Plasmodium falciparum is dependent on de novo myo‐inositol biosynthesis for assembly of GPI glycolipids and infectivity

ArticleinMolecular Microbiology 91(4) · December 2013with100 Reads
Impact Factor: 4.42 · DOI: 10.1111/mmi.12496 · Source: PubMed
Abstract

Intraerythrocytic stages of the malaria parasite, Plasmodium falciparum, are thought to be dependent on de novo synthesis of phosphatidylinositol, as red blood cells (RBC) lack the capacity to synthesize this phospholipid. The myo-inositol headgroup of PI can either be synthesized de novo or scavenged from the RBC. An untargeted metabolite profiling of P. falciparum infected RBC showed that trophozoite and schizont stages accumulate high levels of myo-inositol-3-phosphate, indicating increased de novo biosynthesis of myo-inositol from glucose-6-phosphate. Metabolic labelling studies with (13) C-U-glucose in the presence and absence of exogenous inositol confirmed that de novo myo-inositol synthesis occurs in parallel with myo-inositol salvage pathways. Unexpectedly, while both endogenous and scavenged myo-inositol was used to synthesize bulk PI, only de novo-synthesized myo-inositol was incorporated into GPI glycolipids. Moreover, gene disruption studies suggested that the INO1 gene, encoding myo-inositol 3-phosphate synthase, is essential in asexual parasite stages. Together these findings suggest that P. falciparum asexual stages are critically dependent on de novo myo-inositol biosynthesis for assembly of a sub-pool of PI species and GPI biosynthesis. These findings highlight unexpected complexity in phospholipid biosynthesis in P. falciparum and a lack of redundancy in some nutrient salvage versus endogenous biosynthesis pathways.

Full-text

Available from: James I Macrae, Mar 12, 2014
Plasmodium falciparum
is dependent on
de novo myo
-inositol
biosynthesis for assembly of GPI glycolipids and infectivity
James I. MacRae,
1†
Sash Lopaticki,
2
Alexander G. Maier,
2‡
Thusitha Rupasinghe,
3
Amsha Nahid,
3
Alan F. Cowman
2
and
Malcolm J. McConville
1
*
1
Department of Biochemistry and Molecular Biology,
Bio21 Institute of Molecular Science and Biotechnology,
30 Flemington Road, University of Melbourne,
Melbourne, Vic. 3010, Australia.
2
Division of Infection and Immunity, The Walter and
Eliza Hall Institute of Medical Research, Parkville, Vic.
3052, Australia.
3
Metabolomics Australia, Bio21 Institute of Molecular
Science and Biotechnology, 30 Flemington Road,
University of Melbourne, Melbourne, Vic. 3010,
Australia.
Summary
Intra-erythrocytic stages of the malaria parasite, Plas-
modium falciparum, are thought to be dependent on de
novo synthesis of phosphatidylinositol, as red blood
cells (RBC) lack the capacity to synthesize this phos-
pholipid. The myo-inositol headgroup of PI can either
be synthesized de novo or scavenged from the RBC.
An untargeted metabolite profiling of P. falciparum
infected RBC showed that trophozoite and schizont
stages accumulate high levels of myo-inositol-3-
phosphate, indicating increased de novo biosynthesis
of myo-inositol from glucose 6-phosphate. Metabolic
labelling studies with
13
C-U-glucose in the presence
and absence of exogenous inositol confirmed that de
novo myo-inositol synthesis occurs in parallel with
myo-inositol salvage pathways. Unexpectedly, while
both endogenous and scavenged myo-inositol was
used to synthesize bulk PI, only de novo-synthesized
myo-inositol was incorporated into GPI glycolipids.
Moreover, gene disruption studies suggested that
the INO1 gene, encoding myo-inositol 3-phosphate
synthase, is essential in asexual parasite stages.
Together these findings suggest that P. falciparum
asexual stages are critically dependent on de novo
myo-inositol biosynthesis for assembly of a sub-pool
of PI species and GPI biosynthesis. These findings
highlight unexpected complexity in phospholipid bio-
synthesis in P. falciparum and a lack of redundancy in
some nutrient salvage versus endogenous biosynthe-
sis pathways.
Introduction
Malaria remains a major global health challenge, with
almost half of the world’s population at risk. More than 200
million cases of malaria occur each year resulting in 0.6
million deaths (Miller et al., 2013; WHO, 2013). There is no
effective, safe anti-malaria vaccine and resistance to front-
line drugs is an on-going threat (Dondorp et al., 2009).
Several species of Plasmodium are capable of causing
malaria in humans, with Plasmodium falciparum being the
most important in terms of morbidity and mortality. Disease
is associated with the development of asexual stages that
undergo repeated cycles of invasion and rapid intracellular
replication in red blood cells (RBCs). Infection of RBCs is
initiated by the merozoite stage, which subsequently dif-
ferentiates through several intermediate developmental
stages (ring, trophozoite and schizont) within the parasite-
induced parasitophorous vacuole to produce 16–32 new
merozoites over a 48 hour period (Boddey and Cowman,
2013). This developmental cycle requires the synthesis of
new membrane for production of daughter parasites, as
well as the maintenance of the surrounding parasito-
phorous vacuole and an extensive system of membrane
tubules and cisternae in the RBC cytoplasm that are used
to export parasite proteins and lipids to the RBC plasma
membrane (Besteiro et al., 2010). Additional membrane
lipids may also be needed to sustain increased shedding
of membrane vesicles from the P. falciparum-infected
RBC (Mantel et al., 2013; Regev-Rudzki et al., 2013). This
demand for new membrane is dependent on the upregu-
lation of multiple pathways of phospholipid biosynthesis in
intra-erythrocytic parasite stages as well as the salvage of
essential precursors from the host cell (Besteiro et al.,
2010). The dissection of these pathways has already led
Accepted 15 December, 2013. *For correspondence. E-mail
malcolmm@unimelb.edu.au; Tel. (+61) 3 8344 2342; Fax (+61) 3
9348 1421. Present addresses:
The National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK;
Research
School of Biology, The Australian National University, Acton, ACT
0200, Australia.
Molecular Microbiology (2014) 91(4), 762–776 doi:10.1111/mmi.12496
First published online 16 January 2014
© 2013 John Wiley & Sons Ltd
Page 1
to the development of novel inhibitors that target asexual
stages (Ben Mamoun et al., 2010).
Most studies on phospholipid biosynthesis in P. falcipa-
rum have focussed on the major phospholipid classes,
phosphatidylcholine and phosphatidylethanolamine, and
less is know about the synthesis of phosphatidylinositol
(PI). While accounting for less than 10% of the total mem-
brane phospholipid (Botté et al., 2013), it fulfils a number
of essential functions in intra-erythrocytic stages. In addi-
tion to contributing to bulk lipid composition of mem-
branes, PI is the precursor for free and protein-linked
glycosylphosphatidylinositol (GPI) glycolipids, as well as
complex phosphoinositides. Free GPI glycolipids and
GPI-anchored proteins dominate the surface of asexual
stages (Gerold et al., 1994; Naik et al., 2000; Sanders
et al., 2005), have important immunomodulatory proper-
ties and mediate essential host-parasite interactions
(Sanders et al., 2005; Debierre-Grockiego and Schwarz,
2010), suggesting that GPI biosynthesis is essential for
virulence. PI is also phosphorylated to form phospho-
inositides, such as PI-3-phosphate (PI3P) and PI4P, that
are proposed to have important roles in regulating protein
traffic from the parasite to the RBC membrane, as well as
the biogenesis of essential parasite organelles, such as
the food vacuole and the apicoplast (Tawk et al., 2010;
2011; Bhattacharjee et al., 2012a,b).
RBC contain very low levels of PI and lack the
enzymes needed for the de novo synthesis of PI (Allan,
1982), suggesting that intra-erythrocytic stages of P. fal-
ciparum are largely dependent on endogenous PI syn-
thesis (Elabbadi et al., 1994). De novo PI synthesis
involves the condensation of myo-inositol with CDP-
diacylglycerol by the membrane-bound enzyme, PI syn-
thase (PIS), located in the ER or other organelles in the
secretory pathway (Elabbadi et al., 1994; Wengelnik and
Vial, 2007; Michell, 2008). The myo-inositol head-group
could either be scavenged from the infected red blood
cell or synthesized de novo in a two-step pathway involv-
ing the initial conversion of glucose 6-phosphate to
myo-inositol 3-phosphate and the subsequent de-
phosphorylation of myo-inositol 3-phosphate to myo-
inositol (Majumder et al., 2003; Fischbach et al., 2006).
The P. falciparum genome encodes a putative myo-
inositol synthase (INO1) and two putative inositol-
phosphate phosphatases (IMPase), that catalyse the two
steps in de novo synthesis pathway and all three genes
are transcribed in intra-erythrocytic stages (Le Roch
et al., 2003; Besteiro et al., 2010). On the other hand,
infected RBCs and the surrounding serum contain rela-
tively high levels of myo-inositol (>200 μM) and exog-
enous
3
H-inositol is incorporated into PI lipids in
P. falciparum intra-erythrocytic asexual stages indicating
that salvage pathways are active (Elabbadi et al., 1994).
Whether myo-inositol salvage and de novo synthesis rep-
resent parallel or redundant mechanisms for generating
myo-inositol for PI synthesis remains undefined.
In this study, we have reassessed myo-inositol metabo-
lism in these parasites using metabolite profiling,
13
C-
glucose stable isotope labelling and genetic approaches.
We find that P. falciparum intra-erythrocytic stages accu-
mulate high levels of inositol 3-phosphate (Ino3P) and
that de novo-synthesized myo-inositol is incorporated into
both bulk PI as well as a subcellular pool of PI that is used
for GPI glycolipid biosynthesis. In contrast, scavenged
myo-inositol is primarily used for synthesis of bulk PI. Our
findings highlight unanticipated complexity in the inositol
metabolism of these parasites and suggest that enzymes
involved in myo-inositol biosynthesis are novel target of
new antimalarial drugs.
Results
Stage-specific differences in inositol metabolism of
P. falciparum
Metabolite profiling was used to measure changes in the
level of intracellular polar metabolites in P. falciparum
asexual RBC stages over the 48 h cycle. Synchronized
infected RBC cultures (8% parasitaemia) were sampled
at various time points and polar metabolites analysed
by GC-MS. Of the 250 metabolite peaks detected, 100
metabolites were identified by reference to authentic
standards. The metabolite profiles of unfractionated cul-
tures containing infected (iRBC) and uninfected (uRBC)
RBC, clearly changed during the development of intracel-
lular parasite stages through ring, trophozoite and schizont
stages, as shown by multivariate principal component
analysis (PCA) analysis (Fig. 1A). These cultures were
also clearly distinguished from RBCs that had never been
exposed to P. falciparum (nRBC in Fig. 1A). To further
identify metabolite changes associated with parasite
development, polar metabolite levels in trophozoite- and
schizont-infected RBC and corresponding uninfected RBC
isolated from the same culture were quantified by GC-MS
(Fig. 1B). All stages were again clearly separated from
each other (Fig. 1B), indicating marked changes in
metabolite levels of different late asexual stages. Univari-
ate analysis of uRBCs and iRBCs from trophozoite-
enriched (Fig. 1C) and schizont-enriched (Fig. 1D)
cultures identified a number of metabolites that were sig-
nificantly upregulated during infection. Consistent with a
number of recent studies (Olszewski et al., 2009; Teng
et al., 2009; Macrae et al., 2013; Sana et al., 2013), the
steady-state concentrations of intermediates in glycolysis
(e.g. fructose 6-phosphate, α-glycerophosphate and phos-
phoenolpyruvate), the pentose phosphate pathway (e.g.
ribose 5-phosphate and sedo-heptulose 7-phosphate),
and the TCA cycle (e.g. succinate, fumarate and malate)
Inositol metabolism in
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Fig. 1. Multivariate analysis of P. falciparum-infected RBC cultures.
A. Polar metabolites were extracted from synchronized P. falciparum 3D7 RBC cultures (8% parasitaemia) enriched for ring (ring-iRBC),
trophozoite (troph-iRBC), and schizont (schiz-iRBC) stages and analysed by GC/MS. Principal component analysis (PCA) of the data matrix
generated by progressive clustering and deconvolution of GC-MS chromatograms. The x and y axes represent the principal components
accounting for the greatest (29.8%) and second greatest (22.4%) variability respectively.
B. Polar metabolites were extracted from infected and uninfected RBCs (iRBC/uRBC) obtained from trophozoite (troph) and schizont (schiz)
enriched synchronized cultures. The x and y axes of the PCA plots represent the principal components accounting for 30.1% and 21.0%
variability respectively.
C and D. Targeted Z-score plots of selected metabolites in trophozoite (C) and schizont (D) iRBCs. Plotted z-scores show the mean and
standard deviations of individual metabolites in 8 replicate analyses normalized to the appropriate uRBC sample set (also 8 replicates). Filled
grey circles refer to metabolite levels in uRBCs (which typically cluster within 5 standard deviations of the mean) while filled blue circles show
metabolite levels in trophozoite- and schizont-infected RBCs (as SD from the mean of uRBC). Metabolite levels that deviate from the mean by
>5 SD are considered significant. Note that the z-score plots are truncated at 25 SD for clarity.
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were all increased in iRBCs (Fig. 1C and D). As shown
recently, the mitochondrial catabolism of glutamine in the
TCA may require transaminase reactions involving the
non-proteinogenic amino acid γ-aminobutyric acid, which
was present at elevated levels in trophozoite/schizont-
infected RBC (Fig. 1C and D).
Unexpectedly, levels of Ino3P, a putative intermediate in
the pathway of de novo myo-inositol biosynthesis, were
also highly elevated in iRBC (Fig. 1C and D). Ino3P was
present at <10 nmol per 10
8
nRBCs and ring-iRBCs, but
increased to 380 and 450 nmol per 10
8
parasitized cells in
trophozoite- and schizont-iRBCs respectively (Fig. 2).
Increased Ino3P levels was only observed in iRBCs, and
not in uRBCs isolated from the same culture or in nRBC
(Fig. 2). In contrast, both iRBCs and uRBCs isolated
from the same culture contained similar levels of non-
phosphorylated myo-inositol (Fig. 2). The levels of myo-
inositol in iRBC and uRBC were substantially higher than in
RBC that had never been exposed to P. falciparum (Fig. 2).
As RBC lack the enzymes needed for de novo myo-inositol
synthesis, these results suggest that factors secreted or
released from iRBC can lead to increased uptake of myo-
inositol by uninfected RBC in the same culture.
De novo myo-inositol biosynthesis is upregulated in
P. falciparum-infected RBCs at mid- and late-stages
of infection
Ino3P can be synthesized from Glc6P, the canonical
pathway of de novo myo-inositol biosynthesis (Fig. 3A), or
by direct phosphorylation of myo-inositol (Stephens et al.,
1990; Shi et al., 2005). To distinguish between these
possibilities, synchronized P. falciparum-iRBCs containing
ring, trophozoite or schizont stages were pulse-labelled
with
13
C-U-glucose (6 mM) for 2 h and the incorporation of
13
C into intermediates in the de novo myo-inositol pathway
(Glc6P, Ino3P and myo-inositol) quantified by GC-MS
(Fig. 3B, Table S1). The rate of turnover of Glc6P, Ino3P
and myo-inositol increased progressively as parasites
developed from ring to trophozoite and schizont stages
(Fig. 3B). Significantly, the level of
13
C-enrichment in Ino3P
in trophozoite/schizont stages was comparable to that of
Glc6P indicating that Ino3P is primarily or exclusively syn-
thesized from glucose 6-phosphate (Fig. 3B, Table S1). In
contrast, levels of enrichment in myo-inositol were approxi-
mately 20–40% of that found in Ino3P. These data suggest
that de novo synthesis of Ino3P and myo-inositol is sub-
stantially upregulated in trophozoite/schizont stages, but
that approximately 60% of cellular myo-inositol is derived
from exogenous sources under these growth conditions.
Transcription of the INO1 gene, encoding the single
Ino3P synthase, is increased in P. falciparum trophozoite
and schizont asexual stages (Bozdech et al., 2003). To
determine whether INO1 activity is indeed upregulated in
these stages, cell-free extracts of ring, trophozoite- and
schizont-infected RBC were incubated with
13
C-glucose
6-phosphate and conversion to
13
C-Ino3P measured at
various time points over 4 hours by GC-MS. Consistent
with the in vivo labelling studies, RBC cultures enriched
for ring stages contained very low levels of INO1 activity,
while INO1 activity was highly elevated in RBC containing
trophozoite and schizont stages (Fig. 3C). Uninfected
RBCs had no measurable INO1 activity (Fig. 3C).
The level of
13
C-enrichment in myo-inositol was always
lower than in Ino3P, likely reflecting the uptake of unla-
belled myo-inositol, which is present at 0.2 mM in RPMI
medium. To investigate whether the de novo synthesis of
myo-inositol is repressed when exogenous supplies of
myo-inositol are increased, trophozoite-iRBCs were
labelled with
13
C-U-glucose in the presence of 0.2 mM or
4mM myo-inositol. Incubation in the presence of 4 mM
myo-inositol led to a sevenfold increase in intracellular
levels of myo-inositol and a concomitant decrease in
13
C-
enrichment in this pool (Fig. 3D). In marked contrast,
increased levels of exogenous myo-inositol had no effect
on either the abundance of Ino3P or its metabolic labelling
with
13
C-glucose (Fig. 3D). Collectively, these data show
that de novo synthesis of Ino3P is highly upregulated in
Fig. 2. Inositol metabolite levels in infected
and uninfected RBC. Polar metabolites were
isolated from synchronized P. falciparum
cultures (8% parasitaemia) containing
unfractionated ring-infected RBC (Ring),
magnet-purified trophozoite-RBC (Troph) or
schizont-RBC (Schiz) stages, uninfected RBC
(uRBC, purified from the schizont-infected
culture), and RBCs that had never been
exposed to parasites (nRBC). Intracellular
pool sizes of myo-inositol (myo-Ino) and
inositol 3-phosphate (Ino3P) in 10
8
cell
equivalents were determined by GC-MS. In
the box-whisker plots, thick horizontal lines
indicate the median abundance, boxes the
interquartile range, whiskers extreme
non-outliers, and circles outliers. n = 5–8.
Inositol metabolism in
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trophozoite and schizont stages and that flux into this
pathway is not repressed by exogenous inositol, even
when the uptake of exogenous myo-inositol is increased.
De novo-synthesized and salvaged myo-inositol are
used to synthesize PI, but only de novo synthesized
myo-inositol is incorporated into GPIs
The upregulation of de novo myo-inositol biosynthesis in
trophozoite and schizont stages likely reflects a require-
ment for bulk membrane phospholipids, as well as the
synthesis of phosphoinositides and GPI glycolipids. PI
levels increased twofold in ring-iRBCs compared with
background (non-infected RBCs) and by 7-fold in
trophozoite- and schizont-iRBCs as determined by GC-MS
of unfractionated RBC cultures (Fig. 4A) and LC-MS analy-
sis of total lipids of non-infected and trophozoite-iRBCs
(Table S2). Nineteen different PI molecular species, con-
taining fatty acids of different length and degree of satura-
tion were detected in the LC-MS analyses (Table S3,
Fig. S1). To investigate whether the de novo-synthesized
or scavenged myo-inositol was used to synthesize bulk PI,
trophozoite-iRBCs were labelled with
13
C-U-glucose and
incorporation of
13
C-inositol into PI was monitored by
LC-MS (Fig. 4B, Table S3). Precursor ion scanning for PI
molecular species containing unlabelled or labelled myo-
inositol head-groups (loss of m/z 241 or 247 respectively)
indicated that both scavenged and de novo synthesized
myo-inositol was incorporated into major PI species
(Fig. 4B, lower two panels). Additional PI isotopomers
(containing 3 or 9
13
C atoms) were also detected, corre-
sponding to molecular species with a labelled glycerol
backbone (Fig. 4B). Significantly, PI molecular species
containing labelled fatty acid components were not
detected (data not shown), consistent with asexual RBC
stages having minimal de novo fatty acid biosynthesis
(Tarun et al., 2009). No labelling of either the inositol or
glycerol backbone PI pools in non-infected RBC were
detected, consistent with the absence of active PI synthe-
sis in these host cells (data not shown).
To investigate whether the source of myo-inositol used
for PI synthesis is regulated by the availability of exog-
enous sources, trophozoite-infected RBCs were labelled
with
13
C-glucose in the presence or absence of 4 mM myo-
inositol.
13
C-enrichment in PI was greatly reduced in the
presence of exogenous myo-inositol (Fig. 4C), confirming
that exogenous myo-inositol can displace the need for
endogenous Ino3P/myo-inositol for bulk PI synthesis. The
Fig. 3. Biosynthesis of inositol metabolites
in vivo.
A. De novo biosynthetic pathway of myo-inositol.
The conversion of glucose 6-phosphate (Glc6P)
to inositol 3-phosphate (Ino3P) and myo-inositol
(myo-Ino) is catalysed by inositol 3-phosphate
synthase (INO1) and inositol monophosphatase
(IMPase) respectively.
B. Synchronized P. falciparum RBC cultures (8%
parasitaemia) were pulse-labelled with
13
C-glucose stages for 2 h at different stages of
development. Enrichment of
13
C in Glc6P (black
bars), Ino3P (grey bars), and myo-inositol (white
bars) (relative to exogenous glucose) in
unfractionated ring-infected RBCs (Ring) and
purified trophozoite- (Troph) and schizont-
infected (Schiz) RBCs was determined by
GC-MS. n = 5–8. Data for uRBCs and nRBCs is
shown in Table S1.
C. Levels of INO1 activity in synchronized
P. falciparum RBC cultures. Cell lysates (5 × 10
6
cell equivalents) were incubated with
13
C-Glc6P
and synthesis of
13
C-Ino3P determined by
GC-MS. Ino3P synthesis is shown as pmol per
minute, and is based on initial rate kinetics in a
time-course experiment. Negligible activity was
detected in RBCs that had never been exposed
to parasites (nRBC). n = 4 and error bars
indicate standard deviation.
D. Synchronized P. falciparum RBC cultures
containing trophozoite stages were labelled with
13
C-glucose with or without supplementation of
the medium with 4 mM myo-inositol. The
abundance and
13
C-enrichment in Ino3P and
myo-inositol was determined by GC-MS. n = 4
and error bars indicate standard deviation.
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incorporation of exogenous myo-inositol into PI was further
confirmed by metabolic labelling of trophozoite-iRBCs with
3
H-myo-inositol. Label was incorporated exclusively into
a lipid species that co-migrated with authentic PI and
was sensitive to PI-specific phospholipase C but not
GPI-specific phospholipase D (Fig. 5A), consistent with
the known-specificities of these two lipases. Unexpectedly,
these experiments showed that exogenous
3
H-myo-
inositol was not detectably incorporated into GPI precur-
sors (Fig. 5A). In contrast, in parallel labelling studies,
3
H-glucosamine, a component of the conserved glycan
core of GPI glycolipids, was rapidly incorporated into the
expected intermediates, indicating that GPI biosynthesis
was active under these conditions (Fig. 5A). To investigate
whether GPI glycolipids are assembled on PI species
containing de novo-synthesized myo-inositol, trophozoite-
iRBCs were metabolically labelled with
13
C-U-glucose for
2 h and the glycan head-groups of purified GPIs were
analysed by GC-MS. Comparable levels of
13
C-enrichment
were observed in both the myo-inositol moiety and the
mannose residues of the glycan backbone of free GPI
glycolipids (Fig. 5B). These results show that GPI glycolip-
ids are selectively assembled on a sub-pool of PI species
that contain de novo-synthesized, rather than salvaged
myo-inositol.
P. falciparum INO1 is likely to be essential for
intracellular development
Our data suggested that de novo myo-inositol biosynthe-
sis may be specifically required for synthesis of PI pools
destined for GPI biosynthesis. To investigate whether this
de novo biosynthetic pathway is essential, we attempted
to disrupt the P. falciparum PF3D7_0511800, encoding
INO1 in the presence of exogenous myo-inositol. All
attempts to insert a non-functional gene cassette into the
INO1 locus were unsuccessful (Fig. 6). In contrast, inser-
tion of a HA/STREP-tag into the locus was successful
(Fig. 6) indicating that it is possible to target this locus.
These results provide strong evidence that INO1 provides
an essential pool of myo-inositol and PI that cannot be
by-passed by the presence of exogenous inositol.
Fig. 4. Stage-specific changes in PI synthesis.
A. Synchronized P. falciparum RBC cultures were harvested at different stages of the 48 h cycle and total PI levels determined by analysis of
inositol content in the organic (lipid) phase. Abbreviations as above. n = 4–6 and error bars indicate standard deviation.
B. Synchronized P. falciparum-infected RBC cultures were labelled with
13
C-glucose for 16 h and total lipids analysed by LC-MS. Precursor
scanning for m/z 241 or m/z 247 was used to quantify relative abundance of PI molecular species containing an unlabelled and
13
C
6
-inositol
head-groups respectively. Selected species (i.e. C34:1 and C36:1 are indicated). The chromatograms are representative of 4 separate
experiments.
C. Abundance of all PI species and relative abundance of
13
C-PI species following growth in the presence or absence of 4 mM exogenous
myo-inositol. n = 4 and error bars indicate standard deviation.
Inositol metabolism in
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Discussion
RBC lack the capacity to synthesize PI and intra-
erythrocytic stages of P. falciparum are predicted to be
dependent on de novo PI synthesis for survival within this
niche. In this study, we show that asexual parasite stages
utilize myo-inositol that has been scavenged from the host
as well as endogenously synthesized myo-inositol for the
synthesis of bulk membrane PI. However, only de novo-
synthesized myo-inositol is incorporated into a sub-pool of
PI species that are used for GPI biosynthesis in the ER.
These analyses indicate that the pathway of de novo
synthesis of myo-inositol, which is highly upregulated in
asexual stages, cannot be by-passed by myo-inositol
salvage pathways and is critical for the assembly of essen-
tial surface glycolipids and GPI-anchored proteins.
The upregulation of Ino3P synthesis in trophozoite and
schizont stages was initially detected in an untargeted
metabolomic analysis of unfractionated and purified
infected RBC. These analyses revealed a dramatic
increase in the intracellular concentration of Ino3P in
infected RBC cells. The accumulation of Ino3P has also
recently been reported in asynchronized P. falciparum
RBC stages (Sana et al., 2013). In vivo and in vitro
13
C-
glucose labelling studies demonstrated that Ino3P was
synthesized from glucose 6-phosphate, rather than the
phosphorylation of exogenous myo-inositol, and subse-
quently dephosphorylated to form myo-inositol, reflecting
operation of the canonical pathway of myo-inositol biosyn-
thesis. De novo synthesized myo-inositol was incorporated
into both bulk PI as well as newly synthesized GPI glycolip-
ids. In contrast, exogenous myo-inositol was only incorpo-
rated into bulk PI. The latter conclusion was supported by
the findings that (1) exogenous
3
H-myo-inositol was incor-
porated into bulk PI but not GPI in short term labelling
studies, (2) addition of exogenous myo-inositol to P. falci-
parum RBC cultures reduced the extent to which endog-
enous Ino3P was incorporated into bulk PI, but had no
effect on incorporation into the GPI pool, and (3) compa-
rable levels of
13
C-enrichment occur in the myo-inositol and
mannose residues of GPI head-groups in
13
C-glucose-fed
parasites, indicating that newly synthesized GPIs contain
predominantly or exclusively de novo-synthesized myo-
inositol. Collectively, these results suggest that synthesis
of endogenous Ino3P is required to sustain levels of PI
in the endoplasmic reticulum that are used for GPI biosyn-
thesis, providing an explanation for the apparent essen-
tiality of the INO1 gene in the presence of a robust
myo-inositol salvage pathway.
Related findings have been made in the insect and
mammalian-infective stages of Trypanosoma brucei, the
causative agent of human African trypanosomiasis (HAT)
or Nagana in cattle. RNAi-mediated downregulation of
either INO1 or the single H
+
-inositol transporter in these
Fig. 5. Endogenous Ino3P, and not exogenous myo-inositol is
incorporated into GPI glycolipids.
A. P. falciparum-infected RBCs were metabolically labelled with
3
H-GlcN or
3
H-myo-inositol for 24 h and labelled inositol
(glyco)lipids resolved by HPTLC. Equivalent cell equivalents
(1.25 × 10
7
) were treated with PI-PLC, GPI-PLD or a buffer control
prior to HPTLC analysis. Labelled glycolipid species were detected
by fluorography, and assigned the following structures based on
previous studies (Gerold 1994): glucosamine-acyl-PI (GlcN-acyl-PI),
glucosamine-acyl-PI (GlcN-PI), mannose
4
-glucosamine-acyl-PI
(Man
4
GlcN-acyl-PI), ethanolaminephosphate-mannose
4
-
glucosamine-acyl-PI (EtNP-Man
4
GlcN-acyl-PI) and PI. A band
with a slower HPTLC mobility was generated following GPI-PLD
treatment of the
3
H-inositol labelled lipids. This species was has the
expected HPTLC mobility of lyso-PI and may have been generated
by other lipases in the serum.
B. P. falciparum infected RBCs were labelled with 13 mM
13
C-glucose (2 h post synchronization) and harvested when
parasites reached late trophozoite-stage (28 h). A total lipid extract
was subjected to hydrophobic interaction chromatography to
resolve PI species (two acyl chains) from GPI glycolipids (three
acyl chains). Enrichment of
13
C into the mannose and inositol
moieties of the GPI fraction were determined by GC-MS after
strong aqueous (6 M HCl, 100°C, 24 h) and methanolysis (0.5
methanolic-HCl, 80°C, 24 h) respectively. Error bars indicate
standard deviation (n = 4).
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Fig. 6. Targeted replacement of P. falciparum INO1.
A. Schematic representation showing the targeted replacement of the INO1 gene (PF3D7_0511800) in the wild-type cells with a non-functional
copy (hDHFR) from a plasmid and the resulting disrupted locus. Restriction sites for AgeI (A), EcoRV (E), KpnI (K) and XbaI (X) are indicated,
along with the expected fragment sizes following double-digestion.
B. Southern blot of the wild-type and INO1 mutant cell lines. DNA from wild-type (3D7) and mutant cells (3D7Δ PF3D7_0511800) were
digested with AgeI/KpnI or EcoRV/XbaI and probed as indicated. Similar results were obtained when the same gene was disrupted in the CS2
strain was attempted (results not shown).
C. Schematic representation showing the targeted insertion of a 3× HA/Strep tag onto the 3-end of the INO1 gene in wild-type cells from a
plasmid and the resulting disrupted locus.
D. A Western blot using biotin (anti-streptavidin antibodies) indicated expression of the HA/STREP-tagged copy of INO1 in the mutant cell line.
PfHSP70 is shown as a loading control.
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parasites resulted in loss of viability or reduced growth
respectively (Martin and Smith, 2006a,b; Gonzalez-
Salgado et al., 2012). Reminiscent of the situation in
P. falciparum, repression of INO1 expression in T. brucei
bloodstream stages had little effect on synthesis of bulk
PI but resulted in reduced synthesis of GPI precursors
(Martin and Smith, 2006a,b). Conversely, repression of
inositol transporter expression had little effect on GPI
biosynthesis but resulted in reduced bulk PI synthesis
(Gonzalez-Salgado et al., 2012). In T. brucei, a single
isoform of PI synthase is compartmentalized to the ER
and Golgi, and an H
+
/inositol transporter is targeted to the
Golgi apparatus as well as the plasma membrane (Martin
and Smith, 2006b; Gonzalez-Salgado et al., 2012). Based
on the localization of these enzymes and transporters, it
has been proposed that cytoplasmic pools of myo-inositol,
comprising both scavenged and endogenous myo-inositol
are transported into the lumen of the Golgi and used
by the Golgi PI synthase to make bulk PI and related
lipids (Martin and Smith, 2006a; Gonzalez-Salgado et al.,
2012). In contrast, the ER-located PI synthase, which may
be orientated with its catalytic domain on the cytosolic
(Fischl et al., 1986) or luminal face of the ER, is proposed
to only have access to de novo-synthesized myo-inositol.
Key elements of this model remain to be defined, includ-
ing the precise topology of the ER/Golgi PI synthases and
the mechanism by which de novo-synthesized myo-
inositol is channelled to the ER isoform of PI synthase.
The compartmentalization of inositol and PI synthesis
may be achieved in different ways in T. brucei and P. falci-
parum. Similar to T. brucei, P. falciparum encodes for only
one isoform of PIS (PF3D7_1315600), suggesting that this
enzyme has a dual location or that different pools of PI
are synthesized within the ER. The presence of a single
isoform of PIS also indicates a common membrane topol-
ogy for all PI synthetic reactions. While there is little infor-
mation on the topological orientation of PI synthases in
other organisms in vivo (Fischl et al., 1986), the presence
of myo-inositol membrane transporters in the Golgi mem-
branes of T. brucei and the Golgi/ER membrane of humans
cells, supports a luminal sidedness for this reaction (Di
Daniel et al., 2009; Gonzalez-Salgado et al., 2012). In
contrast to T. brucei, which expresses a single isoform of
the inositol-phosphate phosphatase (IMPase), that cataly-
ses the second step in the INO1 pathway of myo-inositol
synthesis, P. falciparum expresses two IMPases. These
proteins contain conserved synaptojanin-like IMPase
domains and are predicted to be targeted to the cyto-
plasm (PF3D7_0705500) or intracellular membranes
(PF3D7_0802500) respectively. Cytosolic and membrane-
bound forms of IMPase are also predicted to occur in
other organisms (Vadnal et al., 1992; Miller and Allemann,
2007). Whether these proteins have redundant functions in
myo-inositol synthesis or are involved in channelling differ-
ent pools of myo-inositol to the ER or Golgi-located PI
synthase remains to be determined. However, the fact that
only one IMPase is required for metabolic compartmen-
talization in T. brucei implies that the two P. falciparum
IMPases also have a common cytoplasmic topology. Taken
together, we propose a revised model in which Ino3P is
selectively channelled to the ER PIS via an IMPase/myo-
inositol transporter (Fig. 7). PI generated in the ER is
subsequently flipped to the cytoplasmic face where it is
utilized by the initial enzymes in GPI biosynthesis that
utilize the sugar donor UDP-GlcNac and have catalytic
domains orientated on the cytoplasmic side of the ER
(Chang et al., 2002). However, further work is needed to
determine the localization of myo-inositol transporters and
IMPase isoforms and validate this model.
Interestingly, there is evidence that ER pools of PI are
also phosphorylated to generate a functionally important
sub-pool of PI3P in apicomplexan parasites (Tawk et al.,
2011). In both Plasmodium spp. and Toxoplasma gondii,
PI3P has been localized to the cytoplasmic face of
ER-derived membrane vesicles and shown to be required
for apicoplast biogenesis (Tawk et al., 2010; 2011). PI3P is
also proposed to accumulate in the lumen of the ER in
Plasmodium RBC stages where it might be involved in
Fig. 7. Hypothetical model for metabolic compartmentalization of
PI biosynthesis in P. falciparum. P. falciparum trophozoite and
schizont stages upregulate myo-inositol synthesis, via Ino3P, for
bulk membrane PI and GPI glycolipid biosynthesis. These stages
can also scavenge exogenous myo-inositol from the host, although
exogenous myo-inositol is only used to synthesize bulk PI. A similar
metabolic compartmentalization of inositol metabolism and PI
synthesis occurs in African trypanosomes (Martin and Smith,
2006a; Gonzalez-Salgado et al., 2012). Metabolic
compartmentalization in these parasites may be achieved by the
sequestration of PI-synthase to the lumen of the ER and Golgi and
single/multiple isoforms of IMPase to the cytoplasmic leaflet of the
ER in conjunction with an, as yet to be identified, myo-inositol
transporter. Multiple transporters are required for the transport of
glucose and myo-inositol across the RBC plasma membrane
(RBC-PM), parasitophorous vacuolar membrane (PVM) and
parasite plasma membrane (Pf PM).
770
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regulating the export of secretory proteins (Bhattacharjee
et al., 2012a,b). However, other models have been pro-
posed (Rommisch, 2012) and the extent to which PI3P is
transported into the lumen of ER remains to be determined.
Our findings highlight significant differences in inositol
metabolism in different eukaryotic pathogens (Reynolds,
2009). In common with P. falciparum asexual stages, a
number of other protozoan and fungal pathogens, such as
Leishmania mexicana (Ilg, 2002) and Candida albicans
(Chen et al., 2008) can generate all of their myo-inositol
requirements via the INO1-dependent pathway and prolif-
erate normally in the absence of exogenous myo-inositol.
Cryptococcus neoformans is also prototrophic with regard
to myo-inositol. However, this fungal pathogen can also
catabolize myo-inositol as a major carbon sources and is
dependent on myo-inositol uptake for survival in myo-
inositol-rich niches, such as the brain, within its animal host
(Xue et al., 2010; Wang et al., 2011). In contrast, T. brucei
procyclic forms cannot survive without an exogenous
supply of myo-inositol, despite having a functional INO1-
dependent pathway (Gonzalez-Salgado et al., 2012). It is
possible that myo-inositol uptake in T. brucei procyclic
forms is required to spare endogenous Ino3P for ER-
directed PI biosynthesis and production of GPI anchor
precursors that in turn are needed for expression of the
dominant free glycoinositolphospholipids and procyclin
glycoproteins. On the other hand, the apicomplexan para-
site, Toxoplasma gondii, lacks an annotated INO1 gene
and is predicted to be completely dependent on exogenous
myo-inositol for inositol-lipid synthesis. How T. gondii regu-
lates the synthesis of different pools of PI needed for bulk
lipid and GPI biosynthesis remains to be determined.
INO1 is commonly considered to be the rate-limiting step
in de novo myo-inositol biosynthesis. However, in P. falci-
parum, the down-stream IMPases, rather than INO1,
appear to be rate-limiting as shown by the rapid labelling
and accumulation of Ino3P in
13
C-glucose-fed parasites
and the comparatively slow chase of
13
C- into free myo-
inositol. The accumulation of Ino3P in late RBC parasite
stages was striking and may be needed to sustain a high
flux into the essential ER pathway of PI synthesis (Fig. 6).
Alternatively the accumulated Ino3P pool may constitute
an internal ‘reserve’ of myo-inositol which can be used to
maintain inositol-lipid biosynthesis under conditions when
either exogenous inositol or glucose are limiting. Unlike
non-phosphorylated myo-inositol, Ino3P is effectively
trapped within the parasites and not susceptible to reverse
transport by diffusion-driven plasma membrane facilitative
transporters. Ino3P could also have additional roles, inde-
pendent of inositol-lipid biosynthesis. In particular, Ino3P
synthesis is closely coupled to the formation of glucose
6-phosphate, and the size of the Ino3P pool could reflect
nutrient availability and the energy status of asexual
stages. Ino3P could also be converted to more complex
inositol polyphosphates (Stritzke et al., 2012) which have
been shown to regulate multiple cellular processes in other
eukaryotes, including cell signalling, cell cycle control, host
cell invasion and parasite maturation (Irvine and Schell,
2001; Luo et al., 2011). Finally, there is accumulating evi-
dence that the P. falciparum INO1 pathway and PI synthe-
sis might be primarily regulated at the post-transcriptional
level. In particular, enzymes involved in PI metabolism
were highly enriched in the P. falciparum schizont phos-
phoproteome (Lasonder et al., 2012). Similarly, the yeast
INO1 enzyme has also recently been shown to be regu-
lated by phosphorylation (Deranieh et al., 2013). Collec-
tively, these findings suggest that inositol metabolism and
lipid biosynthesis is highly regulated both at the level of
enzyme activity and subcellular compartmentalization. The
dependence of P. falciparum asexual stages on the INO1
pathway makes these regulatory mechanisms attractive
targets for new drugs.
Experimental procedures
Cell culture
Plasmodium falciparum 3D7 parasites were cultured in 4%
haematocrit with RPMI 1640 and glucose-free RPMI media
(both Sigma) as appropriate, supplemented with hypoxan-
thine (52 mg l
1
, Calbiochem), 0.5% AlbuMAX (Gibco),
sodium hydrogen carbonate (0.21%, BDH) and gentamicin
(0.58 mg l
1
, Pfizer), as described previously (Trager and
Jensen, 1976). Parasite synchronization was achieved by
repeated sorbitol lysis (Lambros and Vanderberg, 1979).
Infection was assessed by light microscopy. Human blood
was kindly donated by the Red Cross Blood Service (Mel-
bourne, Australia).
Saponin lysis of RBC membranes
P. falciparum infected RBCs were harvested by centrifugation
(1500 rpm, 5 min, room temperature) and the cell pellet
resuspended in 0.15% saponin (Kodak) in PBS (10 min, on
ice) before washing twice in cold PBS (pH 7.4). Cell prepa-
rations were freeze-thawed before use.
Metabolite extraction of P. falciparum-infected and
uninfected RBCs
P. falciparum-infected/uninfected RBC cultures ( 8% parasi-
taemia) were centrifuged (1500 rpm, 10 min, room tempera-
ture) and the cell pellet resuspended in fresh medium which
was subsequently rapidly chilled to 0°C by immersion of the
tube in a dry ice/ethanol slurry (Saunders et al., 2011; Macrae
et al., 2012). In some experiments, mature trophozoite- or
schizont-infected RBCs were purified from uninfected and
ring stage trophozoite-infected RBCs by magnet purification
(see below). The chilled RBCs (10
8
) were harvested by cen-
trifugation (1500 rpm, 10 min, 0°C), washed three times with
ice cold PBS (pH 7.4) and extracted in chloroform/methanol/
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water (1:3:1 v/v), containing scyllo-inositol (1 nmol) as inter-
nal standard (60°C, 20 min). Polar and apolar metabolites
were separated by phase partitioning (Saunders et al., 2011).
Polar metabolite extracts were methoximated and trimeth-
ylsilyl (TMS) derivitized, prior to analysis by GC-MS (Saunders
et al., 2011). Apolar metabolite extracts were subjected to
methanolysis prior to TMS derivitization and analysis by
GC-MS (McConville et al., 1990). GC-MS was performed with
a Hewlett-Packard 6890-5973 system. Derivitized samples
were infected onto a DB-5MS + DG column (30 m × 0.25 mm,
with 10 m inert gap, J&W, Agilent Technologies) in splitless
mode (injection temperature 270°C for methoximation-derived
samples and 280°C for methanolysis-derived samples) using
helium as the carrier gas. For methoximated polar metabolite
analyses, the initial oven temperature was 70°C (2 min), fol-
lowed by temperature gradients to 295°C at 12.5°C per
minute, and from 295°C to 320°C at 25°C per minute. The final
temperature was held for 3 min. For analysis of fatty acid
methyl esters, the initial oven temperature was 80°C (2 min),
followed by temperature gradients to 140°C at 30°C per
minute, from 140°C to 250°C at 5°C per minute, and from
250°C to 265°C at 15°C per minute. The final temperature was
held for 10 min. Data analysis was performed using Chemsta-
tion software (MSD Chemstation D.01.02.16, Agilent Tech-
nologies). Metabolites were identified by comparison of
retention times and ion fragmentation patterns with authentic
standards. Quantification of metabolites was calculated using
the formula: amount metabolite (nmol) = (area metabolite(s)/
area scyllo-inositol) × (1 nmol scyllo-inositol/
metabolite
MRRF
sI
);
where
metabolite
MRRF
sI
is the molar relative response factor
determined from the mean value of: area appropriate metabo-
lite peak/area scyllo-inositol peak (1:1 standards).
Magnet-purification of trophozoite- and schizont-stage
P. falciparum-infected RBCs
P. falciparum-infected/uninfected cell cultures were centri-
fuged, resuspended in fresh medium and metabolically
quenched, as described above. Mature trophozoite- and
schizont-infected RBCs were purified from uninfected and
ring-stage infected RBCs by passage through a CS Column on
a VarioMACS magnetic cell separator (Miltenyi Biotec) at
4°C as described previously (Trang et al., 2004). Mature
trophozoite- and schizont-infected RBCs were eluted with 1/5
culture volume of ice-cold medium.
Stable isotope labelling of P. falciparum-infected RBCs
P. falciparum RBC cultures were synchronized by repeated
sorbitol lysis (Lambros and Vanderberg, 1979) and suspended
in fresh RPMI medium containing 6 mM
13
C-U-glucose
(Spectra Stable Isotopes). After incubation for 2 h, cultures
were harvested and metabolites extracted as above. Parallel
incubations and extractions were performed on uninfected
RBCs.
13
C-enrichment in different polar metabolites was quan-
tified by GC-MS analysis as previously described (Saunders
et al., 2011), and fractional enrichment based on the calcu-
lated ratio of
12
C-/
13
C-glucose in the medium. In some experi-
ments, the RPMI medium (containing 0.2 mM myo-inositol)
was supplemented with an additional 4 mM myo-inositol.
Statistical analyses
GC-MS chromatograms (n = 5–8 for any given sample)
were processed with AnalyzerPro (SpectralWorks) or PyMS
(http://code.google.com/p/pyms/). Data were normalized by
centring samples using the median values and scaled by
interquartile range (IQR). Missing values were imputed as
half of the minimum abundance detected in other groups.
PCAs were generated using Simca-P 11 software (Umetrics)
while Z-transformation (using PyMS) was performed in order
to scale each metabolite in the infected-RCS according to its
corresponding value in the uninfected-RBC.
Box-whisker plots were generated using R statistics soft-
ware (http://www.R-project.org).
PI quantification
Synchronized P. falciparum-infected RBCs (at 17 % parasi-
taemia) enriched for ring, trophozoite and schizont stages, and
non-infected RBCs were metabolically quenched and har-
vested as described above. RBC (2 × 10
8
) were extracted
in chloroform/methanol/water (1:3:1 v/v), centrifuged to
remove insoluble material and the supernatant dried
under nitrogen and phase partitioned in butan-1-ol/water
(200 μl : 200 μl). An internal standard of 1 nmol scyllo-inositol
(sI) was added to the PI-containing apolar phase and subse-
quently dried, methoximated and analysed by GC-MS as
described above.
Measurement of in vitro INO1 activity
A two stage assay was developed in which
13
C-glucose was
quantitatively converted to
13
C-glucose 6-phosphate with puri-
fied hexose kinase, and then incubated with P. falciparum
lysates to generate Ino3P. The conversion of
13
C-glucose to
13
C-glucose 6-phosphate was achieved by incubating 300 μM
13
C-U-Glc with hexokinase (66 μgml
1
, Sigma) in assay buffer
(1 mM ATP (Sigma), 2.5 mM MgCl
2
(BDH, AnalaR), 100 mM
TrisHCl (pH 7.5, Sigma), 14 mM NH
4
Cl (Ajax), 0.8 mM NAD
+
(Sigma)) at 37°C for 10 min and the hexose kinase denatured
by boiling for 10 min. P. falciparum lysates were prepared by
suspending unfractionated infected RBCs or non-infected
RBCs in ice-cold lysis buffer [1 mM NaHEPES (pH 7.4,
Sigma), 2 mM ethylene glycol tetraacetic acid (Sigma), 2 mM
dithiothreitol (Biovectra)] for 10 min. Aliquots (50 μl) of
13
C-
glucose 6-phosphate and cell lysate [5 × 10
7
RBC equivalents
(5 × 10
6
infected RBC)] were incubated at 37°C and the
reactions stopped after 0, 0.5, 1, 5, 10, 30, 60, 120, 180 and
240 by boiling for 5 min. Polar products were recovered after
extraction of the mixture in chloroform/methanol/water (1:3:3
v/v, containing 1 nmol scyllo-inositol), and the synthesis of
13
C-Ino3P quantified by GC-MS.
Extraction, purification and analysis of radiolabelled GPI
anchor intermediates
Synchronized P. falciparum RBC cultures were metabolically
labelled with
3
H-myo-inositol or
3
H-glucosamine [2.5 μCi ml
1
],
starting at 12 h in the 48 h asexual cycle. Infected RBC were
harvested after 24 h (late trophozoite stage) by magnet puri-
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fication and GPI glycolipids extracted as described previously
(McConville and Blackwell, 1991). Briefly, cell pellets were
lyophilized and lipids were extracted twice in chloroform/
methanol/water (1:2:0.8, v/v) for >1 h at room temperature.
Combined supernatants were dried under nitrogen and gly-
colipids recovered after 1-butanol/water (2:1 v/v) phase parti-
tioning. The 1-butanol phases were dried and analysed by high
performance thin layer chromatography (HPTLC) on Silica Gel
60 plates (Merck) developed in chloroform/methanol/13 M
ammonia/1 M ammonium acetate/water (180:140:9:9:2.3
v/v). Radioactive species were visualized by fluorography
after spraying with EN
3
HANCE (PerkinElmer Life Sciences)
and the major GPI species identified from their HPTLC migra-
tion, as reported previously (Gerold et al., 1994).
Enzyme digestions
Radiolabelled lipids were incubated with PI-PLC (146 mU,
ICN) in 25 mM triethanolamine (pH 7.4), 5 mM ethylenedi-
aminetetraacetic acid, 0.08% Triton X-100 (Sigma) or with
rabbit serum (2 μl, a source of serum GPI-PLD) in 50 mM
Tris-HCl (pH 7.4), 10 mM sodium chloride, 2.6 mM calcium
chloride, 0.1% Nonidet P40 (ICN), at 37°C for 16 h. For the
GPI-PLD digests, additional 2 μl aliquots of rabbit serum
were added after 1 h and 2 h. The digests were phase parti-
tioned in butan-1-ol/water (1:1, v/v) and the apolar phase
dried under nitrogen for HPTLC analysis.
Analysis of GPI glycolipids
Synchronized P. falciparum RBC cultures were metabolically
labelled with
13
C-glucose (26 h), and infected RBC contain-
ing late trophozoite stages were isolated by magnet separa-
tion. Total lipid was extracted in chloroform/methanol/water
(1:2:0.8, v/v) and recovered in the organic phase after butan-
1-ol/water partitioning. Lipid (2 × 10
8
cell equivalents) were
dried under nitrogen, resuspended in 10% propan-1-ol/
100 mM NH
4
Ac and loaded onto a column of octyl-
Sepharose (50 mm × 11 mm) equilibrated in the same buffer.
Phospholipids (containing two acyl-chains) and resolved
from later eluting GPI glycolipids (containing three acyl
chains) by elution of the column with a 10–60% propan-1-ol
gradient over 20 ml. Fractions (1 ml) were collected, dried
under nitrogen, and then subjected to methanolysis (50%) or
strong acid hydrolysis (50%). For the latter, samples were
dried in a glass capillary tube, and hydrolysed with 6 M
hydrochloric acid at 110°C for 16 h under vacuum. The
samples were dried under nitrogen, washed with toluene and
methanol to remove residual acid and released myo-inositol
TMS derivitized before TMS derivation prior to GC-MS analy-
sis. Inositol was quantified by selective ion monitoring (SIM)
for ions at m/z 318–323.
Liquid chromatography-mass spectrometry
(LC-MS) analysis
Total lipids were extracted from magnet-purified P. falciparum-
infected RBC containing late trophozoite stages. Lipid extracts
were suspended in 1-butanol/10 mM ammonium formate
in methanol (1:1, 100 μl) and 5 μl aliquots injected onto
a Ascentis Express RP Amide C18 + Amide column
(50 mm × 2.1 mm × 2.7 μm Supelco) on an Agilent 1200 LC.
Samples were eluted with a gradient of water/methanol/
tetrahydrofuran (50:20:30, v/v) to water/methanol/tetrahy-
drofuran (5:20:75, v/v), at 0.2 ml min
1
over 5 min and the final
buffer held for 3 min. Eluted material was analysed by elec-
trospray ionization-mass spectrometry (ESI-MS) using an
Agilent QTOF 6520 (for full scans) and an Agilent Triple Quad
6410, for precursor ion and neutral loss scanning and multiple
reaction monitoring (MRM) respectively. Samples were intro-
duced using nanospray tips. Positive precursor ion scanning
was used to identify molecular species of phosphatidylcholine
(PC) (m/z 184.1), sphingomyelin (SM) (m/z 184.1), while
negative precursor ion scanning was used to identify molecu-
lar species of PI (m/z 241) and phosphatidylglycerol (PG) (m/z
153). Neutral loss scanning was used to identify molecular
species of phosphatidylethanolamine (PE) (m/z 141 in positive
ion mode) and phosphatidylserine (PS) (m/z 87 in negative ion
mode). Capillary and fragmentor voltages were 4.4–5.5 kV
and 60–160 V respectively. ESI-MS/MS spectra were
recorded using collision voltages of 20–60 V. In all cases, the
collision gas was nitrogen at 7 l min
1
. Data were analysed
using MassHunter software (Agilent). Accurate mass full
scans (MZmine): Capillary, fragmentor and skimmer voltages
were 4.5 kV, 100 V and 60 V, respectively, and drying gas
temperature was 325°C. In all cases, the collision gas was
nitrogen at 7 l min
1
. Profiling raw data obtained from Agilent
QTOF were converted to mzdata format using Mass Hunter
Qualitative software and imported to MZmine software version
2.4. Raw data was processed by mass detection with the noise
level setting as 1 × 10
4
and chromatogram build with m/z
tolerance at 0.01. Lipid species were identified according to
the accurate mass and retention time.
Gene knock-out studies
A gene disruption construct was generated by insertion of a 5
(563bp) and 3 (529bp) segment of PF3D7_0511800 into
pCC-1 (Maier et al., 2008), with the restriction enzymes SacII/
SpeI and EcoRI/AvrII. The segments were amplified from 3D7
genomic DNA using oligonucleotide primers Aw915/Aw916
and Aw917/Aw918 respectively. Primer pair aw919/920 was
used to amplify a targeting sequence for 3 replacement, which
was inserted into the pHAST vector (SacI/XhoI). Integration of
the plasmid into the genome resulted in the expression of a
3xHA and Strep-tagged INO1 protein.
Oligonucleotides and DNA analysis: The following oligonu-
cleotides were used:
Aw915: atcccgcgGTTCAAATATTAAGAGACTC
Aw916: gatactagTGGAGCATATGCATGTTCTTTATCC
Aw917: atcgaattcGATTGTTTAGTTCGTAACAAAGTTTTTG
Aw918: gatcctaggCCTGTTCCAAAGTAAAAGTCGAGGAGG
Aw919: atcccgcgGTGACTATGTACGTGCTAATG
Aw920: gatctcgagAATCATATATGGTAAATCAATATGAGC
Underlined are restriction sites introduced for cloning pur-
poses. Genomic DNA was prepared with the DNeasy Tissue
Kit (Qiagen) and subjected to Southern Blot analysis using the
Roche DIG system following the manufacturer’s instructions.
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Page 12
Acknowledgements
We thank Ms Monica Brown at the Walter and Eliza Hall
Institute of Medical Research for technical assistance, Pro-
fessor Andrew Holmes for providing authentic myo-inositol
3-phosphate, Dr Maria Doyle (Melbourne University) for dis-
cussions, and the Australian Red Cross Blood service (Mel-
bourne), for provision of serum. This work was supported by
program (APP406601) and project (APP1006024) grants and
the Independent Research Institute Infrastructure Support
Scheme from the National Health and Medical Research
Council of Australia (NHMRC) and the Victorian State Gov-
ernment Operational Infrastructure Support Scheme. J.I.M.
was supported by a Royal Society Travelling Fellowship.
M.J.M. is an NHMRC Principal Research Fellow. A.F.C. is an
International Scholar of the Howard Hughes Medical Institute.
A.G.M. is an ARC Australian Research Fellow. None of the
authors have a conflict of interest with the work reported in
this study.
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    • "The recent identification of the T. brucei TbHMIT in the Golgi [92,93] may explain these early findings. In addition, the dependence of GPI synthesis from de novo synthesized but not imported myo-inositol has recently also been observed in Plasmodium parasites [95]. Further evidence that the topology of yeast Pis1 is different from that of its trypanosome homolog can be deduced from "
    [Show abstract] [Hide abstract] ABSTRACT: Glycerophospholipids are the principal fabric of cellular membranes. The pathways by which these lipids are synthesized were elucidated mainly through the work of Kennedy and colleagues in the late 1950s and early 1960s. Subsequently, attention turned to cell biological aspects of lipids: Where in the cell are lipids synthesized? How are lipids integrated into membranes to form a bilayer? How are they sorted and transported from their site of synthesis to other cellular destinations? These topics, collectively termed ‘lipid topogenesis’, were the subject of a review article in 1981 by Bell, Ballas and Coleman. We now assess what has been learned about early events of lipid topogenesis, i.e. “lipid synthesis, the integration of lipids into membranes, and lipid translocation across membranes”, in the 35 years since the publication of this important review. We highlight the recent elucidation of the X-ray structures of key membrane enzymes of glycerophospholipid synthesis, progress on identifying lipid scramblase proteins needed to equilibrate lipids across membranes, and new complexities in the subcellular location and membrane topology of phosphatidylinositol synthesis revealed through a comparison of two unicellular model eukaryotes.
    Full-text · Article · Mar 2016 · Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
    • "Myo-inositol 1-phosphate synthase is one of the enzymes in the myo-inositol group to synthesize phospholipid from red blood cell. Recently, a study showed that P. falciparum asexual stages are critically dependent on de novo myo-inositol biosynthesis (Macrae et al., 2014 ). No any inhibitor has been studied for ferrodoxim reductase-like protein. "
    [Show abstract] [Hide abstract] ABSTRACT: Detoxification of hemoglobin byproducts or free heme is an essential step and considered potential targets for anti-malaria drug development. However, most of anti-malaria drugs are no longer effective due to the emergence and spread of the drug resistant malaria parasites. Therefore, it is an urgent need to identify potential new targets and even for target combinations for effective malaria drug design. In this work, we reconstructed the metabolic networks of Plasmodium falciparum and human red blood cells for the simulation of steady mass and flux flows of the parasite's metabolites under the blood environment by flux balance analysis (FBA). The integrated model, namely iPF-RBC-713, was then adjusted into two stage-specific metabolic models, which first was for the pathological stage metabolic model of the parasite when invaded the red blood cell without any treatment and second was for the treatment stage of the parasite when a drug acted by inhibiting the hemozoin formation and caused high production rate of heme toxicity. The process of identifying target combinations consisted of two main steps. Firstly, the optimal fluxes of reactions in both the pathological and treatment stages were computed and compared to determine the change of fluxes. Corresponding enzymes of the reactions with zero fluxes in the treatment stage but non-zero fluxes in the pathological stage were predicted as a preliminary list of potential targets in inhibiting heme detoxification. Secondly, the combinations of all possible targets listed in the first step were examined to search for the best promising target combinations resulting in more effective inhibition of the detoxification to kill the malaria parasites. Finally, twenty-three enzymes were identified as a preliminary list of candidate targets which mostly were in pyruvate metabolism and citrate cycle. The optimal set of multiple targets for blocking the detoxification was a set of heme ligase, adenosine transporter, myo-inositol 1-phosphate synthase, ferrodoxim reductase-like protein and guanine transporter. In conclusion, the method has shown an effective and efficient way to identify target combinations which are obviously useful in the development of novel antimalarial drug combinations.
    No preview · Article · Dec 2015 · Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases
    • "The second major class of lipids observed was sphingolipids, which possess a sphingosine backbone linked to FAs via amide bonds and play critical roles in both membrane structure and signaling (Gault et al., 2010). Sphingomyelin (SM), a structural sphingolipid that aids the biogenesis and maintenance of the tubulovesicular network of membranes (Lauer et al., 1997), was the third most abundant lipid overall, consistent with other studies (Botté et al., 2013; Macrae et al., 2014) (Figure 1). Like other structural lipids, SM levels remained relatively static throughout the IDC (Figure 1). "
    [Show abstract] [Hide abstract] ABSTRACT: During its life cycle, Plasmodium falciparum undergoes rapid proliferation fueled by de novo synthesis and acquisition of host cell lipids. Consistent with this essential role, Plasmodium lipid synthesis enzymes are emerging as potential drug targets. To explore their broader potential for therapeutic interventions, we assayed the global lipid landscape during P. falciparum sexual and asexual blood stage (ABS) development. Using liquid chromatography-mass spectrometry, we analyzed 304 lipids constituting 24 classes in ABS parasites, infected red blood cell (RBC)-derived microvesicles, gametocytes, and uninfected RBCs. Ten lipid classes were previously uncharacterized in P. falciparum, and 70%-75% of the lipid classes exhibited changes in abundance during ABS and gametocyte development. Utilizing compounds that target lipid metabolism, we affirmed the essentiality of major classes, including triacylglycerols. These studies highlight the interplay between host and parasite lipid metabolism and provide a comprehensive analysis of P. falciparum lipids with candidate pathways for drug discovery efforts.
    No preview · Article · Sep 2015 · Cell host & microbe
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