 1A). This conserved
domain contains two hydrophobic sphingolipid binding
domains (aa residues 13–41 and 133–151) along with a
conserved palmitoylation site, both of which contribute
to membrane association (Morrow et al., 2002; Morrow
and Parton, 2005). The required post-translational
palmitoylation of flotillins at the N-terminus follows their
synthesis on ribosomes followed by an unusual Golgiindependent trafficking pathway that targets the protein
to the plasma membrane (Morrow et al., 2002). The
C-terminal end of the E. huxleyi flotillin-like protein possessed a predicted α-helix coil (aa residues 266–286;
Fig. 1A), which is a characteristic of all classic flotillins and
is thought to oligomerize with other flotillin molecules
(Morrow and Parton, 2005; Solis et al., 2007). The presence of three adjacent α-helix coils within this domain
allow for the oligomerization of flotillin in higher
eukaryotes (Solis et al., 2007). Exposed within this protein
are lysine residues thought to facilitate a high-affinity,
stable covalent linkage between flotillin monomers (Solis
et al., 2007). Modelling of the tertiary structure of the
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
4
S. L. Rose et al.
E.huxleyi cell abundance
(cells ml-1)
3.0E+06
A
2.5E+06
Uninfected Control
2.0E+06
+EhV86
1.5E+06
1.0E+06
5.0E+05
0.0E+00
EhV abundance
(virus ml-1)
1.0E+09
B
1.0E+08
1.0E+07
1.0E+06
0
24
48
72
96
Time (hours post infection)
Fig. 2. Dynamics of EhV86 infection of E. huxleyi CCMP1516.
Time course of host (A) and virus (B) abundance over 96 h for
uninfected control and EhV86-infected (+EhV86) cultures. Error
bars represent standard deviation for triplicate measurements and,
where not discernible, are smaller than symbol size.
E. huxleyi flotillin-like protein using Phyre2 and PyMOL
software packages (Fig. 1B) successfully visualized
this dimerizing α-helix coil tail. Furthermore, Western
immunoblot analysis with a monoclonal antibody raised
against human flotillin-2 (NCBI Accession #Q14254) successfully detected the expression of a prominent 67 kDa
band in E. huxleyi CCMP1516, consistent with the predicted dimer size of the flotillin-like protein (Fig. 1C).
Immunoblot analysis also successfully detected the predicted 42 kDa human flotillin in human A-375 whole cell
lysate of malignant melanoma cells, thereby serving as a
positive control for our analysis.
Lipid raft dynamics, isolation and characterization
We investigated lipid raft dynamics and composition both
in control host cells (uninfected) and during host–virus
interactions at 2, 24, 48 and 72 h post-infection (hpi) to not
only determine the lipid raft landscape in healthy cells, but
to also determine if and how lipid rafts are altered by virus
infection. We first confirmed successful lytic infection of
E. huxleyi CCMP1516 with EhV86, which resulted in a
notable reduction of host cell abundance over 72 h compared with uninfected control cells (Fig. 2A); infected cell
populations only reached host cell abundances of
∼1.25 × 106 cells ml−1 at 72 h, while uninfected cell abundance reached 2.5 × 106 cells ml−1. Uninfected E. huxleyi
cells continued to increase in abundance over the time
course of the experiment. EhV86 abundance increased
by over two orders of magnitude in infected cultures
between 24 and 72 hpi (Fig. 2B), concomitant with
decreases in host growth (Fig. 2A).
We developed a purification method, based on
published techniques (Radeva and Sharom, 2004;
Macdonald and Pike, 2005) to isolate lipid rafts from
higher eukaryotes, employing lysis and homogenization
of cells in a non-ionic detergent (Brij-96) followed by
ultracentrifugation in a discontinuous (5–45%) OptiPrep
density gradient (Fig. 3A and B). Detergent-resistant lipid
rafts from higher eukaryotic cells have been isolated using
the detergent Triton X-100 followed by sucrose gradient
fractionation, with fractions enriched in sphingolipids, cholesterol, flotillin and GPI-anchored proteins distributed in
the top few, less dense fractions (Munro, 2003; Pike,
2003; Radeva and Sharom, 2004; Macdonald and Pike,
2005). Here, Brij-96 detergent treatment was used to
produce ‘outside-in’ DRMs for subsequent identification
and characterization of lipid raft-associated lipids and proteins found on the extracellular plasma membrane of
E. huxleyi uninfected and EhV86-infected cell populations. Direct visualization of lipid vesicles in select fractions via transmission electron microscopy (TEM) showed
the presence of prominent, 0.2 μm DRM vesicles in
buoyant Fraction 2 (1.055 g cm−3) for both uninfected controls and EhV86-infected samples (Fig. 3C). This is consistent with published characteristics of DRMs in higher
eukaryotes (Pike, 2003; Radeva and Sharom, 2004). No
lipid vesicles were seen in higher density fractions for
either treatment.
Polymerase chain reaction (PCR) amplification of the
EhV86 major capsid protein (MCP) was also used as a
proxy to identify the distribution of EhVs (and associated
DNA) in OptiPrep density gradient fractions from EhV86infected cell populations at 2, 24, 48 and 72 hpi (Fig. 4).
This helped provide an initial characterization of the
OptiPrep density fractions and important context on
where to expect viruses (and their associated GSLs and
proteins). Successful amplification of the expected 248 bp
MCP amplicon was only detected in fractions 6–12 for
both 2 and 24 hpi, indicating the selective presence of
EhVs in higher density, soluble fractions (Fig. 4). The dramatic increase in EhV abundance (10- to 100-fold) during
late lytic phase (48–72 hpi; Fig. 1) was clearly reflected in
the increased intensity MCP amplification in fractions
6–12 (Fig. 4). At the same time, a weaker, yet notable,
amplification signal of EhV MCPs was also seen in light
DRM fractions (1–5) indicative of an accumulation of
EhVs with distinct lipid characteristics. Our MCP amplicon
data could represent the distribution of intact EhV virions
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Lipid rafts in Emiliania huxleyi
A
Optiprep™ gradient
g/cm3
1.055
1.055
1.060
1.065
1.065
1.190
1.250
1.260
1.290
1.300
1.310
1.360
Fraction 1
5%
(39,000 x g, 4°C, 16 h)
35%
Fraction 12
45% +
E. huxleyi cell lysate
B
C
control
+EhV86
F2
F2
0.2
0.2
F3
5
Fig. 3. Lipid raft isolation and characterization
in E. huxleyi CCMP1516.
A. Schematic of the OptiPrep density gradient
procedure used to isolate lipid raft fractions.
Corresponding fraction densities (g cm−3) are
indicated.
B. Picture of OptiPrep gradients
corresponding to the 2 (#1), 24 (#2), 48 (#3)
and 72 (#4) h samples for control E. huxleyi
cultures. Gradients were also performed for
EhV86-infected cell populations at the same
time points (not shown).
C. Direct visualization of lipid vesicles in
select fractions (F2, F3, F4 and F11) via
transmission electron microscopy (TEM).
Images in the left column correspond to
uninfected control cells; image in right
column corresponds to fraction 2 for
EhV86-infected-cells. Note the presence of
prominent detergent-resistant lipid vesicles
only in Fractions 2 and 3. No lipid vesicles
were observed in lower fractions for either
treatment.
0.2
F4
0.2
F11
0.2
or the released EhV genomic DNA within the OptiPrep
fractions.
We also tried to locate and quantify EhV abundance in
OptiPrep fractions via SYBR-Green staining and flow
cytometry, but were unable to clearly discern the distinct
EhV population signal in gradient fractions (data not
shown). Brij-96 treatment of a freshly generated EhV86
lysate was found to compromise the SYBR-Green, flow
cytometry signal of EhVs; it wasn’t as focused as
untreated lysates and had a detectable increase in the
520 nm fluorescence signal (Supporting Information
Fig. S1). Nonetheless, most EhVs (60.5% of the
OptiPrep fraction
Fig. 4. Distribution of EhVs in OptiPrep
density gradients. PCR of the EhV MCP (see
Experimental procedures) was used as a
proxy to identify the distribution of EhVs (and
associated DNA) in density gradients of
EhV86-infected cell fractions for 2, 24, 48 and
72 hpi post infection samples. Successful
amplification of the expected 248 base pair
amplicon was detected in fractions for each
time point. Note that at early time points
(2–24 hpi), MCP was distributed between
fractions 6–10. Subsequent time points (48
and 72 hpi) displayed intense amplicons in
Fractions 6–12 and weaker amplification in
Fractions 1–5; this corresponded with 10- to
100-fold increase in EhV abundance (see
Fig. 1B). EhV86 viral lysate served as a
positive control (‘v’; upper panel). No template
(nt) and genomic DNA for Emiliania huxleyi
(h) were used as negative controls (upper
panel). Molecular weight (bp) markers are
indicated.
MW nt
h
v
1
2
3
4
5
6
7
8
9
10 11 12
2
300
24
300
48
300
72
Hours post infection
300
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
6
S. L. Rose et al.
population) localized within the diagnostic side scatter
(SSC) vs 520 nm fluorescence gate (Supporting Information Fig. S1) and were likely intact. Viruses subjected to
OptiPrep ultracentrifugation gradients have reported
buoyant densities of 1.14–1.22 g cm−3 (Lawrence and
Steward, 2010), including a purified Aureococcus
anaphagefferens virus (AaV), a large dsDNA virus infecting the Pelagophyte Aureococcus anophageferrens that,
like EhV, is a member of the nucleocytoplasmic large DNA
virus (NCLDV) family (M. Moniruzzaman, G.R. LeCleir,
C.M. Brown, C.J. Gobler, W.H. Wilson, K.D. Bidle, and
S.W. Wilhelm, in preparation). Based on this information,
one would expect intact EhVs to be concentrated between
Fractions 5 and 8 (1.065–1.260 g cm−3). Notably, this
indeed corresponds with distribution of a majority of EhV
MCP amplicons in Fig. 4 (especially for 2 and 24 hpi time
points). Given reported buoyant densities of DNA at 1.69–
1.71 g cm−3 (Smith, 1977), the presence of EhV MCP
amplicons in more dense fractions (Fractions 10–12) at
later time points (48–72 hpi) suggests that some EhVs
had indeed been disrupted and released genomic DNA.
Taken together, these flow cytometry, PCR and density
data argue that most EhVs (and associated proteins)
were not present in more buoyant fractions (e.g. Fraction
2) containing DRMs and suggest that a subpopulation of
EhVs have a membrane envelope structure (Mackinder
et al., 2009) that is susceptible to disruption and
solubilization by non-ionic detergent treatment.
EhV replication appears to be uniquely characterized
by two distinct modes of infection, consisting of a ‘chroniclike’ phase through budding and a lytic phase (Mackinder
et al., 2009). Most EhV progeny emerge through lytic
phase [Fig. 1; (Bidle et al., 2007; Mackinder et al., 2009;
Vardi et al., 2009)], as is evidenced by the concomitant
disappearance of host cells (= lysis) and exponential
increase in EhVs (Fig. 1). EhVs produced through these
two fundamentally different modes might be expected to
produce distinct virus morphologies and detergent sensitivities, with lytic EhVs perhaps possessing an inner membrane like the other Phycodnaviridae (Van Etten et al.,
2002). Fulton and colleagues (2014) recently updated
the two-step model for EhV acquisition of lipids originally
proposed by Mackinder and colleagues (2009), and suggested that EhVs acquire an inner membrane in association with intracellular lipid bodies and then acquire an
outer membrane, which is potentially more GSL rich and
presumably more detergent resistant, via budding through
lipid rafts in the host plasma membrane. Together with the
results of Fulton and colleagues (2014), our findings point
towards a bimodal distribution of EhVs, where one
subpopulation emerges from the host via budding and
possess a detergent-resistant outer membrane, while
another detergent-susceptible subpopulation is released
via host lysis and lacks an outer membrane.
Anti-flotillin immunoblot analysis was performed on 2
and 48 h control and EhV86-infected E. huxleyi cell
extracts to identify flotillin-containing fractions. Strong
immunohybridization was observed to a prominent
67 kDa flotillin-like protein in both E. huxleyi control and
EhV86-infected fractions at both time points (Fig. 5).
Uninfected E. huxleyi fractions showed strong immunohybridization of the 67 kDa flotillin-like protein among
a broad range of low density, DRM-containing fractions
1–5 (Fig. 5, upper panels). The pattern of anti-flotillin
immunohybridization was dramatically different in EhV86infected cell populations, instead being characterized
by a much more defined and focused hybridization signal
in fractions 2 and 3 at 2 hpi and 48 hpi respectively
(Fig. 5, lower panels). These results clearly demonstrate
that the interaction of E. huxleyi host cells with EhV86
virions, even for short time periods (2 hpi), has a pronounced effect on the physical attributes of flotillinassociated DRMs. Taken together, our physical (buoyant
densities, TEM, SYBR-Green), molecular (distribution of
MCP amplicons) and biochemical (anti-flotillin western
immunoblot analysis) results highlighted fraction 2 as a
strong candidate for subsequent characterization of the
lipidome and proteome associated with isolated lipid rafts.
Distribution and composition of GSLs
GSLs are polar lipids that critically ‘lubricate’ E. huxleyi–
EhV interactions both in culture (Vardi et al., 2009; Bidle
and Vardi, 2011) and in natural systems (Vardi et al.,
2009; 2012). Indeed, host-specific glycosphingolipids
(hGSLs), consisting of palmitoyl-based GSLs (Vardi et al.,
2012) and viral glycosphingolipids (vGSLs), consisting of
myristoyl-based GSLs (Vardi et al., 2009; 2012), play
important functional roles in the infection of E. huxleyi and
even serve to trace both host and virus populations
respectively (Vardi et al., 2012). Furthermore, a novel
class of sialic acid glycosphingolipids (sGSLs) with
2-Keto-3-deoxynonic acid as the headgroup appear to be
promising biomarkers for E. huxleyi populations susceptible to EhV infection (Fulton et al., 2014). Knowing that
GSLs are classically thought to play prominent roles in
lipid rafts of higher eukaryotes, we examined their relationship to DRMs in E. huxleyi and determined which
GSLs, if any, coincide with the flotillin biochemical protein
marker of lipid rafts.
Using high-performance liquid chromatography-triple
quadrapole mass spectrometry, we examined the abundance and chemical identity of GSLs in the OptiPrep
fractions from control and EhV86-infected cell populations
at 2, 48 and 72 h (Fig. 6). A variety of GSL species were
detected in our analysis, which were grouped into four
general component classes: ‘hGSLs’, hGSLs with a d19:3
long-chain base (LCB) and either a C22:3 hydroxy- or
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Lipid rafts in Emiliania huxleyi
OptiPrep fractions
hc
1
2
3
4
5
6
7
8
9
7
OptiPrep fractions
10 11 12
hc 1
2
3
4
5
6
7
50
8
9
10 11 12
control
37
+EhV86
50
37
2h
48 h
Fig. 5. Distribution of the E. huxleyi flotillin-like protein in OptiPrep density gradients. Anti-flotillin Western immunoblot analysis of density
gradient fractions at 2 and 48 h for uninfected (control; top row) and EhV86-infected (+EhV86; bottom row) cells. Note that, while the
prominent flotillin-like protein is distributed among most control fractions, it is concentrated within fractions 1–5. In contrast, EhV86-infection
considerably focused this protein to within fractions 2–3. Flotillin control immunoblot analysis recognized the predicted 42 kDa human flotillin
(NCBI Accession# AAF17215; 253 aa) in human A-375 whole cell lysate of malignant melanoma cells. Molecular weight markers (MW; kDa)
are indicated.
C22:2 hydroxy-fatty acid (Vardi et al., 2012); ‘vGSLs’,
EhV-derived, myristoyl-based GSLs with C15–C24 saturated fatty acids and C20–C24 monounsaturated fatty
acids (Vardi et al., 2009) that increased substantially with
EhV infection; ‘sGSLs’, sGSLs (monosialylceramides)
containing a palmitoyl-based LCB (d18:2 or d18:1) and
a C22:0 fatty acid (Fulton et al., 2014), that increased
with infection; and ‘rGSLs’, raft GSLs with a glycosyl
headgroup and a primary neutral mass loss of −162 Da,
indicative of an LCB similar to vGSL (Vardi et al., 2009).
rGSLs were detected as a sequence with relatively low
molecular weights (m/z 704, 718, 732 and 746) that likely
differed in fatty acid chain length, and based on their early
elution times, we predict that the rGSL LCB was longer,
saturated and less hydroxylated than that of vGSL; rGSLs
did not increase with infection. Total GSL concentration
(normalized to the glycolipid recovery standard DNP-PE;
see Experimental procedures) was generally confined to
higher density, lower fractions (6–12; Fig. 6), indicating
that most GSLs were not associated with DRMs. We were
specifically interested in the relative percentage of individual GSL classes in each fraction to get a better sense
of their individual distribution and whether any specific
classes are enriched in flotillin-associated DRMs in both
control and EhV-challenged cells.
Among the different GSL classes in uninfected control
cultures (Fig. 6; left column), hGSLs were generally confined to the lower fractions (5–12) and comprised a rela-
tively high percentage (30–98%) of GSLs in uninfected
control cells within each of these more dense fractions.
The sGSL class, which was only recently described
(Fulton et al., 2014), showed a more even distribution in
OptiPrep density gradients than hGSLs, but there was
evidence of a high relative sGSL percentage in more
buoyant DRMs, with sGSLs making up to 94% and 72% of
the GSLs in fractions 1 and 2 at 72 h. rGSLs showed a
very different distribution in the OptiPrep density gradients
(Fig. 6) instead being preferentially concentrated in the
upper, low density fractions (1–5) and accounting for up to
87% of the GSLs in these respective fractions. These host
rGSLs clearly behaved differently in the density gradients
than the other three GSL classes, and more closely corresponded to other lipid raft physical (presence of DRMs,
Fig. 3) and biochemical markers (distribution of flotillin,
Fig. 4) and, thereby, more likely represent the lipid raft
lipidome. Not surprisingly, no chemical signatures of
‘vGSLs’ were detected in uninfected control cells at all
time points suggesting that this class of GSLs is indeed
derived from EhV biosynthetic machinery (Monier et al.,
2009; Vardi et al., 2009).
Viral infection triggered a massive upregulation (3–10fold) in the normalized total GSL production at 48 h and
especially 72 h post-infection, with most GSLs being
confined to fractions 5–10 (Fig. 6, right column; Supporting Information Table S1). Our results further support
existing data that EhVs induce GSL biosynthesis during
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
8
S. L. Rose et al.
Control
Total
Fraction GSL*
2h
48 h
72 h
+ EhV86
% GSLa
hGSL†
vGSL‡
sGSL#
Total
GSL*
rGSL§
% GSLa
hGSL†
vGSL‡
sGSL#
rGSL§
1
0.0
11
0
19
70
0.1
11
0
13
76
2
3
4
5
6
7
8
0.0
0.1
0.1
0.3
1.2
2.1
1.9
19
0
31
52
62
86
68
0
0
0
0
0
0
0
16
13
30
13
32
7
31
66
87
40
34
6
7
1
0.2
0.0
0.1
0.3
0.1
1.2
1.5
5
0
0
29
15
92
74
0
0
0
0
0
0
23
12
16
6
8
0
6
0
84
84
94
63
85
2
3
9
10
11
12
2.3
2.3
1.0
0.5
70
64
62
70
0
0
0
0
27
34
37
25
2
1
1
5
1.6
2.5
1.6
0.7
98
78
37
85
0
18
53
0
0
3
4
1
2
1
7
13
1
2
3
4
5
6
7
8
9
10
11
12
0.1
18
0
1
81
0.4
5
80
0
15
0.1
0.1
0.1
0.6
4.9
7.4
9.3
21
9
35
90
68
68
60
0
0
0
0
0
0
0
21
48
50
9
32
32
40
58
44
15
2
1
0
1
0.7
0.2
0.7
1.9
14.6
16.5
13.6
16
18
4
50
51
61
55
78
53
54
35
42
36
41
1
11
4
1
6
3
5
6
19
38
25
1
0
0
10.0
7.2
3.3
2.3
56
76
76
71
0
0
0
0
44
24
24
29
0
0
0
0
18.7
9.6
6.0
3.6
44
61
60
59
37
27
38
29
18
13
2
12
0
0
0
0
1
2
3
4
5
6
7
8
9
10
11
12
0.1
6
0
94
0
6.0
2
94
1
3
90
94
94
68
83
70
59
2
4
1
11
5
14
21
0
0
0
0
0
0
0
68
71
74
74
11
9
4
13
0
0
0
0
0.5
0.3
0.6
7.7
15.4
12.0
12.5
2
53
11
33
68
59
60
0
0
0
0
0
0
0
72
46
40
67
32
40
39
26
1
49
0
0
1
1
25.6
12.1
10.8
47.9
100.0
70.6
63.2
8
3
5
21
13
17
20
14.5
12.7
4.2
3.7
50
54
47
55
0
0
0
0
50
46
53
44
0
0
0
2
73.2
49.3
15.0
9.6
21
20
22
13
a - calculated based on realtive peak areas of individual GSL classes
* - expressed as the normalized (%) peak area, compared to a DNP-PE recovery standard (see Experimental procedures)
† - based on the d19:3 long chain base and either a C22:3 hydroxy- or C22:2 hydroxy-fatty acid (Vardi et al., 2012)
‡ - EhV-derived myristoyl-based GSLs with C15-C24 saturated fatty acids and C20-C24 monounsaturated fatty acids (Vardi et al., 2009)
# - GSL with a sialyl (KDN) headgroup, d18:1 or d18:2 LCB and C22:0 fatty acid (Fulton et al., 2014)
§ - Low molecular weight GSLs with a glycosyl headgroup; differ from vGSLs in the LCB length and number and/or positions of hydroxyls
Fig. 6. Distribution of total glycosphingolipids (GSLs) and the corresponding relative percentages of individual GSL classes (hGSLs, vGSLs,
sGSLs and rGSLs) in OptiPrep density gradient fractions (1–12) for uninfected control and EhV86-infected Emiliania huxleyi CCMP1516 at 2,
48 and 72 h. Definitions of the four GSL classes are provided.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Lipid rafts in Emiliania huxleyi
a
9
c
Table 1. Top 20 protein hits detected in proteomic analysis of lipid rafts for uninfected Emiliania huxleyi cells at 2 h.
Protein IDb
Protein description
Protein log e
# of unique peptides
detected
Protein
MW (kDa)
444491
363433
444671
366130
432658
440685d
436031d
446310
441334
358662
420185
433847
360139
246130
439254d
46509
434557
444211d
460471
447939d
Outer membrane protein porin
Band_7; flotillin-like protein
Protein of unknown functione
Chlorophyll a/b binding protein
Mitochondrial import receptor subunit
High affinity nitrate transporter
Delta-carbonic anhydrase
Chlorophyll a/b binding protein
Type 1 actin
Chloroplast light harvesting protein
Chloroplast light harvesting protein
Chloroplast light harvesting protein
Mitochondrial substrate carrier
Histone H4
Nitrite transporter
Chloroplast light harvesting protein
Protein of unknown functione
Protein of unknown functione
Protein of unknown functione
Na+/Ca2 ± K + exchanger
−160.6
−109.1
−73.5
−72.4
−67
−61.1
−58.9
−58.2
−56.2
−55.9
−53.1
−53
−49.9
−46.8
−35.5
−35.2
−31.8
−27.8
−26.8
−19.8
19
14
11
9
7
9
8
6
8
5
3
7
6
7
5
5
4
4
4
3
31.6
33.5
19.6
20.2
30.9
73.3
72.4
22.6
41.6
21.9
15.6
30
36.7
11.4
29.3
18
16.5
30.6
31.2
67.9
a.
b.
c.
d.
e.
Based on rank order of Protein log e.
JGI (filtered best models; http://genome.jgi-psf.org/).
Fraction #2 from 2 h.
Contains predicted transmembrane domains.
No Gene Ontology, Conserved Domains, or BlastP hits with e-value < 0.
infection (Vardi et al., 2009; 2012; Bidle and Vardi, 2011;
Fulton et al., 2014). The hGSL distribution and percent
contribution at 2 hpi was even more concentrated in
denser gradient fractions (6–10) compared with control
cells. Infection with EhV86 subsequently downregulated
hGSL production at 48 hpi and even more so at 72 hpi
(peak lytic phase), whereby hGSLs comprised only
13–22% of GSLs in fractions 5–12. For comparison,
hGSLs accounted for 47–68% of GSLs for uninfected
control fractions at this time point. Likewise, virus infection
downregulated the relative contribution of sGSL and rGSL
species, especially at 72 hpi, where rGSLs were almost
completely removed from much of the fractionated
lipidome and sGSLs were < 20%. sGSLs were, however,
relatively enriched in buoyant DRM fractions (1–5) at
2 hpi. This fractionation with DRMs (fractions 1–5) was
even more apparent for rGSLs at 2 hpi comprising
63–94% of the fraction-specific GSLs. In stark contrast,
vGSLs dominated the GSL composition as infection progressed, and were generally distributed throughout all 12
fractions at 48 and 72 hpi (Supporting Information
Table S1). At 72 hpi, vGSLs accounted for an impressive
59–94% of GSLs in all fractions. This distribution of
vGSLs corresponded quite well with our PCR-based
amplification of the EhV MCP gene, indicating that
genomic DNA and viral membranes (which consist largely
of vGSLs) co-localized in our density gradients lending
further support that most EhVs were intact.
Characterizing the lipid raft proteome
Given the physical (via TEM), biochemical (via flotillin
immunoreactivity) and lipid (GSL species) distribution patterns, we targeted fraction 2 from both control and EhV86infected samples at 2 hpi for proteomic analyses in order
to provide insight into the key protein players in initial
viral–host interactions (Allen et al., 2006b; Mackinder
et al., 2009) and provide a better understanding of the cell
surface proteins that serve to mediate viral infection and
host cell response. The liquid chromatography-tandem
mass spectrometry (LC-MS/MS) peptide fragment data
were searched against the common Repository of Adventitious Proteins (cRAP; http://www.thegpm.org/crap/),
which consists of proteins commonly found in proteomics
experiments through unavoidable contamination with
common laboratory proteins and/or protein standards,
and the respective E. huxleyi CCMP1516 and EhV86
annotated proteome databases with amino acid sequences parsed from Uniprot database. A total of 86 proteins
were identified in the uninfected control E. huxleyi cells
sample, whereas 116 total proteins were found in the
EhV86-infected sample. An analysis of the top 20 protein
hits (protein log-e rank order) for both control and EhV86infected samples (Tables 1–2 respectively) indentified
many common proteins, most notably a Band_7, flotillinlike protein (Protein ID 363433; log e = −109.1; 34 unique
peptides) with the predicted SPFH-containing domain,
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
10
S. L. Rose et al.
Table 2. Top 20 protein hitsa detected in proteomic analysis of lipid raftsc for EhV86-infected E. huxleyi cells at 2 h post infection.
Protein IDb
Protein description
Protein log e
# of unique
peptides
detected
444491
363433
444671
366130
439740d
432658
442092
440685d
437063
436031d
446310
441334
358662
420185
433847
360139
246130
439254d
46509
75032d
Outer membrane protein porin
Band_7; flotillin-like protein
Protein of unknown functione
Chlorophyll a/b binding protein
membrane-bound proton-translocating pyrophosphatase
Mitochondrial import receptor subunit
Heat shock protein 70
High affinity nitrate transporter
Mitochondrial carrier protein
Delta-carbonic anhydrase
Chlorophyll a/b binding protein
Type 1 actin
Chloroplast light harvesting protein
Chloroplast light harvesting protein
Chloroplast light harvesting protein
Mitochondrial substrate carrier
Histone H4
Nitrite transporter
Chloroplast light harvesting protein
H ± translocating pyrophosphatase
−160.6
−109.1
−73.5
−72.4
−71.2
−67
−62
−61.1
−59.8
−58.9
−58.2
−56.2
−55.9
−53.1
−53
−49.9
−46.8
−35.5
−35.2
−32.9
19
14
11
9
9
7
9
9
7
8
6
8
5
3
7
6
7
5
5
5
a.
b.
c.
d.
e.
Protein
MW (kDa)
31.6
33.5
19.6
20.2
93.7
30.9
71.8
73.3
33.3
72.4
22.6
41.6
21.9
15.6
30
36.7
11.4
29.3
18
75.8
Based on rank order of Protein log e.
JGI (filtered best models; http://genome.jgi-psf.org/).
Fraction #2 from 2 h.
Contains predicted transmembrane domains.
No Gene Ontology, Conserved Domains, or BlastP hits with e-value < 0.
thus corroborating our anti-flotillin Western blot analyses
and further supporting fraction 2 as a representative bona
fide lipid raft fraction. Our proteomic analysis also identified several known membrane proteins, with predicted
transmembrane domains, including an outer membrane
porin, nitrate/nitrite transporters, an import receptor, a
sodium/calcium exchange membrane protein and a substrate carrier protein. Carrier proteins are integral membrane proteins that are involved in the movement of ions,
small molecules or macromolecules, such as another
protein, across biological membranes. These all provided
confidence in the membrane-containing nature of our lipid
raft proteome. Interestingly, we also detected a variety of
chlorophyll a/b binding proteins among our top 20 protein
hits. The chlorophyll pigment distribution in OptiPrep
density gradients did not coincide with lipid raft, DRM
fractions (Supporting Information Fig. S2), indicating that
these fractions were not contaminated with bulk chloroplast material. Instead, these findings suggest that lipid
rafts (and associated proteins) are also features of
subcellular organelle membranes, not surprising given the
roles that lipid rafts play in normal cell trafficking.
We wanted to better differentiate the ‘basal’ host lipid
raft proteome from a virus-manipulated one by identifying
proteins that were uniquely associated with respective
uninfected (Supporting Information Table S2) and EhV86infected (Tables S3) cell states. We were particularly
interested in the pool of stress-, PCD- and/or innate
immunity-related proteins associated with lipid rafts given
the established connections of these cellular processes
with EhV infection (Bidle et al., 2007; Vardi et al., 2009;
Bidle and Vardi, 2011; Bidle and Kwityn, 2012). Although
our analysis admittedly included many singleton protein
fragments with low Blast-e confidence scores, it nonetheless revealed that E. huxleyi cells not only have a diverse,
basal lipid raft proteome composition, but that EhVs fundamentally alter this composition during early infection
processes.
The ‘basal’ lipid raft proteome (Supporting Information
Table S2) of healthy E. huxleyi cells encompassed a wide
range of proteins, whose homologues and evolutionary
conserved domains are widespread in higher eukaryotes
and are involved in the regulation of normal cell function.
These included nutrient transporters, DNA- and RNAbinding proteins, protein kinases and phosphatases,
a cell division regulator protein, fibronectin, ferredoxin,
tetratricopeptide repeat proteins and ankyrins. We also
identified a WD40-containing Sec31-like secretory protein
that is integral to specialized membrane domains and act
as transporters, scaffolds or adaptors to mediate protein–
protein interactions, intracellular trafficking or vesicle
transport (Mohler et al., 2002; Cortajarena and Regan,
2006; Suman et al., 2011). Proteins with notable stress
and defence domain functions were also associated with
healthy E. huxleyi lipid rafts, such as phosphoinositidebinding Phox, a U-Box/Ring finger, patatin and RAP RNA-
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Lipid rafts in Emiliania huxleyi
binding domains. Phox-like proteins possess an ability to
generate superoxide (Kawahara and Lambeth, 2007) and
play an important role in host defence through a
microbiocidal complex. U-box domain proteins, most commonly found in plants, act as negative regulators in cell
death signalling (Zeng et al., 2008). Patatin proteins, a
group of glycoproteins also found in plants, have lipolytic
activity and participate in defence and signal transduction
pathways (Banerji and Flieger, 2004). RAP RNA-binding
domains are important in various parasite–host cell interactions in a variety of eukaryotic host species.
Of the 73 proteins that were uniquely associated with
lipid rafts of the 2 hpi EhV86-infected cell state (Supporting Information Table S3), several notable candidates
have putative function in lipid trafficking, signal transduction processes, pathogen defence and innate immune
response. CRAL/TRIO domain-containing proteins regulate intracellular trafficking of hydrophobic ligands by
binding small lipophilic molecules (Panagabko et al.,
2003). The calmodulin-binding DENN/MADD domaincontaining proteins are involved in mitogen-activated
protein (MAP) kinase induction (Schievella et al., 1997), a
known mediator of stress-induced PCD, and they serve
as regulators of Rab GTPases (Marat et al., 2011), which
control membrane trafficking. Proline-rich extensins
(PRICHEXTENSN) function in the signal transduction of
pathogen defence upon compromised cell wall structure
(Silva and Goring, 2002; Sanabria et al., 2010). Toll interleukin 1 receptor (TIR) and leucine rich repeat (LRR)
domain proteins, which are often connected through a
nucleotide-binding (NB) domain, collectively mediate
pathogen recognition/resistance and activate host cell
defence responses (Peart et al., 2005; Swiderski et al.,
2009). TIR-NB-LRR proteins have actually been shown to
specially recognize viral membrane proteins through
ligand–receptor interaction resulting in the stressinduced, plant hypersensitive PCD response (Heath,
2000; Nimchuk et al., 2003). Membrane-associated
proteophosphoglycans and regulator of chromosome
condensation 1 proteins are also particularly interesting
candidates given their respective involvement in parasite
evasion of host cell immune systems (Aebischer et al.,
1999) and regulation of parasite virulence through controls on efficient nuclear trafficking (Frankel et al., 2007).
The detection of an EhV86 C-type lectin 1 domaincontaining membrane protein (ehv149; Q4A2Y5) among
the EhV86-infected lipid rafts directly implicates lipid
raft domains as viral entry and/or exit sites. This protein
was among the 23 putative, transmembrane domaincontaining proteins detected in the mature EhV virion
proteome (Allen et al., 2008). Denaturing polyacrylamide
gel electrophoresis of the mature EhV virion proteome
detected a prominent ∼40 kDa band (Allen et al., 2008)
that could be composed of a combination of four proteins:
11
ehv067, ehv100, ehv149 and ehv175 with predicted
weights of 41.9, 40.0, 40.0, 40.6 kDa respectively. Consequently, EhV virions could conceivably be enriched in
ehv149. We wanted to constrain that ehv149 and other
mature EhV proteins would not migrate to buoyant fractions on their own accord upon Brij-96 treatment. The
most abundant EhV protein is the MCP (Allen et al.,
2008), accounting for ∼40% of total virome protein mass in
Phycodnaviridae (Nandhagopal et al., 2002). If EhV proteins, like ehv149, migrated to Fraction #2 on their own
accord, the MCP protein should have been detected in our
proteomic analysis, but it was not. Furthermore, it is highly
unlikely that any protein would migrate to Fraction #2 on
its own accord, based on the inherent biophysical relationship between protein buoyant densities and molecular
weight. Using a comprehensive biophysical dataset of
protein mass density of proteins, Fischer and colleagues
(2004) showed that the mass density of proteins ranges
from 1.34 g cm−3 (∼160 kDa proteins) to 1.42 g cm−3
(< 20 kDa proteins), inversely scaling with molecular
weight. Proteins of ∼40 kDa have an inherent mass
density of ∼1.37 g cm−3 that would place ehv149 at the
bottom of the OptiPrep gradient (see Fig. 3A), along
with all other free proteins. The only conceivable way
that ehv149 could migrate to buoyant Fraction #2
(1.055 g cm−3) is if it was associated with DRMs/lipid rafts.
The C-type lectin-containing proteins are classic ligandbinding partners for toll-like receptors/TIR (Neilan et al.,
1999). C-type lectin-containing proteins, involved in cell–
cell adhesion and endocytosis, have been reported in
poxviruses and African swine fever virus mediating the
early events involving host cell attachment and subsequent internalization (Neilan et al., 1999; Cambi et al.,
2005). Microarray analysis has classified expression of
ehv149 transcripts to a 2 hpi ‘window’ (Allen et al.,
2006b), and laser confocal microscopy has confirmed an
early stage (< 4 hpi), ‘chronic-like’ replication and release
of EhV virions (Mackinder et al., 2009), so it is unclear if
our proteomic analysis represents entry or exit of EhVs.
Nonetheless, it implicates a possible protein–protein
(TIR-NB-LRR/C-type Lectin) specific binding interaction
between E. huxleyi CCMP1516 and EhV86 for the successful attachment to host cell membranes for viral
entry or exit through lipid rafts (Neilan et al., 1999;
Nimchuk et al., 2003; Cambi et al., 2005; Swiderski et al.,
2009). Notably, the other C-type lectin 2 domaincontaining protein (ehV060) in the mature EhV proteome
(Allen et al., 2008) was not detected in our lipid raft
proteome analysis so it is unclear whether it also interacts
with lipid rafts. A truncated version of this particular
protein (ehv060) resides in EhV163 (Allen et al., 2006b),
another Coccolithovirus that, unlike EhV86, is unable
to infect E. huxleyi CCMP2090 (previously E. huxleyi
CCMP1516B). In light of our lipid raft proteome data, it is
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
12
S. L. Rose et al.
tempting to speculate that the interactions of these C-type
lectin-containing proteins with lipid raft proteins are key
conduits to successful infection and targets of cellular
resistance mechanisms.
or flow cytometry (details below). EhV86 was propagated
using Ehux1516 as previously described (Bidle et al., 2007).
Virus abundance was determined by SYBR Gold staining and
analytical flow cytometry (details below).
Virus infection
Conclusion
We demonstrated the successful isolation of lipid rafts
using host-derived GSLs and flotillin as respective lipid
and protein biomarkers, in order to characterize raftassociated proteins involved in host–virus interactions.
Our data provide the first direct biomolecular evidence of
EhV interaction with lipid rafts, whereby they profoundly
alter and shape their lipid and protein composition, in part
through the induction of PCD- and innate immune
response-related proteins. Further research on the functionality of raft-associated proteins and their response to
virus infection will elucidate cross-talk pathways between
these proteins and others triggering downstream PCD
and/or innate immunity pathways, and/or in facilitating
viral entry and exit strategies. The application of methods
used in this study to various sensitive and resistant strains
of E. huxleyi to EhV86 infection will likely further implicate
raft-associated proteins as critical determinants host–viral
interactions.
Experimental procedures
Bioinformatic analysis
The completed Emiliania huxleyi CCMP1516 genome
assembly [http://genome.jgi-psf.org/Emihu1/Emihu1.home
.html; v1.0; (Read et al., 2013)] was used to search, identify
and characterize the E. huxleyi flotillin-like protein (Protein ID
363433). The EhV86 genome (accession# NC 007346) was
accessed through the National Center for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/). BLASTP (http://
blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1990) was
used for initial protein characterizations, with subsequent
protein sequence analyses used the Simple Modular Architecture Research Tool (http://smart.embl.de/) (Shultz et al.,
1998; Letunic et al., 2012). Secondary and tertiary structure
analyses employed Protein Homology/Analog Y Recognition
Engine V2.0 (PHYRE2) (http://www.sbg.bio.ic.ac.uk/phyre2/
html/page.cgi?id=index) (Kelly and Sternberg, 2009) for
protein fold recognition and PyMOL molecular visualization system (http://www.pymol.org/) for modelling threedimensional structures.
Host and virus growth and maintenance
Emiliania huxleyi strain CCMP1516 (Ehux1516) was
obtained from the Provasoli-Guillard Center for Culture for
Marine Phytoplankton and grown in f/2-Si (minus silica) under
14:10 (L:D) cycle, 200 μmol photons m−2 s−1 at 18°C with
constant aeration. Cell abundance was determined using a
Coulter Multisizer II (Beckman Coulter, Fullerton, CA, USA)
Experiments were designed to harvest biomass from
both uninfected, ‘control’ Ehux1516 and EhV86-infected
Ehux1516 at 2, 24, 48 and 72 h post-infection (hpi). Exponentially growing cultures (5 L; ∼2 × 105 cells per ml) of
Ehux1516 were infected with viral strain EhV86 at a multiplicity of infection (MOI) of 2. A control culture without addition
of EhV86 was performed in parallel. Cultures were grown
according to the conditions mentioned above. For each time
point, 1 L from each culture was harvested via vacuum filtration, snap frozen in liquid N2 and stored at −80°C until processed. Samples were also collected for cell and viral counts
for each time point and analysed daily by analytical flow
cytometry (details below).
Flow cytometry analysis
Samples from each time point were counted on an Influx
Mariner 209 s Flow Cytometer and High Speed Cell Sorter
(BD Biosciences, San Jose, CA, USA) at the Rutgers
Microbial Flow Sort Lab (http://marine.rutgers.edu/flowsort/
index.html). Emiliani huxleyi cells were counted using chlorophyll fluorescence (692/40 nm) after triggering with forward
scatter (granularity). EhV86 abundance was determined by
fixation and staining with SYBR Gold, as previously
described (Brussaard et al., 2000). Briefly, samples (40 μl)
were fixed in 1% glutaraldehyde at 4°C for 15 min, −80°C for
5 min, and then room temperature, followed by staining in
0.5X SYBR Gold (Life Technologies, Grand Island, NY, USA)
in TE buffer [10 mM Tris-Cl (pH 7.8), 1 mM EDTA)] at 80°C
for 10 min in the dark. Stained samples were then counted
using green fluorescence (520 nm) after triggering with SSC
(size).
Lipid raft isolation
Frozen pellets from 2, 24, 48 and 72 h time points were
brought to 4°C on ice and suspended in 1 ml lysis buffer
(0.5% Brij-96, 25 mM Tris-HCl pH 8.0, 140 mM NaCl pH 8.0,
1 mM PMSF), which used anon-ionic detergent (Brij-96;
Sigma-Aldrich, St. Louis, MO, USA) to solubilize all membrane and a protease inhibitor to prevent protein degradation.
Samples were placed on ice for 30 min followed by lysate
clarification for 30 min via centrifugation at 4°C (4000 g). The
resulting supernatants were removed and stored at −20°C for
use in OptiPrep (Sigma) density gradient centrifugation. Corresponding pellets were stored at −80°C.
DRM fractions from total cell lysates were isolated using
5–35% discontinuous OptiPrep density gradients. The supernatant from each time point was added to OptiPrep to obtain
a 45% solution and placed at the bottom of 13 ml ultra-clear
centrifuge tubes (Beckman #344059). The 45% OptiPrep/
sample mixture was sequentially overlaid with 35% and 5%
OptiPrep solutions respectively. A blank density gradient (with
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Lipid rafts in Emiliania huxleyi
no added cell lysate to the 45% OptiPrep solution) was run in
parallel. Density gradients were centrifuged using a SW40 Ti
Beckman rotor at 39 000 × g, 4°C, for 16 h (protocol adapted
from Macdonald and Pike, 2005). Twelve 1 ml fractions were
collected by pipette in a top down fashion. Individual fractions
(1–12) were collected from each time point (200 μl) and
stored at −80°C for subsequent proteomic, lipidomic, Western
immunoblotting, PCR and TEM analyses.
Western blot analysis
Equal volumes of density gradient subsamples were loaded
and separated by SDS-PAGE (Criterion TGX 4–20% gels;
Bio-Rad Laboratories, Inc., Hercules, CA, USA) and
eletrophoretically transferred to PVDF membranes in transfer buffer (200 ml methanol, 3.03 g Tris, 14.4 g glycine per
1 L of MilliQ H20) at 100 V for 1 h on ice. Membranes were
probed with an anti-human flotillin 2 monoclonal antibody
(1:500; #sc-25507; Santa Cruz Biotechnology, Santa Cruz,
CA, USA) followed by polyclonal goat anti-rabbit secondary
antibody (1:20 000; #sc-2004; Santa Cruz Biotechnology)
and horseradish peroxidase chemiluminescence detection
(SuperSignal; Pierce, Rockford, IL, USA) using ChemiDoc
MP System imager and Image Lab Software version 4.0.1
(Bio-Rad Laboratories, Inc.). Protein Plus (Bio-Rad #161–
0374) was used as a protein molecular weight standard.
Human A-275 whole cell lysate of malignant melanoma
cells (#sc-3811; Santa Cruz Biotechnology) was used as a
positive control.
TEM
TEM was used to visualize the presence and size of DRM
vesicles. Fractions were dialysed against 200 mM Hepes
buffer (pH 7.5). Dialysis of each fraction (200 μl) was
achieved using a Pierce Slide-A-Lyzer 10 K (10 000 mwco)
dialysis cassette with 0.1–0.5 ml sample volume (Thermo
Scientific, Rockford, IL, USA) following manufacturer’s protocol. Samples were prepared for TEM using an adapted negative staining method. Formvar/carbon-coated mesh grids
(Electron Microscopy Sciences, Hatfield, PA, USA) were subjected to ultraviolet light to remove static interference enhancing sample binding then placed on top of the dialysed sample
for 1 min. Excess sample was wicked off using Whatman
paper (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA)
and then placed sample side down onto 1% uranyl acetate
solution for 1 min. Excess stain was wicked off and grids
were allowed to air dry 5–10 min before visualization.
Samples were visualized at the Rutgers Electron Imaging
Facility (http://cbn.rutgers.edu/emlab/emlab.html) using a
JEOL 100 CX Electron microscope (JOEL Ltd, Tokyo, Japan)
operating at 80 kV. Digitized images were viewed and captured using a Gatan CCD 673-0200 camera (Gatan Inc,
Warrendale, PA, USA) connected to two high-resolution
video monitors.
PCR amplification
PCR was used to amplify the EhV MCP using previously
published primer sequences [(Schroeder et al., 2003); EhV-
13
MCP-F1, 5′-GTC TTC GTA CCA GAA GCA CTC GCT-3′;
EhV-MCP-R1 5′-ACG CCT CGG TGT ACG CAC CCT CA-3′].
PCR reaction conditions were as follows: 5 μl of each fraction
was directly added to a 15 μl reaction cocktail mixture containing 1 U RedTaq DNA polymerase (Sigma), 1 × PCR reaction buffer (Promega, Madison, WI, USA), 0.25 mM dNTPs
and 30 pmol of each primer. PCR reactions were run in a
Mastercycler EPgradient S (Eppendorf, NY, USA) with an
initial high temperature cycle to lyse intact viral capsids (8°C,
65°C, 97°C × 3 cycles) followed by an initial denaturing step
of 94°C (5 min) and 30 cycles of denaturation at 94°C (30 s),
annealing at 55°C (30 s), and extension at 72°C (45 s). PCR
amplicons were run on a 1% agarose gel (w/v) in 1 × TAE at
80 V for 1.5 h, stained with ethidium bromide (0.5 μg ml−1)
and visualized using ChemiDoc MP System imager and
Image Lab Software version 4.0.1 (Bio-Rad Laboratories). EZ
Load 100 bp molecular weight marker (Bio-Rad; Cat #170–
8352) was used as a DNA sizing standard. Purified E. huxleyi
genomic DNA and fresh EhV86 viral lysate served as negative and positive template controls respectively.
Lipidomic analysis
Subsamples (100 μl) from OptiPrep gradient fractions were
analysed for intact polar lipids using a Thermo TSQ Vantage
triple quadrupole mass spectrometer with electrospray ionization at the Woods Hole Oceanographic Institution (Woods
Hole, MA, USA). Lipids were extracted using a modified
Bligh–Dyer method, as previously described (Bligh and Dyer,
1959; Popendorf et al., 2013). Cellular polar membrane lipids
were separated by high-performance liquid chromatography
(HPLC) as described (Sturt et al., 2004; Popendorf et al.,
2013) using an Agilent 1200 HPLC. Brij-96 and OptiPrep
reagents at high concentrations interfere with the detection of
IPL molecular ions using typical ion trap MS (ITMS) methods
(Sturt et al., 2004; Van Mooy et al., 2011) thereby necessitating detection and quantitation by characteristic MS to MS2
neutral losses by triple quadrapole mass spectrometry
(Popendorf et al., 2011; 2013). The total lipid extracts from
infected and uninfected E. huxleyi CCMP1516 cultures were
used for the initial identification and tuning parameters of
hGSL (neutral loss of 180 Da), vGSL (−162 Da) and sGSL
(−268 Da), which were detected previously by HPLC-ESIITMS (Vardi et al., 2009; Vardi et al., 2012; Fulton et al., 2014
#2218}. rGSL was detected only in low density raft fractions
using the parameters for vGSL, but due to its relatively early
elution time, it is thought to be structurally distinct from vGSL.
A subset of the samples were analysed using identical HPLC
conditions on a Thermo LTQ FT Ultra high-resolution Fouriertransform ion cyclotron resonance mass spectrometer for
confirmation of the elemental formulas of hGSL, vGSL and
sGSL molecular ions and MS2 fragment ions.
Chlorophyll a was detected as an early eluting peak
at the beginning of the intact polar lipid normal phase chromatographic method. It was identified by its characteristic
photodiode array ultraviolet (UV)-visible spectrum with
absorbance maxima at 431 nm (Soret band) and 665 nm (Qy
band), as well as smaller peaks at 617, 580 and 535 nm. We
did not detect pheophytin a or any other chlorophyll degradation products and assume that any degradation from sample
storage in dichloromethane under Argon or low pigment
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
14
S. L. Rose et al.
yields due to inefficient extraction by the Bligh and Dyer
method are proportional in all samples. We present relative
quantitation based on chromatographic peak area integration
at 665 nm. UV-visible light spectra showed that there were
no interfering compounds absorbing in the Qy band. The peak
areas were normalized to our internal recovery standard,
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(2,4dintrophenyl) (DNP-PE), which elutes later and has an absorbance maximum at 340 nm.
Proteomic analysis
Frozen lipid raft fractions were sent to the Biological Mass
Spectrometry Facility co-run by the UMDNJ-Robert Wood
Johnson Medical School and Rutgers’ Center for Advanced
Biotechnology and Medicine (http://www3.cabm.rutgers.edu/
home.php) for proteomic analysis via LC-MS/MS. Proteins
were concentrated by vacuum aspiration (200 μl samples
were reduced to 20 μl) and run ∼1 cm into a Novex gel
Bis-Tris 10% gel (Life Technologies, Carlsbad, CA, USA).
The entire gel band was excised and proteins therein were
reduced, carboxymethylated and digested with trypsin using
standard facility protocols. Peptides were extracted, solubilized in 0.1% trifluoroacetic acid and analysed by nanoLCMS/MS using a RSLC system (Dionex, Sunnyvale, CA, USA)
interfaced with a Velos-LTQ-Orbitrap (ThermoFisher, San
Jose, CA, USA). Samples were loaded onto a self-packed
100 μm × 2 cm trap packed with Magic C18AQ, 5 μm 200 A
(Michrom Bioresources, Auburn, CA, USA) and washed with
‘Buffer A’ (0.2% formic acid) for 5 min with flow rate of
10 μl min−1. The trap was brought in-line with the homemade
analytical column (Magic C18AQ, 3 μm 200 A, 75 μm ×
50 cm) and peptides fractionated at 300 nL min−1 using
multi-step gradients of ‘Buffer B’ (0.16% formic acid 80%
acetonitrile) consisting of 4–25% over 60 min and 25–55%
over 30 min). Mass spectrometry data were acquired using a
data-dependent acquisition procedure with a cyclic series of
a full scan acquired in Orbitrap with resolution of 60 000
followed by MSMS scans (acquired in linear ion trap) of 20
most intense ions with a repeat count of two and the dynamic
exclusion duration of 60 s.
The LC-MS/MS data were searched against the complete
annotated E. huxleyi CCMP1516 (filtered best models; http://
genome.jgi-psf.org/) and EhV86 protein databases, with the
latter being parsed from Uniprot database and cRAP (http://
www.thegpm.org/crap/). Analyses used a local version of the
Global Proteome Machine (GPM cyclone, Beavis Informatics,
Winnipeg, Canada) with carbamidomethyl on cysteine as
fixed modification and oxidation of methionine and tryptophan
as variable modifications using a 10 ppm precursor ion tolerance and a 0.4 Da fragment ion tolerance.
Acknowledgements
We thank Haiyan Zheng at the Rutgers University Biological
Mass Spectrometry Facility for assistance in proteomic analyses and Dr. Helen Fredricks (Woods Hole Oceanographic
Institution) for her assistance and guidance with HPLC/
TQ-MS analyses. This study was supported by funding from
the National Science Foundation (OCE-1061883 to KDB and
BVM) and in part by The Gordon and Betty Moore Foundation. The authors have no conflicts of interest with regard to
this research.
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Supporting information
Additional Supporting Information may be found in the online
version of this article at the publisher’s web-site:
Fig. S1. Flow cytometry plots showing the relationship
between side scatter (SSC) and fluorescence (520 nm) for
SYBR-Green-stained, untreated (upper panel) or Brij-treated
(lower panel) EhV virions.
Fig. S2. Chlorophyll a distribution in OptiPrep density gradient fractions (1–12) for uninfected control and EhV86-infected
Emiliania huxleyi at 2, 48 and 72 h post infection. Chlorophyll
a was detected as an early eluting peak at the beginning of the
intact polar lipid normal phase chromatographic method (see
Experimental procedures). Note that Chlorophyll a distribution
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology
Lipid rafts in Emiliania huxleyi
is preferentially enriched in fractions 5–10 and does not
overlap with buoyant DRM, lipid raft fractions.
Table S1. Detailed breakdown of the fraction distribution of
vGSL species# in OptiPrep density gradients for EhV86infected E. huxleyi cells at 2, 48 and 72 h post infection.
#
17
Table S2. List of proteins uniquely associated with lipid
rafts‡ of control Emiliania huxleyi cells.
Table S3. List of proteins uniquely associated# with lipid
rafts‡ for EhV86-infected Emiliania huxleyi cells at 2 h post
infection.
© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology