BAB VIII - Biochemistry and Metabolism of Toxoplasma Gondii Lipid Synthesis and Uptake

C H A P T E R
8
Biochemistry and metabolism of
Toxoplasma gondii: lipid synthesis
and uptake
Isabelle Coppens1 and Cyrille Botté2
1
Department of Molecular Microbiology and Immunology, Johns Hopkins University Bloomberg School
of Public Health, Baltimore, MD, United States 2Apicolipid Team, Institute for Advanced Biosciences,
CNRS UMR5309, Université Grenoble Alpes, Grenoble, France
8.1 Introduction fatty acids and phosphatidic acid, are impor-

tant signal transducers involved in several cel-
Lipid inventory in Toxoplasma gondii reveals lular and metabolic pathways. Bioinformatic
the presence of neutral and polar lipids, as tools, for example, Kyoto Encyclopedia of
found in any eukaryotic organism. Lipid Genes and Genomes framework have delin-
metabolism in the parasite is essential for the eated the metabolic maps for lipid synthesis in
production of infectious progeny, signal trans- T. gondii and have concomitantly revealed aux-
duction for proper interactions with mamma- otrophies for several lipid species. In addition,
lian cells, and the long-term persistence in the recent lipidomic and fluxomic approaches
host. Among lipids, glycero(phospho)lipids, have unveiled that some phospholipid species
sterols, and sphingolipids are major building have a mixed composition, containing one fatty
blocks of biological membranes, whose compo- acyl chain synthesized by the parasite and the
sition and homeostasis define the physical other one coming from the host cell as a result
properties and functions of the compartment of scavenging activities. Toxoplasma has also
where they reside. Some lipids, in the form of uncommon lipid molecular species that can
triacylglycerols, cholesteryl esters, and other serve as signatures, indicating the presence of
neutral lipids usually found in lipid droplets unique biosynthetic enzymes and transloca-
(LDs), are also important storage molecules for tors/transporters for lipid acquisition. The dis-
T. gondii and used for energetic purposes or covery of the apicoplast and the unique fatty
lipotoxicity prevention. Other lipids, especially acid synthetic pathways operational in this
Toxoplasma Gondii
DOI: https://doi.org/10.1016/B978-0-12-815041-2.00008-6 367 © 2020 Elsevier Ltd. All rights reserved.
368 8. Biochemistry and metabolism of Toxoplasma gondii: lipid synthesis and uptake
organelle has highlighted that the parasite chain occurs by condensing another malonyl-
synthesizes prokaryotic-like lipids that are ACP with the acyl-ACP and repeating the reac-
essential for its survival. This raises the pros- tion cycle.
pect that lipid homeostatic, trafficking, and In nature, there are two basic types of FAS
remodeling pathways in the parasite may architectures based on their origin. The proto-
abound in potential drug targets. As a prime typical FASI, found in vertebrates and fungi, is
example, fatty acid biosynthetic pathways are an associated system consisting of a mega, sin-
being successfully exploited as antimicrobial gle gene that encodes a multifunctional protein
targets against Toxoplasma infections. This that contains all of the enzymatic reaction cen-
chapter summarizes the different classes of ters capable of producing a fatty acid molecule
lipids present in Toxoplasma, as a result of bio- (Smith et al., 2003). In contrast, bacteria, algae,
synthesis in the parasite and/or salvage from plants, organisms bearing chloroplast-like
the host cell. organelles including Apicomplexa, most mito-
chondria, and some lower eukaryotes express
multiple enzymes that act as one complex from
a prokaryotic origin (White et al., 2005). This
8.2 Fatty acids
dissociated system named FASII encodes each
enzymatic component that catalyzes a single
8.2.1 Fatty acid biosynthetic pathways—
step in the pathway. FASI is thought to have
generalities evolved by the fusion of the prokaryotic type II
Fatty acid synthesis (FAS) is a critical ana- complex into a single protein.
bolic pathway in most organisms. In addition The multifunctional protein of FASI is usu-
to being the hydrophobic building blocks of ally localized in the cytosol of mammalian
membrane lipids, fatty acids are important cells. In plants, photosynthetic and plastid-
energy storage molecules, and fatty acyl deri- bearing organisms, FASII, is located in the
vatives possess a variety of physiological func- plastid (chloroplast) that is derived from a
tions, including posttranslational modification cyanobacterial endosymbiont, for example,
of numerous proteins. The fundamental pro- the apicoplast in Toxoplasma. The genes for
cess of fatty acid biosynthesis is highly con- these enzymes are all encoded in the nuclear
served among species and involves major genome, and the proteins are posttranslation-
metabolites in the central carbon metabolism ally exported to the plastid, as it is common
as substrates, including acetyl-CoA. The central with plastid enzymes due to a massive lateral
feature of the pathway is the sequential exten- plastid genome transfer during evolution
sion of an alkanoic chain, two carbons at a (McFadden, 1999). Some FASII intermediates
time, by a series of decarboxylative condensa- are used in the synthesis of key cellular consti-
tion reactions. This process is generally initi- tuents and cofactors, such as lipoic acid that is
ated with the carboxylation of acetyl-CoA to essential for the proper function of most dehy-
yield malonyl-CoA (Smith et al., 2003). The drogenase complexes (such as pyruvate dehy-
malonate group of malonyl-CoA is transferred drogenases, a central component of the central
to the phosphopantetheine prosthetic group of carbon metabolism). This enormous diversity
a small, acidic protein or protein domain, of FASII products is possible because the
called the acyl carrier protein (ACP). Malonyl- acyl-ACP intermediates (activated forms of
ACP is then condensed with acetyl-CoA, fatty acids) are diffusible entities that are traf-
reduced, dehydrated, and reduced once again ficked and diverted into many biosynthetic
yielding an acyl-ACP. The elongation of the pathways.
Toxoplasma Gondii
8.2 Fatty acids 369
In addition, some fatty acids can further be parasite must encounter different nutritional
elongated into (very) long-chain fatty acids and metabolic challenges. It is no surprise
chains by individual membrane-bound that T. gondii has evolved to express a broad
enzymes, named elongases (ELO), usually set of fatty acid-related genes for de novo
located in the endoplasmic reticulum (ER). The synthesis (Mazumdar and Striepen, 2007).
synthesis of very long-chain fatty acids is a Bioinformatic, genetic, and biochemical stud-
ubiquitous system is found in different organ- ies document that T. gondii expresses three
isms and cell types, and specific “elongated” fatty acid synthetic pathways localized to
fatty acids serve commonly as building blocks different cellular compartments (Waller et al.,
of sphingolipids, but they can be also constitu- 1998; Seeber, 2003; Ramakrishnan et al., 2012):
ents of glycerophospholipids, triacylglycerols a cytosolic FASI-like pathway; a FASII present
(TAG), and steryl- and wax-esters (Jakobsson in the apicoplast producing myristic (C14:0)
et al., 2006; Uttaro, 2006). and palmitic (C14:0) acids, in addition to
lipoic acid; and an elaborate fatty acid elonga-
tion pathway compartmentalized in the ER
and responsible for the production of very
8.2.2 Fatty acid synthesis in Toxoplasma long-chain monounsaturated fatty acids
Toxoplasma has the great ability to infect (Fig. 8.1). The FASI pathway consists of a
virtually any warm-blooded animals and to single large polypeptide that harbors the
multiply in any type of nucleated cells as their ACP, FabD, FabH, FabG, FabZ, and FabI
hosts. In these different environments the activities.
FIGURE 8.1 Fatty acid salvage and biosynthetic pathways in Toxoplasma. The parasite harbors three fatty acid syn-
thetic pathways: (A) Cytosolic-located FASI. (B) Apicoplast-localized FASII producing myristic and palmitic acid in addi-
tion to lipoic acid. (C) The parasite is also able to scavenge several fatty acids from the environment. (D) ER-associated
elongase system that synthesizes very long-chain monounsaturated fatty acids using the activity of ELO. Major products
are highlighted in red. The parasite is also able to scavenge several fatty acids from the environment. Ac, Acetate; ELO,
elongases; ER, endoplasmic reticulum; hcell, host cell; Mal, malonate; PV, parasitophorous vacuole. Source: Adapted from
Ramakrishnan, S., Docampo, M.D., Macrae, J.I., Pujol, F.M., Brooks, C.F., van Dooren, G.G., et al., 2012. Apicoplast and endoplasmic
reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii. J. Biol. Chem. 287, 4957 4971.
Toxoplasma Gondii
In Toxoplasma the apicoplast possesses a attached biotin. The first step of the ACC-
complete prokaryotic FASII. This pathway is catalyzed reaction is an ATP-dependent
essential during the tachyzoite (proliferative) transfer of the carboxyl group from bicarbonate
life stage because it provides the bulk of fatty to the biotin residue (first half-reaction).
acids used for the synthesis of major mem- The carboxyl group is then transferred to
brane lipid classes (Mazumdar et al., 2006; acetyl-CoA producing malonyl-CoA (second
Ramakrishnan et al., 2012; Amiar et al., 2016; half-reaction). Malonyl-CoA is used for de
Sidik et al., 2016). T. gondii uses glucose as a novo fatty acid biosynthesis as well as in fatty
major source for energetic purposes. The apico- acid elongation. Incubation of radiolabeled
plastic FASII is dependent on the import of malonyl-CoA with T. gondii extracts results in
host glucose and the parasite’s glycolytic path- the production of C16:0. Based on recent lipi-
way to generate the proper substrate for FAS, domics studies, C16:0 is likely produced
that is, acetyl-ACP (Ramakrishnan et al., 2012; through the elongase pathway (Ramakrishnan
Amiar et al., 2016). Glucose and acetyl-CoA do et al., 2012; Dubois et al., 2018). Intriguingly, T.
not enter the apicoplast to fuel FASII (as gondii expresses a second ACC, most likely
initially thought), but instead two triose phos- cytosolic although having the multidomain
phate glycolytic products, that is, dihydroxyac- type as the ACC prototype found in the cyto-
etone phosphate and phsophoenolpyruvate plasm of eukaryotes and in plastids of some
(PEP), are imported into the apicoplast by the plants. This cytosolic ACC may generate sub-
apicoplast phosphate transporter, named strates for the cytosolic FASI but experimental
TgAPT (Lim et al., 2009; Ramakrishnan et al., evidence is still missing.
2012; Amiar et al., 2016). Recent stable isotope Subsequently to the ACC activity, acetyl-
labeling assays combined to lipidomics confirm CoA, and malonyl-CoA are transferred to an
that the apicoplast metabolizes glycolytic pro- ACP, which transfers the nascent fatty acid
ducts (Ramakrishnan et al., 2012; Amiar et al., chain to the different enzymes of the pathway,
2016). Imported PEP can then be converted through the actions of acetyl-CoA ACP transa-
into pyruvate and finally to acetyl-CoA by the cylase and malonyl-CoA:ACP transacylase
apicoplastic pyruvate kinase, named TgPKII, (FabD), respectively. β-Ketoacyl:ACP is then
and the apicoplast pyruvate dehydrogenase synthesized from acetyl-ACP and malonyl-ACP
complex, named TgPDH (Oppenheim et al., by a β-ketoacyl:ACP synthase (FabH). β-Ketoacyl-
2014; Tymoshenko et al., 2015b; Amiar et al., ACP is reduced by β-ketoacyl:ACP reductase
2016; Dubois et al., 2018). One of the subunits (FabG) to form β-hydroxyacyl-ACP that is dehy-
of the TgPDH complex, E2 PDH, must be drated by β-hydroxyacyl-ACP dehydrase (FabZ)
lipoylated de novo in the apicoplast to be func- to form α,β-trans enoyl-ACP. This is further
tional (Mazumdar et al., 2006). reduced to butyryl-ACP by the action of enoyl-
In the next major step of FASII in T. gondii, ACP reductase (FabI). This cycle occurs up to
acetyl-CoA is carboxylated to form malonyl- 6 8 times in T. gondii (Ramakrishnan et al.,
CoA by an acetyl coenzyme A carboxylase 2012; Amiar et al., 2016; Dubois et al., 2018).
(ACC) using bicarbonate as a source of the car- ACP plays a central role in fatty acid biosynthe-
boxyl group, biotin as a cofactor, and ATP as a sis by holding the forming acyl chain, whereas
source of energy (Jelenska et al., 2001). Indeed, FabH and FabZ are involved in the condensa-
ACC consists of three major functional tion and dehydration steps, respectively, of
domains: the biotin carboxylase domain, the acetyl addition during acyl chain elongation.
carboxyltransferase domain, and the biotin car- Loss of FASII severely compromises the rep-
boxyl carrier domain containing covalently lication and the virulence of Toxoplasma
Toxoplasma Gondii
8.2 Fatty acids 371
(Mazumdar et al., 2006; Ramakrishnan et al., the target for existing antibiotics and herbicides
2012; Martins-Duarte et al., 2016; Amiar et al., (Roberts et al., 2003; Sonda and Hehl, 2006;
2016). In particular, genetic disruption of ACP Goodman and McFadden, 2007; Martins-
leads to defects in apicoplast biogenesis, with Duarte et al., 2009), although some promising
loss of the organelle. Stable isotope labeling molecules against FASII in Apicomplexa have
using 13C-U-glucose as a precursor for FASII been shown to have off-target effects (Botté
reveals that the pathway relies on glycolytic et al., 2012). In vitro and in vivo tests with
intermediates and not on import of acetate to selected aryloxyphenoxypropionate herbicides
generate fatty acid products (Ramakrishnan show that the carboxyltransferase domain of
et al., 2012; Amiar et al., 2016), and that the the apicoplast T. gondii ACC is the binding tar-
main products of FASII are medium to long get for this class of inhibitors (Jelenska et al.,
fatty acid chains, mainly C12:0 (dodecanoate), 2002). Expectedly, the cytosolic form of T. gondii
C14:0 (myristate), C16:0, and C18:0 (stearate). ACC and human ACC are resistant to aryloxy-
Comprehensive fluxomics using 13C-U-glucose phenoxypropionates. Triclosan is also a potent
and 13C-acetate in combination with mass inhibitor of type II FabI (Baldock et al., 1996).
spectrometry-based lipidomics, performed to This compound restricts the growth of T. gondii
assess whether C12:0, C14:0, C16:0, and C18:0 in vitro (McLeod et al., 2001). Triclosan blocks
produced by FASII are important for parasite the incorporation of radioactive acetate into the
growth, shows that these fatty acids are used fatty acids of Toxoplasma and specifically inhi-
to generate the bulk of major phospholipid bits FASII. Morphological analyses on
classes, especially phosphatidylcholine (PC), triclosan-treated parasites reveal that this com-
phosphatidylinositol (PI), and phosphatidyl- pound affects apicoplast inheritance and para-
ethanolamine (PE) for membrane biosynthesis site division by preventing cytokinesis
and organelle biogenesis (Amiar et al., 2016; completion, resulting in incomplete daughter
see below for detailed mechanisms in phos- cell budding (Martins-Duarte et al., 2016).
pholipid synthesis by FASII products). Long-chain fatty acid supplementation in the
In addition to providing medium-long fatty medium rescues the cytokinesis and prolifera-
acids, FASII is required for de novo synthesis tion defects of FASII inhibition, which confirms
of lipoic acid, a short fatty acid chain, C8:0. that FASII is essential to generate lipid sub-
Lipoic acid is an essential cofactor for oxidative strates. Thiolactomycin, a fungal secondary
decarboxylases and is usually involved in the metabolite (Oishi et al., 1982) selectively inhi-
response to oxidative stress in eukaryotic sys- bits type II FabH of T. gondii (Waller et al., 1998;
tems. ACP-knockdown parasites are impaired Martins-Duarte et al., 2009). Thiolactomycin
in protein lipoylation by the apicoplast PDH decreases rapidly the growth of this parasite.
(pyruvate dehydrogenase complex), the sole Cerulenin, a metabolite of Cephalosporium caeru-
source of the metabolic precursor acetyl-CoA. lens, is an inhibitor of both types I and II FabH
In particular, the apicoplast produces lipoic (Waller et al., 1998; Heath et al., 2001).
acid that is required for the functional mainte- Cerulenin is found to act synergistically with
nance of FASII and is thus important for mem- triclosan in inhibiting FAS II in the related
brane biogenesis and progeny formation. malaria parasite (Waller et al., 1998; Botté et al.,
Fundamentally different from the cytosolic 2012). Thiolactomycin and cerulenin represent
type I pathway of the mammalian host, FASII potential drugs that may also affect the two
in T. gondii has a tremendous potential for the FAS in T. gondii, and therefore growth.
development of parasite-specific inhibitors. If the apicoplast represents a significant
Many components of this pathway are already source of fatty acids, the latter products can
Toxoplasma Gondii
further be modified in the ER of Toxoplasma hydroxyacyl-CoA dehydratase and enoyl-CoA

(Ramakrishnan et al., 2012; Dubois et al., 2018). reductase.
The existence of membrane contact sites In T. gondii the initial substrate for the
between the apicoplast outermost membrane ER-based elongation pathway is acetyl-CoA
and the ER as shown at the ultrastructural level produced by two enzymes: a mitochondrial cit-
suggests a nonvesicular trafficking of lipid rate lyase, named TgACL, and a single cyto-
between the two organelles (Tomavo et al., solic acetyl-CoA synthetase, named TgACS,
2009), although the two organelles share phos- that uses acetate as substrate (Tymoshenko
phatidylinositol phosphate vesicular-based et al., 2015; Sidik et al., 2016; Dubois et al.,
trafficking pathways sustaining apicoplast bio- 2018). Neither single knockout of TgACL nor
genesis (Tawk et al., 2011). The synthesis of TgACS affects the parasite growth, but para-
very long-chain fatty acids is primarily sites in which TgACL has been silenced and
dependent on the fatty acid elongation system TgACS ablated are not viable, suggesting that
comprising three ELO, two reductases, and these enzymes have redundant functions for
a dehydratase expressed by T. gondii the parasite. Based on 13C-stable isotope label-
(Ramakrishnan et al., 2012). Metabolic labeling ing combined with mass spectrometry-based
studies with radioactive glucose show that lipidomics analyses, TgACS is involved in pro-
intracellular parasites synthesize a range of viding acetyl-CoA for the fatty elongation
long to very long-chain fatty acids pathway in the ER to generate very long fatty
(C14:0 26:1). The enzymatic steps involved in acids (C18:0, C18:1, C20:0, C20:1, C22:0, and
the fatty acid elongation process are similar to C24:0). Disruption of TgACS has a minor effect
those in FASII, but the growing chain is held on the global fatty acid composition, likely due
by CoA instead of ACP. Genetic ablation of to the metabolic flux changes induced to com-
each individual fatty acid elongase (ELO-A, pensate for its loss. The precise role of TgACL
ELO-B, and ELO-C) does not result in global in fatty acid elongation is currently unknown.
growth defect despite observations of
decreased amounts of very long fatty acids for
most single elongase-deficient parasites. This
suggests functional redundancy between these
8.2.3 Fatty acid salvage by Toxoplasma
complexes and/or that other fatty acid biosyn- In addition to producing fatty acids through
thetic or salvage mechanisms compensate three biosynthetic pathways, Toxoplasma
for the loss of individual ELO complexes. imports selected fatty acids from the host cell
Nevertheless, conditional knockdown of the (Tomavo et al., 1989; Quittnat et al., 2004;
nonredundant hydroxyacyl-CoA dehydratase Polonais and Soldati-Favre, 2010; Charron and
and enoyl-CoA reductase enzymes in the ELO Sibley, 2002; Ramakrishnan et al., 2012; Hu
pathway severely repressed intracellular para- et al., 2017; Nolan et al., 2017, 2018; Pernas
site growth (Ramakrishnan et al., 2015). et al., 2018). Exogenous fatty acids are used for
13
C-U-glucose and 13C-acetate labeling and incorporation into complex lipids. Intracellular
comprehensive lipidomics analyses of these T. gondii scavenges the fluorescent fatty acid
mutants show selective defect in synthesis of analog 5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-
unsaturated long to very long-chain fatty acids s-indacene-3-nonanoic acid (C4-BODIPY-C9)
and depletion of PI and PE species containing from the medium and delivers this lipid to the
unsaturated long to very long-chain fatty acids. parasitophorous vacuole (PV) membrane,
Supplementing the media with these fatty Golgi/ER (Charron and Sibley, 2002) and
acids is insufficient to compensate the loss of thin tubules filling the lumen forming the
Toxoplasma Gondii
8.2 Fatty acids 373
intravacuolar network (IVN) (Sibley et al., 1995). growth arrest and lipid-induced damage, con-
In infected cells pulsed with BODIPY-FL-C 12 firming a major disconnect between fatty acid
(FL C12), a 12-carbon saturated fatty acid was uptake and the parasite’s cellular lipid require-
covalently bound to the BODIPY fluorophore at ments. However, exogenous fatty acids are
its hydrophobic end (hence equivalent to a required to generate parasite membrane phos-
long-chain fatty acid), FL C12 is rerouted to the pholipids and sustain survival (Amiar et al.,
PV (Pernas et al., 2018). Another study showed 2016). Therefore the parasite needs to maintain
that upon incubation of mammalian cells for a balance in fatty acid fluxes between the two
24 hours with the nonmetabolized fatty acid 4,4- sources, salvage, and synthesis. Exogenous
difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza- fatty acids can also be acquired from scav-
s-indacene (BODIPY493/503) that accumulates in enged phospholipids that are then recycled by
LDs, followed by infection with Toxoplasma, the the parasite (Charron and Sibley 2002; Amiar
signal for BODIPY493/503 is detected within et al., 2016).
parasite LD, indicating that Toxoplasma is able Extracellular parasites incorporate C16:0
to retrieve fatty acids from host LD for storage and C14:0 into GPI anchors, such as that of
(Nolan et al., 2017). This selective compartmen- SAG1 (Tomavo et al., 1989). Based on a chemi-
talization of diverted lipids reflects sorting cal proteomic approach, a comprehensive anal-
activities mediated by the parasite to adeptly ysis of palmitoylated proteins in T. gondii,
distribute exogenous lipids into proper orga- identifies 282 cytosolic, membrane-associated
nelles. Intracellular and extracellular T. gondii or transmembrane proteins, involved in motil-
are competent to accrue various free fatty acids ity (myosin light chain 1, myosin A), cell mor-
from their environment, including C16:0, oleic phology (PhIL1), and host-cell invasion (apical
(C18:1), stearic (C18:0), linoleic (C18:1), arachi- membrane antigen 1, AMA1) (Foe et al., 2015).
donic (C20:4) acids, with a preferential internali- Treating tachyzoites with the palmitoyl acyl-
zation of C16:0 (Quittnat et al., 2004), and also transferase (PAT) inhibitor, 2-bromopalmitate,
the polyunsaturated fatty acids C18:2, C20:4, inhibits motility and host-cell invasion and
and C20:5 that the parasite is unable to synthe- disrupts parasite morphology (Alonso et al.,
size (Amiar et al., 2016). Within the parasite, 2012). A rhoptry-localized PAT, named
exogenous fatty acids are manufactured into TgDHHC7, has been characterized and func-
TAG (Quittnat et al., 2004), cholesteryl esters tions to properly affix the rhoptries at the api-
(Nishikawa et al., 2005; Lige et al., 2013), and cal end of the parasite (Beck et al., 2013).
phospholipids (Amiar et al., 2016). Unsaturated Conditional disruption of TgDHHC7 results in
fatty acids [C18:1, palmitoleate (C16:1), linoleate defects in rhoptry localization and function.
(C18:2)] added at physiological concentrations This indicates that palmitoylation is ubiquitous
accumulate in large LD in Toxoplasma and throughout the T. gondii proteome and that
impair parasite replication, whereas saturated palmitate is a critical fatty acid for the para-
fatty acids (C16:0, C18:0) neither stimulate LD site’s survival.
formation nor impact growth (Nolan et al., After the uptake of butyric acid (C4:0) by
2018). Examination of parasite growth defects the parasite, this short fatty acid chain is ana-
with 0.4 mM oleate shows massive lipid depos- bolized to generate PC (Charron and Sibley,
its outside LD, indicating enzymatic inadequa- 2002). Similarly, most phospholipid classes
cies for storing neutral lipids in LD in response (PC, PI, and PE) require fatty acids both
to the copious salvage of oleate. Toxoplasma scavenged and produced through FASII
exposure to 0.5 mM oleate leads to irreversible (Amiar et al., 2016). Interestingly, the reduced
Toxoplasma Gondii
growth of T. gondii consecutive to ablation of role of host mitochondria-PV association is still

type II fatty acid synthase activities is partially unknown but when TgACBP2-deficient type II
restored by addition of long-chain fatty acids parasites are transfected with MAF-1, they par-
in the medium (Ramakrishnan et al., 2012), tially regain virulence, suggesting a metabolic
confirming that both salvaged and de novo network and lipid exchange between host mito-
synthesized fatty acids have compensatory chondria and the PV.
activities, although neither sources can be fully Obviously, the diversity and redundancy of
disrupted. the fatty acid pathways might be taken as an
Conserved among bacteria, archaea, and indication that the availability of the correct
eukaryotes, the sterol-carrier protein-2 (SCP-2) fatty acids is an essential determinant for suc-
is a nonspecific lipid transfer protein for phos- cessful adaptation of the parasite to various
pholipids, fatty acids, and fatty acyl-CoA, host cells. If fatty acid uptake is as essential as
(Gallegos et al., 2001). A D-bifunctional protein FAS for the parasite growth and pathogenesis,
consisting of one N-terminal D-3-hydroxyacyl- fatty acid homeostatic pathways hold
CoA dehydrogenase domain fused to two tan- promise for much more Toxoplasma-specific
dem SCP-2 domains, TgHAD-2SCP-2, has been drug targets.
identified in Toxoplasma and promotes the circu-
lation of cholesterol, phospholipids, and fatty
acids between parasite organelles and the
plasma membrane (Lige et al., 2009). The para-
8.2.4 Fatty acid fluxes in Toxoplasma
site expresses TgACBP1, a cytosolic acyl-CoA T. gondii synthesizes some phospholipids,
binding protein believed to be a fatty acid trans- made on a “patchwork” of fatty acids that are
porter, whose disruption causes dysfunction of synthesized by FASII and/or salvaged from the
fatty acid uptake from host cells (Fu et al., host (Amiar et al., 2016). Mechanisms allowing
2018). 13C-labeling assays combined with gas the parasite to strike a balance between the two
chromatography-mass spectrometry (GC MS) different sources of fatty acids remain largely
show that double disruption of TgACBP1 and unknown. The advent of 13C-U-glucose based
TgHAD-2SCP-2 leads to reduced synthesis rates stable isotope labeling, allowing the determina-
of C18:0, C22:1, and C24:1. Another acyl-CoA tion of apicoplast-derived (labeled) versus scav-
transporter, TgACBP2, localized to the parasite enged host fatty acids, has surpassed major
mitochondrion may provide acyl chains for lacunae. MS/MS analysis reveals that some
phosphatidic acid and phosphatidylglycerol major phospholipid species in the parasite con-
(PG) and also be involved in the synthesis of tain one 13C-labeled fatty acid moiety and one
cardiolipins (phospholipid species with a that is unlabeled, thus originating from the host
diphosphatidylglycerol (DPG) structure com- environment, raising the issue how the parasite
bined with four acyl chains, which is the typical could achieve/generate such patchwork lipids
signature lipid class of mammalian mitochon- (Amiar et al., 2016).
dria), based on reduction in the amounts of PA, In the apicoplast, TgATS1 generates lyso-
PG, and cardiolipins in TgACBP2-deficient phosphatidic acid (LPA) using fatty acids
Toxoplasma (Fu et al., 2019). These mutants from synthesized by FASII, which in turn fuels
cystogenic type II strains have attenuated viru- bulk phospholipid biosynthesis reactions.
lence in mice. Type I but not type II strains of Disruption of ATS1 is lethal to Toxoplasma,
Toxoplasma recruit host mitochondria at their PV resulting in significant decrease of C14:0 into
by secreting a mitochondrial association factor-1 bulk synthesis of major phospholipid classes
(MAF-1) localized at the PV membrane. The (Amiar et al., 2016). This implicates the
Toxoplasma Gondii
8.3 Glycerophospholipids 375
importance of apicoplast-derived fatty acid 8.3 Glycerophospholipids
flux within the parasite, which cannot always
be bypassed by fatty acid salvage. The apico- 8.3.1 Phospholipid biosynthetic
plast possesses a second enzyme for PA syn- pathways—generalities
thesis, named ATS2, which construes the fatty
acid scavenging capacities of the parasite. Glycerophospholipids, known as phospholi-
Disruption of TgATS2 results in an increase of pids, are key molecules that contribute to the
polyunsaturated fatty acids that are readily structural definition of cells and that partici-
scavenged, such as C20:4, eicosapentaenoic pate in the regulation of many cellular pro-
acid (C20:5), and docosadienoic acid (C22:6) cesses. Phospholipid metabolism is a major
along with C18:0 as a compensatory mecha- activity that engages cells throughout their
nism for the loss of FASII-derived fatty acids growth (Carman and Zeimetz, 1996). These
(mostly C14:0). TgATS2-deficient parasites amphiphilic lipids insert in cell membranes
import significantly more exogenous lipids, and form into a sheet two molecules thick with
such as PA and PC, which compensates for the the fat-soluble portions inside, shielded on
inability of the parasite to synthesize these both sides by the water-soluble portions. This
lipids adequately (Katris, Botte, and Dass, stable structure provides the cell membrane
unpublished). with its integrity. In mammalian cells the most
Fatty acids can also be enzymatically abundant glycerophospholipids are PC, PE, PI,
recycled for membrane homeostasis. phosphatidylserine (PS), PG, and cardiolipin.
Phospholipases participate in lipid turnover by All phospholipids are synthesized de novo
generating lysophospholipids and free fatty from the unique and central precursor PA and
acid moieties for recycling/reshuffling to vari- its two direct downstream products, diacylgly-
ous membranous compartments within the par- cerol (DAG) and CDP-DAG. PA is synthesized
asite. Toxoplasma has an apicoplastic patatin-like de novo by the sequential esterification of
phospholipase, named TgPL2, which is acyl-CoA/ACP onto a glycerol-3-phosphate
involved in recycling of fatty acids by generat- backbone by acylglycerol-3-phosphate acyl-
ing lysophosphatidylcholine from PC (Lévêque transferase (AGPAT/ATS1), forming LPA and
et al., 2017). Phospholipases (host and parasite an acylglycerol-3-phosphate acyltransferase
derived) are also involved in recruiting fatty (AGPAT/ATS2) forming PA from LPA. PS, PI,
acids and/or lipids from host LD that localize PG, and cardiolipins are synthesized from the
near and within the PV (Nolan et al., 2017). so-called cytidine diphosphate-DAG (CDP-
Another group of enzymes facilitating the remo- DAG) pathway, while PC and PE are synthe-
deling of host versus apicoplast-derived lipids sized from DAG by the Kennedy pathway
are fatty acid transporters and acyl-CoA synthe- where polar heads precursors (CDP-choline
tases. 13C-U-labeling assays combined with and CDP-ethanolamine) are transferred onto a
GC MS show that double disruption of the DAG, made from PA by a phosphatidic acid
acyl-CoA transporter TgACBP1 and of TgSCP2 phosphatase (PAP).
reduces the abundance of major scavenged fatty
acids C18:0, C22:1, and nervonic acid (C24:1)
8.3.2 Phospholipid composition and
(Fu et al., 2018). Understanding this complex,
yet essential, fatty acid metabolism will help in
physiological relevance in Toxoplasma
the identification of key weak points in the par- Quantification of the phospholipid profiles
asite “biological armor” for developing novel of Toxoplasma reveals that PC is the most prev-
therapeutics. alent lipid, accounting for about 75% of total
Toxoplasma Gondii
phospholipids (Gupta et al., 2005). The next lipid classes where most phospholipid classes
most abundant lipids are PE (10%), PI (7.5%), (especially PC, PE, and PI) are composed of
PS (6%), and PA (1%). Compared to mamma- one fatty acid coming from the FASII and one
lian cells, T. gondii has higher levels of PC scavenged from the host.
but lower levels of sphingomyelin and PS PA plays central in Toxoplasma cell cycle and
(Welti et al., 2007). However, a recent high- infectivity, as it controls the secretion of micro-
performance liquid chromatography analysis nemal proteins involved in parasite invasion
reveals the presence of a novel lipid eluting (Bullen et al., 2016). In addition, PA is found in
next to PS and corresponding to phosphatidyl- the PV lumen where it triggers the naturel
threonine (PThr) (Arroyo-Olarte et al., 2015). In egress of the parasite when the host cell
contrast to mammalian in which PThr is a trace becomes a hostile environment (Bisio et al.,
component, this lipid is significantly more 2019). A dynamin-related protein, named
abundant in parasite membranes, and consid- TgDrpC participates in the parasite’s endodyo-
erably more preponderant than PS. The role of geny, which in turn relies on the local PA and
PThr in T. gondii is still unknown, but its pres- its precursor LPA to generating appropriate
ence may be due to high amounts of threonine membrane curvature, thereby assisting in cyto-
scavenged from the host, directly used by a PS kinesis of daughter cells (Katris, Botté,
synthase 2 named TgPSS2. In other systems, unpublished).
PSS2 enzymes usually catalyze PS synthesis The distinctive T. gondii phospholipid pro-
from serine but TgPSS2 in the parasite may file may be particularly suited to the function
have more affinity for threonine than for PS. of parasitic membranes and the interaction of
Being one of the most abundant anionic lipids, the parasite with the host cell and the host’s
PThr may regulate Ca21 homeostatic events. immune system. Some PS molecules are
Calcium signaling is central for Toxoplasma secreted by Toxoplasma in the PV (Gupta et al.,
infection as it governs motility, egression, and 2012). Present at outer leaflet of the plasma
invasion (Lourido and Moreno, 2015), poten- membrane of eukaryotic cells, PS is a major
tially making PThr an important contributor to ligand involved in the uptake of apoptotic cells
these events. (Fadok et al., 2001). Phagocytosis of apoptotic
Compared to eukaryotic cells, T. gondii has a cells by macrophages induces a noninflamma-
wider range of polar lipids with unique lipid tory response based on the exposure of PS that
composition and unusual abundance, such as a leads to TGFβ1 secretion (Fadok et al., 1998).
relatively high level of ceramide phosphoetha- PS exposure on the cell surface has also been
nolamine (B2% of the total polar lipids), which related to evasion mechanisms of parasites, a
has a fatty acid profile solely constituted of 16 concept known as apoptotic mimicry. T. gondii
and 18 carbon species (Welti et al., 2007). The mimics apoptotic cells by exposing PS, induc-
parasite has also a greater amount of short to ing secretion of TGF-β1 by infected activated
medium chain fatty acid incorporated into macrophages that leads to the degradation of
polar lipids. For example, PC containing two inducible nitric oxide synthase and inhibition
saturated acyl chains with 12, 14, or 16 carbons of nitric oxide production, and consequently
makes up over 11% of PC but less than 3% of parasite persistence in macrophages (Santos
the mammalian PC molecular species. This et al., 2011). A PS-negative subpopulation of
specificity is directly linked to the metabolic Toxoplasma enters macrophages by phagocyto-
capacities of the apicoplastic FASII, in which sis and is unable to inhibit nitric oxide synthe-
C12:0, C14:0, and C16:0 are the major products. sis, whereas a PS-positive subpopulation
Furthermore, fluxomics reveals patchwork invade macrophages by active penetration, and
Toxoplasma Gondii
no sign of inflammation is detected in mouse. gene product is predicted to be a glycerol-3-
This indicates that the escape mechanism of phosphate acyltransferase (GPAT) from a
T. gondii is dependent on the exposure of PS, eukaryotic origin and another one, a prokary-
making this lipid essential for a successful otic acylglycerol-phosphate-acyltransferase
infection and survival. Interfering with the PS (AGPAT), both located in the ER. In addition,
biosynthetic pathways would dramatically T. gondii expresses TgATS1 in the apicoplast
reduce the parasite burden in their hosts. (see Section 8.2). The apicoplast-located FASII
Among PI species, T. gondii contains PI 3- and TgATS1, which are primarily used to gen-
monophosphate that is involved in a vesicular erate plastid galactolipids in plants and algae,
trafficking process involved in the biogenesis have been repurposed to generate bulk phos-
of the apicoplast, allowing the fusion of vesi- pholipids for membrane biogenesis in T. gondii.
cles containing nuclear-encoded apicoplast In mammalian systems, DAG is intercon-
proteins with this organelle (Tawk et al., 2011). verted to PA via DAG kinases (DGK) and
Pharmacological studies have revealed that PAPs. In the T. gondii genome, three genes
PC is also a central phospholipid for code for putative PAP: two cytosolic TgPAP1
Toxoplasma physiology. The choline analog N, and TgPAP2 (Bullen et al., 2016), and a
N-dimethylethanolamine is taken up by intra- TgPAP2-like located to the inner membrane
cellular parasites as efficiency as choline complex sutures (Chen et al., 2017), but the bio-
(Gupta et al., 2005). As a result, T. gondii chemical activities of these enzymes remain to
growth is progressively arrested, probably due be characterized. Furthermore, three genes
to dramatic PC depletion and/or toxic phos- encode three putative DGK: TgDGK1 localized
phatidyldimethylethanolamine amassing in to the parasite plasma membrane, TgDGK2 to
parasite membranes. In mammalian cells, dense granules and to the PV, and TgDGK3 to
phosphatidyldimethylethanolamine is nor- micronemes (Bullen et al., 2016). Depletion of
mally produced as a short-live intermediate in TgDGK1 impairs egress and causes parasite
the conversion of PE to PC (Vance and Vance, death. This enzyme contains an acylated
2004). Clearly, dimethylethanolamine interferes pleckstrin-homology domain-containing pro-
with choline uptake and metabolism to PC, tein (APH) on the microneme surface that
resulting in selective alteration in parasite senses PA during microneme secretion and is
membrane morphology at concentrations non- necessary for microneme exocytosis. TgDGK2
toxic for the host cell. This indicates that the is responsible for the production of PA into the
dominance of PC as a major lipid in T. gondii PV lumen (Bisio et al., 2019). PA acts as an
membranes offers great potentialities to dis- intrinsic signal that elicits natural egress
rupt the membrane biogenesis of the parasite. upstream of an atypical guanylate cyclase,
which is composed of a P4-ATPase and two
guanylate cyclase catalytic domains. The
P4-ATPase domain is predicted to be responsi-
8.3.3 Phospholipid synthesis in
ble for sensing of vacuolar levels of PA and
Toxoplasma pH. This suggests the existence of a signaling
As mentioned above, glycerophospholipid platform that responds to an intrinsic lipid
synthesis is initiated by the synthesis of the mediator and extrinsic signals to control pro-
central phospholipid precursor, PA. Toxoplasma grammed and induced egress.
possesses four genes encoding two separate CDP-DAG synthesis is particularly relevant
sets of the acyltransferases required for PA in T. gondii as the parasite has two CDP-DAG
synthesis de novo (Amiar et al., 2016). One pools (Kong et al., 2017). It expresses two
Toxoplasma Gondii
phylogenetically divergent CDS enzymes: the analyses on the TgCDS1-deficient parasites

eukaryotic-type TgCDS1 located in the ER and show declines in PI levels in the Golgi appara-
the prokaryotic-type TgCDS2 residing in the tus while TgCDS2-deficient parasites have less
apicoplast. Conditional knockdown of TgCDS1 PG for the mitochondrion.
severely attenuates parasite growth and results Toxoplasma is also enzymatically equipped
in severe loss of virulence in mice. The residual to synthesize de novo glycerophospholipids
growth of TgCDS1 mutant is abolished by con- via the Kennedy pathway (Fig. 8.2). T. gondii
secutive deletion of TgCDS2. Lipidomics incorporates choline, ethanolamine, and serine
Ethanolamine Choline DME
EK CK
Phosphoethanolamine Phosphocholine PhosphoDME
PET PCT
CDP-ethanolamine CDP-choline CDP-DME
EPT CPT
PEMT
PtdEtn PtdCho PtdDME
PSS1
PSD
PSS2
PtdSer
Serine
FIGURE 8.2 Biosynthetic pathways of three major phospholipids in human and Toxoplasma. The Homo sapiens path-
ways are adapted from literature and that of Toxoplasma gondii are constructed based on the reported enzyme activities and
annotations in the parasite database (www.ToxoDB.org). The common pathways are shown in black; those specific to
human are depicted in green. Initial precursors are shown in blue; the intermediates of lipid synthesis are in black; phos-
pholipids are in red, and the enzymes are in brown color. DME is metabolized via the CDP-choline route and produces PE,
which is not methylated to PC in T. gondii causing the disruption of membrane biogenesis. CDP, Cytidine diphosphate; CK,
choline kinase (forming clusters in the cytosol in green); CPT, CDP-choline phosphotransferase (localized to the ER in
green); DME, dimethylethanolamine; EK, ethanolamine kinase; EPT, CDP-ethanolamine phosphotransferase; PC, phosphati-
dylcholine; PCT, phosphocholine cytidylyltransferase (localized to nucleus in green); PE, phosphatidylethanolamine; PEMT,
phosphatidylethanolamine methyltransferase; PET, phosphoethanolamine cytidylyltransferase; PSD, phosphatidylserine
decarboxylase; PSS, phosphatidylserine synthase; SD, serine decarboxylase. Source: Adapted from Sampels, V., Hartmann, A.,
Dietrich, I., Coppens, I., Sheiner, L., Striepen, B., et al., 2012. Conditional mutagenesis of a novel choline kinase demonstrates plastic-
ity of phosphatidylcholine biogenesis and gene expression in Toxoplasma gondii. J. Biol. Chem. 287, 16289 16299.
Toxoplasma Gondii
into PC and PS, respectively. Unlike its gain in PS. However, TgPTS-deficient parasites
mammalian host (Vance and Vance, 2004), have impaired lytic cycle and virulence in
Toxoplasma does not show any activities from a mice. DPG, a mitochondrial cardiolipin repre-
serine decarboxylase, a PE decarboxylase, and sentative, and PG are produced by Toxoplasma
a PE methyltransferase and thus appears as monitored by metabolic experiments using
incompetent in making PC from serine and/or radioactive acetate (Bisanz et al., 2006).
ethanolamine (Gupta et al., 2005; Sampels Mammalian lecithin: cholesterol acyltransfer-
et al., 2012). Conversely, a parasite choline ase (LCAT) is characterized by dual activity,
kinase has potential to counteract the loss of PLA2, and acyltransferase (Glomset, 1968). This
ethanolamine kinase and to sustain de novo enzyme catalyzes the transacylation of the sn-2
synthesis of PE in T. gondii. The parasite also fatty acid liberated from various phospholipids
harbors a PS decarboxylase route to produce (e.g., PC and PE) to the 3-β-hydroxyl group on
PE from PS. The parasite resilience to a pertur- the A-ring of cholesterol, thereby forming cho-
bation of choline kinase, compositional flexibil- lesteryl esters. Toxoplasma expresses a LCAT
ity of its membranes, and likely redundant homolog TgLCAT. Unlike other LCAT
routes of PE synthesis confer an ability for T. enzymes, TgLCAT is cleaved into two proteo-
gondii to adjust membrane biogenesis in lytic fragments that share the residues of the
response to dissimilar nutrient environments catalytic triad and need to be reassembled to
in host cells. This observed metabolic plasticity reconstitute enzymatic activity (Pszenny et al.,
allows T. gondii to fine-tune membrane biogen- 2016). TgLCAT uses PC as substrate to form
esis according to intracellular niche and may lysophosphatidylcholine that has the potential
have contributed to its evolution as a promis- to disrupt membranes. The released fatty acid is
cuous pathogen. transferred to cholesterol, but with a lower
The parasite produces PE in the mitochon- transesterification activity than mammalian
drion and in the PV by decarboxylation of PS, LCAT. TgLCAT localizes to the plasma mem-
and in the ER by the fusion of CDP- brane on extracellular parasites, contributing to
ethanolamine and DAG (Hartmann et al., Toxoplasma egress, likely by disrupting the host-
2014). PE in the mitochondrion is synthesized cell membrane during exit (Pszenny et al., 2016;
by the PS decarboxylase TgPSD1mt a type I Schultz and Carruthers, 2018). During the intra-
class PSD that harbors a targeting peptide cellular stage of the parasite, TgLCAT is
required for mitochondrial localization. secreted into the PV and distributes to the mem-
Ablation of TgPSD1mt expression leads to branous tubules forming the IVN. The role of
growth impairment in the parasite, however, intravacuolar TgLCAT would be to process host
the PE content remains unchanged, suggesting vesicles and organelles trapped in the PV
the presence of compensatory mechanisms. (Coppens et al., 2006; Romano et al., 2013) by
Toxoplasma secretes a soluble form of PS decar- disrupting their membranes, thus making their
boxylase, TgPSD1, from dense granules releas- nutrient content available to the parasite
ing PE in the PV lumen (Gupta et al., 2012). It (Romano et al., 2017). In support to this hypoth-
remains possible that secreted TgPSD1 could esis, less intact host organelles are detected
reduce externalized PS on host cells, enabling inside the PV of parasite-overexpressing
the evasion of phagocytosis. The parasite TgLCAT, probably as a result of host organelle
expresses TgPTS (see Section 8.2) that produces processing and degradation (Romano et al.,
PThr in the ER (Arroyo-Olarte et al., 2015). 2017), and these overexpressors are more
Genetic disruption of TgPTS abrogates de novo virulent than wild-type parasites (Pszenny
synthesis of PThr and results in a threefold et al., 2016).
Toxoplasma Gondii
In many organisms, patatin-like phospholi- small vesicles, while BODIPY-PA moves to the
pases are involved in numerous cellular func- parasite compartments similar to those contain-
tions, including lipid metabolism and ing fluorescent C4-BODIPY-C9, corresponding
membranes remodeling. T. gondii codes for six presumably to the Golgi/ER and the PV mem-
proteins predicted to bear a patatin-like brane. Interestingly, diversion of BODIPY-PA
domain. TgPL1 is secreted in the PV upon loaded in host cells prior to infection shows a
immune stresses but seems lacking lipase bright fluorescent labeling in the PV membrane.
activities (Mordue et al., 2007; Tobin and Knoll, By contrast, BODIPY-PC is completely excluded
2012). TgPL2 is on the apicoplast and is from parasites that have invaded prelabeled
involved in lipid metabolism. TgPL2 contri- hosts.
butes to the maintenance of this organelle as Intracellular and extracellular T. gondii are
TgPL2-depleted parasites have degenerated able to take up host-derived PA, but only the
apicoplast concomitant to decrease in the parasites inside host cells further metabolize
amounts of PC, PE, and PI consistent to scavenged PA into PC (Charron and Sibley,
reduced FAS from FASII (Lévêque et al., 2017). 2002). T. gondii acquires the phospholipid head
PC is a probable substrate for TgPL2, which group precursors from its environment and
could use it to generate lysophosphatidylcho- use them for the synthesis of major lipids
line and liberate fatty acids available for phos- (Gupta et al., 2005). Labeled serine internalized
pholipid synthesis in the apicoplast. by free parasites is metabolized into PS and PE
after PS decarboxylation, as well as in minor
sphingolipids. PE is the main polar lipid gener-
ated after the uptake of ethanolamine. Like ser-
8.3.4 Phospholipid salvage by
ine, the metabolism of ethanolamine shows a
Toxoplasma time-dependent increase in lipid synthesis that
Quantitative data on the rates of phospho- progressively slows over a 6 hours period. No
lipid syntheses reveal that T. gondii has an ade- significant radioactive PC is detected in
quate synthetic capacity to produce all of the Toxoplasma membranes after incubation in the
PE species, but only 50% of PS and B5% 10% presence of either tritiated serine or ethanol-
of PC as required for a parasite doubling. This amine, suggesting that the parasite has a negli-
indicates that T. gondii must be auxotrophic for gible PE methyltransferase activity (Gupta
PS and PC—or their precursors to acquire et al., 2005). However, another study con-
sufficient amounts of all phospholipids (Gupta ducted on parasites grown in host cells fed
et al., 2005; Charron and Sibley, 2002). with labeled serine or ethanolamine showed
Activities of phospholipid uptake by T. gondii that T. gondii synthesizes PC as a major resul-
have been exemplified by using fluorescent tant end product using these precursors
glycerophospholipid analogs 2-(4,4-difluoro- (Charron and Sibley, 2002). This discrepancy is
5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza- probably ascribed to differences in metabolic
s-indacene-3-pentanoyl)-1-hexadecanoyl-sn- requirements between parasites released from
glycero-3-phosphocholine (BODIPY-PC) and cells or growing inside cells. When host cells
2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza- are loaded with labeled serine or ethanolamine
s-indacene-3-pentanoyl)-1-hexadecanoyl-sn- prior to infection, no subsequent metaboliza-
glycero-3 phosphate (BODIPY-PA) that label par- tion of radioactive serine or ethanolamine is
asite compartments after internalization in host observed (Charron and Sibley, 2002). This may
cells (Charron and Sibley, 2002). BODIPY-PC is be linked to the rapid conversion of serine and
mobilized to plasma membrane and dispersed ethanolamine into PC by mammalian cells and
Toxoplasma Gondii
8.5 Acylglycerol synthesis and storage in Toxoplasma 381
the inability of the parasite to scavenge PC distinct roles: supporting the biosynthesis/deg-
intact from host cells, as corroborated in fluo- radation of phospholipids and regulating the
rescence studies (as stated previously). protein kinase C activity that controls cell
Exposure of parasites to labeled choline or growth. In animals, TAG are thus energy
methylcholine results in the uptake and meta- stores, repositories of fatty acids and precur-
bolization of these compounds into PC sors for phospholipid biosynthesis, and depots
(Charron and Sibley, 2002; Gupta et al., 2005). of signaling molecules (van Blitterswijk and
Choline preaccumulated into host cells before Houssa, 1999). By contrast, in bacteria and
infection is also further metabolized into PC by lower eukaryotes, TAG are solely synthesized
the parasites, in accordance with the compe- during times of stress or resource depletion,
tence of the intravacuolar T. gondii to readily and they are used primarily for phospholipid
take up choline. Various forms of radioactive synthesis. It is not surprising that higher organ-
choline-containing lipids are only observed in isms have developed several pathways for
intravacuolar parasites. This parallels the TAG synthesis and regulation, as compared to
observation showing that the metabolism of unicellular organisms. Commonly, the forma-
choline is increased by about twofold in host- tion of TAG is catalyzed by the activity of
free parasites incubated in an intracellular-type microsomal acyl-CoA:DAG acyltransferases
medium compared to parasites maintained in (DGAT).
an extracellular-type medium. This leads to the
assumption that choline metabolism and PC
synthesis are stimulated in response to para- 8.5 Acylglycerol synthesis and storage in
sitic invasion and replication within host cells. Toxoplasma
In T. gondii, TAG synthesis occurs via the
glycerol-3-phosphate pathway and involves
8.4 Acylglycerols
DGAT homolog TgDGAT1 (Quittnat et al.,
2004). Fatty acids can be incorporated into
8.4.1 Acylglycerol biosynthetic
Toxoplasma DAG, revealing that this latter lipid
pathways—generalities
is the acyl acceptor. TgDGAT1 contains signa-
Bacteria, yeast, plants, and animals all have ture motifs characteristic of the DGAT1 family.
the ability to synthesize acylglycerols, mainly TgDGAT1 is an integral membrane protein
TAG and DAGs, a critical function during peri- localized to the ER. When a Saccharomyces cerevi-
ods of nutritional excess and/or nutritional siae mutant strain lacking neutral lipid produc-
stress (Coleman and Lee, 2004). In higher tion is transformed with TgDGAT1, significant
eukaryotes, TAG are packaged in circulating DGAT activity is reconstituted, resulting in the
lipoproteins for distribution to peripheral tis- biogenesis of cytosolic lipid inclusions. In con-
sues where they can be used immediately or trast to human DGAT1 that lacks fatty acid
stored in cytosolic LD. Such energy-dense TAG specificity, TgDGAT1 preferentially incorpo-
stores can free organisms temporally and spa- rates palmitate into TAG. TAG are stored in
tially from the need for an immediate energy parasite cytosolic LD. Stored TAG may be a res-
supply and provide a reserve depot that can be ervoir of fatty acids utilizable for phospholipid
used when local resources fail or when specific biosynthesis and/or exploitable as respiratory
kinds of fatty acids or lipid precursors are substrates in Toxoplasma although no evidence
required. TAG stores can also be partially for fatty acid β-oxidation machinery exists in
hydrolyzed to form DAG, which performs two the parasite.
Toxoplasma Gondii
The apicoplast lacks typical plastid- Upon supplementation of 0.2 and 0.4 mM
signature galactolipids as evidenced by mass oleate in the culture medium, Toxoplasma acti-
spectrometry-based lipidomic analysis, but vates TgDGAT transcription by 1.5-fold, result-
instead the parasite contains galactosyl- and ing in increased acylgycerol synthesis to control
digalactosyl-ceramides (Marechal et al., 2002; the influx of oleate in the parasite (Nolan et al.,
Welti et al., 2007, Botté et al., 2008). The 2017). However, such an increase in TgDGAT
absence of galactolipids is likely explained by transcripts seems insufficient, as lipids also
the evolution of apicomplexa from autotrophic accumulate outside LD (Nolan et al., 2018). At
photosynthetic organisms to obligate intracel- 0.5 mM oleate, Toxoplasma is puffed with lipids
lular parasites, which led to the loss of and unable to response adequality to excess ole-
galactosyltransferase and eventually to the ate by activating DGAT and stops replicating.
repurposing of apicoplast acyltransferases TAG synthesis is essential for buffering
(Botté et al., 2008; Amiar et al., 2016) excess fatty acids taken up by Toxoplasma and
Toxoplasma infection increases host TAG preventing lipotoxicity in Toxoplasma. TgDGAT
production in infected cells (Hu et al., 2017) shares 31% identity and 49% similarity with
and the number of host LD at the beginning of human DGAT1 (HsDGAT1) (Quittnat et al.,
infection of fibroblasts (Nolan et al., 2017) and 2004). T863 is a selective inhibitor of HsDGAT1,
up to 2 days in skeletal muscle cells (Gomes blocking the acyl-CoA binding site of the
et al., 2014) and macrophages (Mota et al., enzyme, resulting in a blockade of DGAT1-
2014). Increase in host LD would benefit mediated TAG formation (Cao et al., 2011).
Toxoplasma for the copious lipid content of Treatment of Toxoplasma tachyzoites with T863
these organelles. The parasite is able to salvage at subtoxic concentrations for mammalian cells
lipids stored in host LD (Nolan et al., 2017). results in rapid parasite growth arrest (Fig. 8.3)
The LD are also sites of storage and synthesis and impaired TAG storage (Nolan et al., 2018).
of cytokines. Stimulation of host LD biogenesis T863-treated tachyzoites show abnormal ER-
may increase the levels of prostaglandin E2, derived membranous structures that are stock-
contributing to the control of the synthesis of piled in the cytoplasm, likely impeding normal
IL-12 and IFN-γ during infection and decrease endodyogeny. It seems that, in the face of
in nitric oxide (NO) synthesis (Mota et al., TgDGAT inhibition, DAG and fatty acids may
2014; Gomes et al., 2014). It has been proposed be diverted by the parasite to the synthesis of
that increase of IL-12 and IFN-γ, which are PC and PE through the Kennedy pathway. Dual
involved in the repair and homeostasis of mus- addition of OA (0.5 mM) and T863 is synergi-
cle cells after injury, might contribute to the cally detrimental for the parasite. Toxoplasma
maintenance of the chronic phase of T. gondii bradyzoites also contain LD for TAG and cho-
infection in skeleton muscle tissues. Parasite- lesteryl ester storage (Nolan et al., 2018). T863
induced LD accumulation seems linked to the treatment leads to misshapen cysts and reduced
inhibition of host mTOR and JNK signaling LD in bradyzoites (Fig. 8.3), although sensitivity
pathways throughout the secretion of a para- occurs at concentrations higher than those
site component in the host cell (Hu et al., 2017). observed for tachyzoites, perhaps due to the
This process requires the involvement of the lower growth rate and more quiescent metabo-
parasite effector MYR1 that mediates the pas- lism of bradyzoites. Inhibiting TgDGAT1 enzy-
sage of protein throughout the PV membrane. matic activity to interfere with energy storage in
Indeed, no change in neutral lipid storage has Toxoplasma may be a reasonable therapeutic
been observed in fibroblats infected with intervention against toxoplasmosis, including
Myr1-lacking parasites. the control of the chronic stage of infection.
Toxoplasma Gondii
8.6 Sterols and steryl esters 383
the ER (Goldstein and Brown, 1990). The
cholesterol molecule is formed from acetate
units. The acetate units are joined in a series of
reactions to form farnesyl pyrophosphate, a
branch point for the biosynthesis of other iso-
prenoid compounds such as ubiquinone, doli-
chol, and farnesylated proteins. Hydroxy-3-
methylglutaryl-CoA reductase from the meva-
lonate pathway is the rate determining enzyme
for the entire pathway from acetate to
cholesterol.
Deposition of excess cellular cholesterol in
the form of cholesteryl esters is catalyzed by
acyl-CoA:cholesterol acyltransferases (ACAT)
and ER resident enzymes. Native and exoge-
nous cholesterol has several possible fates:
incorporation into membranes, efflux to extra-
cellular acceptors, conversion into cholesteryl
esters, or depending on the cell type, metabo-
lism into bile acids, or steroid hormones. Rates
of cholesterol biosynthesis, LDL internaliza-
tion, and cholesterol esterification are exqui-
sitely sensitive to cellular levels of free
FIGURE 8.3 The treatment of tachyzoites and brady- cholesterol. Three possible mechanisms of cho-
zoites of Toxoplasma with T863. Immunofluorescence
images of tachyzoites (RFP-parasites) and bradyzoites lesterol movement include aqueous diffusion,
(TRITC-lectin for cyst wall) showing misshapen parasites vesicle-mediated transport, and soluble car-
upon incubation with T863. riers, which may work together or separately
to mobilize cholesterol within the cell (Liscum
8.6 Sterols and steryl esters and Underwood, 1995). Evidence has accrued
that biological membranes are made of a
8.6.1 Sterol lipid biosynthetic mosaic of lipids domains. Maintenance of
domain structure is critical for cell function.
pathways—generalities
Cholesterol plays a key role in organizing sig-
Cholesterol is the major sterol molecule naling lipids and proteins within these mem-
ubiquitously present in mammalian cells. This brane domains (Anderson and Jacobson, 2002).
lipid has been selected in the long natural evo-
lution process for its ability to maintain a deli-
cate balance between membrane rigidity (e.g.,
8.6.2 Sterol salvage and transport in
to allow large cell volumes) and membrane flu-
idity (e.g., to allow membrane-embedded pro-
Toxoplasma
teins to function properly; Bretscher and Toxoplasma membranes contain ß-hydroxy-
Munro, 1993). Mammalian cells obtain choles- sterols, as probed using the polyene antibiotic
terol both by internalization of plasma low filipin routinely used to reveal the steady-state
density lipoprotein (LDL) particles or by de distribution of sterols by fluorescence micros-
novo synthesis via the mevalonate pathway in copy (Coppens et al., 2000). A predominantly
Toxoplasma Gondii
parasite staining is located to the plasma lipoproteins stimulates parasite replication

membrane and the rhoptries, apical secretory (Coppens et al., 2000; Nishikawa et al., 2005).
organelles whose content is discharged upon LDL-derived cholesterol levels also play a cru-
parasite invasion (Coppens and Joiner, 2003). cial role in bradyzoite conversion as LDL star-
T. gondii diverts LDL-derived cholesterol vation induces expression of bradyzoite
that has transited through host lysosomes proteins (Ihara and Nishikawa, 2014). A study
(Coppens et al., 2000) as demonstrated after based on gene data mining shows that acute
incubation of infected cells with labeled cho- infection with T. gondii is associated with a
lesterol incorporated into LDL. Cholesterol decrease in cholesterol content in the liver and
movement from lysosomes to the PV requires brain, as a part of the host defense response to
temperatures permissive for vesicular trans- deprive the parasite of cholesterol necessary
port, metabolic energy, and host microtu- for tachyzoite proliferation and development
bules, but no fusion (Sehgal et al., 2005) (Milovanović et al., 2017).
Toxoplasma sequesters cholesterol-filled host Other studies reveal a strong link between
lysosomes within invaginations of the PV toxoplasmosis and several lipid disorders,
membrane (Coppens et al., 2006). Cholesterol including hypercholesterolemia and obesity in
delivery into the PV requires functional host humans (Portugal et al., 2009; Reeves et al.,
Niemann-Pick type C proteins (Sehgal et al., 2013), a correlation supported by data in mice
2005) that mediate cholesterol egress across (Portugal et al., 2004). Increased blood lipopro-
the endo-lysosomal membranes (Sleat et al., tein/cholesterol deposition in the arterial wall
2004). In addition, a host cell P-glycoprotein in atherosclerotic patients is a consequence
transporter, a membrane-bound efflux pump, of a Th1-mediated pro-inflammatory reaction.
is required for cholesterol transport to the PV Toxoplasma induces a dysregulation in host
(Bottova et al., 2009). Cholesterol incorpo- lipid metabolism throughout its scavenging
ration into the parasite is abolished after pro- activities. In the brain a reduction in cholesterol
tease treatment on parasite plasma membrane levels is observed during acute infection, per-
(Sehgal et al., 2005). A lipid-translocating haps as part of the host defensive reaction to
importer of the ATP-binding cassette (ABC) the parasite, potentially serving to decrease
transporter G subfamily (ABCG family) parasite replication through cholesterol depri-
located to the PV and parasite plasma mem- vation. On the other hand, T. gondii infection
brane delivers cholesterol to the parasite inte- elicits a systemic Th1 inflammatory response,
rior (Ehrenman et al., 2010). A D-bifunctional possibly imposing a proatherogenic environ-
protein containing two sterol-carrier protein-2 ment in the host. As a result, the association
domains promotes the circulation of choles- between atherosclerosis and toxoplasmosis in
terol within the parasite, in addition to phos- one individual could change the outcome of
pholipids and fatty acids (Lige et al., 2009). each disease, depending on the availability of
Other parasite ABCG transporters are involved intracellular cholesterol and the intensity of the
in cholesterol and phospholipid movement inflammatory reaction triggered by the para-
from the parasite to the PV and may contribute site. Studying host cholesterol manipulation by
to the expansion of PV membrane size the Toxoplasma cysts could then be relevant for
(Ehrenman et al., 2010). a clinical point of view.
The availability of host cholesterol has a Delivery of sterol analogs incorporated into
direct impact on Toxoplasma development LDL could be an efficient strategy to substi-
inside its PV. LDL deprivation impairs parasite tute the indispensable cholesterol by
growth, whereas the overabundance of these structural-related compound with growth-
Toxoplasma Gondii
8.6 Sterols and steryl esters 385
reducing activity. The sterol analogs 22,26- The loss of individual ACAT can be tolerated
azasterol and 24,25-(R,S)-epiminolanosterol, by the parasite though slower growth com-
inhibitors of sterol-24-methyl transferase pro- pared to wild-type parasites has been
ducing 24-alkyl sterols, have potent and selec- observed, due to increase in toxic-free choles-
tive antiproliferative activity against T. gondii terol within membrane. This suggests that the
(Dantas-Leite et al., 2004; Martins-Duarte parasites can endure the resulting slight differ-
et al., 2009). The molecular mechanism of ences in their cholesteryl ester pools and that
these lipids is unclear since 24-alkyl sterols the ACAT enzymes partially complement each
are not detected in this parasite. It is observed, other. Loss of both ACAT results in synthetic
however, that the rapid accumulation of these lethality, indicating that cholesterol storage is
lipid analogs in diverse membranes alters an important function for T. gondii. Another
maintenance, fusogenicity, and function of the source of cholesteryl esters for the parasite is
parasite organelles. It is reported that selected provided by TgLCAT using the fatty acid
sterol analogs (e.g., cholesteryl chloride, cho- chains liberated from phospholipids to esterify
lestanone, or thiocholesterol) can affect the cholesterol (Pszenny et al., 2016). In addition,
growth of various cholesterol-auxotroph Toxoplasma is able to retrieve neutral lipids
organisms (Clayton, 1964). Their antiprolifera- stored in LD and sequester host LD in the PV
tive properties should promisingly be extended lumen, suggesting that it may have access to
to Toxoplasma. host cholesteryl esters, as a source of choles-
terol (Nolan et al., 2017).
Host LDL and fatty acids scavenged by
Toxoplasma serve as ACAT activators by stimu-
8.6.3 Sterol storage in Toxoplasma lating cholesteryl ester synthesis and LD bio-
Nile red that strongly fluoresces in the pres- genesis in the parasite. Upon supplementation
ence of steryl esters detects the presence of in the medium with 0.2 and 0.4 mM oleate,
cytosolic LD in Toxoplasma (Sonda et al., 2001; Toxoplasma activates TgACAT2 transcription
Charron and Sibley, 2002; Quittnat et al., 2004), by B1.3-fold, resulting in increased cholesteryl
indicating the ability of the parasites for choles- ester synthesis to control the influx of oleate in
terol storage. Of the 21 molecular species the parasite (Nolan et al., 2017). Lipoprotein
detected in Toxoplasma, cholesteryl oleate (42%) depletion causes a progressive consumption of
and palmitate (26%) are the main esters as for material stored in parasite’s LD. Under the
mammalian cells but the parasite also has conditions of excess LDL the activity of choles-
uniquely large amounts of cholesteryl eico- terol esterification is significantly increased,
sanoate (7%). In addition, the parasite contains entailing that the parasites adeptly control the
to a lesser extent, cholesteryl palmitoleate, stea- massive supply of cholesterol by producing the
rate, linoleate, arachidonate, and some polyun- storage form of cholesterol. A Niemann-Pick,
saturated fatty acids. Other cholesteryl ester type C1-related protein in Toxoplasma controls
fatty acid species represented 3.5% of the total the intracellular levels of several lipids (Lige
species (Lige et al., 2011). Toxoplasma is compe- et al., 2009). Parasites lacking the Niemann-
tent to synthesize cholesteryl esters by two Pick I-related protein accumulate LD enriched
ACAT-related enzymes located in the ER, in cholesteryl esters.
TgACAT1, and TgACAT2 (Nishikawa et al., The replication rate of intracellular T. gondii
2005; Lige et al., 2013). Parasite ACATs present correlates with the LDL concentration in the
a broad sterol substrate affinity but preferen- medium. Excess cholesterol diverted by the
tially use palmitate to form cholesteryl esters. parasite is rapidly neutralized and stored in
Toxoplasma Gondii
LD. Blockade of cholesteryl ester synthesis is 8.7.2 Sphingolipid synthesis in

deleterious for the parasite, leading to a rapid Toxoplasma
induction of free cholesterol crystallization in
parasite membranes and rupture of the plasma Our lipidomic analysis of T. gondii revealed
membrane (Nishikawa et al., 2005). ACAT- that the parasite contains .20 species of sphin-
deficient parasites are particularly sensitive to golipids consisting of both saturated and unsat-
ACAT inhibitors (Lige et al., 2013). The higher urated fatty acids (Lige et al., 2011). The parasite
vulnerability of T. gondii toward ACAT inhibi- is capable of the de novo synthesis of sphingoli-
tors compared with mammalian cells is proba- pids, based on inhibitor studies (Sonda et al.,
bly linked to the absence of cholesterol 2005), as well as on metabolic labeling studies
acceptors (mainly lipoproteins) in the PV, of extracellular or released intracellular para-
which are known to desorb excess cholesterol sites (Azzouz et al., 2002; Bisanz et al., 2006).
from membranes. Toxoplasma can readily incorporate sugars and
amino acids as precursors of sphingolipids. De
novo synthesis of ceramide, glycosylated cer-
amide, and sphingomyelin in T. gondii has been
8.7 Sphingolipids demonstrated by metabolic labeling studies
using tritiated serine and galactose. After inter-
8.7.1 Sphingolipid biosynthetic nalization, labeled galactose is metabolized in
pathways—generalities various glycosphingolipids (e.g., di- and trigly-
cosylated ceramide; Azzouz et al., 2002).
Glycosylphosphatidylinositols (GPI) are a Incubation of the parasite with labeled serine
class of glycolipids that are used by a wide leads to the production of ceramide. After
variety of eukaryotic cells to anchor proteins, uptake, labeled glucosamine serves as a GPI
polysaccharides, and small oligosaccharides to glycolipid precursor and is associated with the
the plasma membrane through covalent lin- dominant surface protein, SAG1 (Striepen et al.,
kages (Ferguson and Williams, 1988). 1997; Zinecker et al., 2001).
Comparison of the chemical structures of GPI Toxoplasma has a sphingolipid synthase
membrane anchors of different organisms indi- homolog, named TgSLS, and recombinant
cates that the anchors contain a remarkably TgSLS exhibits an inositol phosphorylceramide
conserved core glycan structure, suggesting synthase activity in vitro (Pratt et al., 2013).
that a common biosynthetic pathway may have The parasite also expresses on the ER a serine
been conserved throughout eukaryotic evolu- palmitoyltransferase, named TgSPT1, that cata-
tion. The transfer of GPI anchors to proteins lyzes the first and rate-limiting step in sphingo-
occurs in the ER with concurrent displacement lipid biosynthesis: the condensation of serine
of a C-terminal hydrophobic peptide, followed and palmitoyl-CoA (Mina et al., 2017). Unlike
by the rapid substitution of the peptide tail by in eukaryotes that express a serine palmitoyl-
the GPI anchor. Ceramides are the principal transferase forming a conserved heterodimeric
lipid components present in sphingomyelin, enzyme complex, TgSPT1 is homodimeric
complex glycolipids, cerebrosides, and ganglio- and evolutionarily related to the prokaryotic
sides (Sharma and Shi, 1999). Ceramides are serine palmitoyltransferase, identified in the
broadly recognized as vital second messengers Sphingomonadaceae as a soluble homodimeric
in the signal transduction process mediated enzyme. TgSPT1 could have been acquired by
by receptors of many cytokines and growth Toxoplasma via lateral gene transfer from a
factors. prokaryote.
Toxoplasma Gondii
8.7 Sphingolipids 387
GPI in T. gondii serve as membrane anchors compounds specifically interfere with GPI
for a large number of plasma membrane pro- biosynthesis in many different pathogenic
teins (Tomavo et al., 1992; Striepen et al., 1997; organisms (de Macedo et al., 2003). Synthesis
Zinecker et al., 2001). Their biosynthetic path- of parasite ceramide is dramatically decreased
way is initiated on the parasite ER with the after incubation of intracellular Toxoplasma
transfer of N-acetylglucosamine to PI involving with either threo-phenyl-2-palmitoylamino-3-
a PI-glycan class A (PIGA)-like protein morpholino-1-propanol, a specific inhibitor of
(Wichroski and Ward, 2003). The GPI core gly- glucosylceramide synthesis, or L-cycloserine
can is then assembled via sequential glycosyla- that blocks the serine palmitoyltransferase
tion of PI (Tomavo et al., 1992). Toxoplasma activity (Azzouz et al., 2002). The antibiotic
PIGA sequence contains a potential transmem- aureobasidin A, a potent inhibitor of inositol
brane domain followed by a stretch of mostly phosphorylceramide that is absent from mam-
hydrophilic residues extending to the C- malian cells, abrogates T. gondii replication by
terminus. A functional copy of PIGA is the severe reduction of total complex sphingo-
required for viability, demonstrating that GPI lipids’ synthesis without noticeable host-cell
biosynthesis is an essential process in T. gondii. alterations (Sonda et al., 2005).
Glycosphingolipids, for example, inositol
phosphorylceramide are synthesized de novo
via the 3-ketosphinganine pathway from serine
8.7.3 Sphingolipid salvage by
and palmitoyl-CoA (Azzouz et al., 2002; Sonda
et al., 2005) with ceramide as an intermediate.
Toxoplasma
Metabolic studies show that T. gondii readily Intracellular T. gondii is also able to retrieve
incorporates radioactive acetate into glycosyl- sphingolipids intact from the culture and accu-
cerebroside, lactosylcerebroside, and globo- mulate the scavenged lipids into the Golgi
triaosylceramide, while only intracellular apparatus. In mammalian cells, exogenous cer-
parasites produce globoside (Bisanz et al., amides concentrate the Golgi complex to be
2006). further metabolized into major sphingolipids
GPI-anchored proteins dominate the surface and glucosylceramides. After incubation with
of T. gondii and are implicated in both host-cell NBD-C6-ceramide the Golgi of intravacuolar
attachment and modulation of the host T. gondii is stained, suggesting that the parasite
immune response (Lekutis et al., 2001). can intercept the ceramide pathway of the host
Although the GPI core glycan is conserved in cell to acquire exogenous ceramides or other
all organisms, some differences in additional sphingolipids manufactured in the host Golgi
modifications to GPI structures and biosyn- (de Melo and de Souza, 1996). A morphological
thetic pathways have been reported for T. gon- study shows that the PV of Toxoplasma prefer-
dii (de Macedo et al., 2003). This indicates that entially localizes near the host Golgi early dur-
the GPI biosynthetic pathway is a potential tar- ing infection and remains closely associated
get for the development of new chemothera- with this organelle throughout infection
peutics against this parasite. Indeed the lethal (Romano et al., 2013). The parasite subverts the
consequences of PIGA disruption in T. gondii structure of the host Golgi, resulting in its frag-
may result from a deficiency in GPI-anchored mentation into numerous ministacks, which
proteins, free GPI, or both (Wichroski and surround the PV, and hijacks host
Ward, 2003). In vitro and in vivo studies reveal Golgi derived vesicles within the PV. The
that sugars and amino acid analogs, synthetic vesicles, marked with Rab14, Rab30, or Rab43
mannoside acceptor substrates and natural colocalize with host-derived sphingolipids in
Toxoplasma Gondii
the vacuolar space. Scavenged sphingolipids suggesting that host organelles in the PV are
contribute to parasite replication since alterations processed to liberate and make available to the
in host sphingolipid metabolism are detrimental parasite their content.
for the parasite’s growth.
Host Rab vesicles enter the PV via invagina-
tions of the vacuolar membrane extending into 8.8 Isoprenoid derivatives
the PV lumen (Fig. 8.4). Some invaginations are
formed following the fusion of an IVN tubule 8.8.1 Isoprenoid biosynthetic
with the vacuolar membrane or are generated pathways—generalities
by host microtubules poking the PV membrane
(Coppens et al., 2006; Romano et al., 2017). The posttranslational modification of proteins
Host vesicles use either type of invagination of by isoprenoid residues such as farnesyl and
the PV membrane as conduits, resulting in geranylgeranyl, is a major mechanism by which
their sequestration into the vacuole, based on cytosolic proteins interact with cellular mem-
EM observations. TgLCAT secreted by dense branes (Swiezewskaa and Danikiewiczb 2005).
granules (Pszenny et al., 2016), localizes to the Isoprenylation is also required for the proper
IVN and seems involved in the degradation of membrane localization and the biological activ-
host intravacuolar vesicles (Romano et al., ity of several cellular proteins implicated in the
2017). Parasites overexpressing TgLCAT con- regulation of DNA replication and cell cycling,
tain fewer host Rab vesicles in the PV, therefore having important roles in the regula-
tion of cell proliferation. Two coexisting isopren-
oid pathways exist in organisms, the mevalonate
pathway present in the cytosol of mammalian
cells (as stated previously) and the recently
described 1-deoxy-D-xylulose-5-phosphate path-
way. This latter pathway seems to be restricted
so far to bacteria, plastids in plants and apico-
plast in Apicomplexa (Jomaa et al., 1999).
8.8.2 Isoprenoid synthesis in Toxoplasma

T. gondii membranes contains both farnesy-
lated and geranylgeranylated proteins (Ibrahim
et al., 2001; Ling et al., 2005). The parasite is
able to make its own isoprenoids (Fig. 8.5).
Enzymes of the 1-deoxy-D-xylulose-5-phosphate
FIGURE 8.4 Proposed model of host Rab vesicle trap- pathway seem to have an apicoplast origin and
ping inside the PV for sphingolipid acquisition. (1)
Transport of sphingolipid-loaded Rab14, Rab30, or Rab43 may contribute to the production of farnesyl
vesicles to the PV using host microtubules (green); (2) and geranylgeranyl molecules for the parasite
docking via parasite proteins; (3) internalization via tubular (Seeber, 2003). Two farnesyl-diphosphate
invaginations; (4) detachment from the PV membrane and synthase homologs, named TgFPPS, have been
encircling by IVN tubules; (5) disruption of Rab vesicle
identified in Toxoplasma and localized to the
membranes by TgLCAT and liberation of sphingolipids in
the PV lumen; and (6) uptake of sphingolipids by the para- mitochondria; this enzyme is bifunctional,
site. IVN, Intravacuolar network; PV, parasitophorous catalyzing the formation of both farnesyl
vacuole. diphosphate and geranylgeranyl diphosphate
Toxoplasma Gondii
8.8 Isoprenoid derivatives 389
FIGURE 8.5 Isoprenoid synthesis and salvage by Toxoplasma gondii. The DOXP pathway localizes to the parasite
apicoplast (green), and the mevalonate pathway is only present in the mammalian host. Green arrows show metabolites
imported from the host cell. TgFPPS synthesizes FPP and GGPP while the host uses two enzymes (FPPS and GGPPS) to
make the same metabolites. Enzymes and their known inhibitors are indicated. Alkyl-BP, alkyl bisphosphonates; DMAPP,
dimethyl allyl diphosphate; Fos, fosmidomycin; FPP, farnesyl diphosphate; G3P, glyceraldehyde 3-phosphate; GGPP, gera-
nylgeranyl diphosphate; GPP, geranyl diphosphate; IPP, isopentenyl diphosphate; N-BP, nitrogen bisphosphonates.
Source: From Li, Z.H., Ramakrishnan, S., Striepen, B., Moreno, S.N.J., 2013. Toxoplasma gondii relies on both host and parasite iso-
prenoids and can be rendered sensitive to atorvastatin. PLoS Pathog. 9, e1003665.
(Ling et al., 2007). Intracellular TgFPPS-deficient for isoprenylation. Peptidomimetics act as

parasites survive without endogenous pro- alternative substrates in vitro, thereby competi-
duction of farnesyl diphosphate and/or gera- tively block protein farnesylation. Clearly,
nylgeranyl diphosphate because they are able specific inhibition of T. gondii protein farnesyl-
to scavenge these lipids from the host cells transferase activity is observed using selected
(Li et al., 2017). modified heptapeptides (Ibrahim et al., 2001).
Prenylated proteins are ubiquitously impor- Bisphosphonates, diphosphate analogs in
tant for cell proliferation regulation. Two cate- which a carbon atom replaces the oxygen atom
gories of protein farnesyltransferase inhibitors bridge between the two phosphorus atoms of
have been described so far (Qian et al., 1997): the diphosphate, are potent inhibitors of
isoprene analogues and peptidomimetics based farnesyl-diphosphate synthase and inhibit the
on the consensus CAAX motif that is required growth of Toxoplasma (Martin et al., 2001) by
Toxoplasma Gondii
inhibiting the parasite farnesyl diphosphate/ genetic capacity to express unique and redun-
geranylgeranyl-diphosphate synthase. Moreover, dant lipid biosynthetic pathways, and it has
in vivo testing of bisphosphonates against developed efficient mechanisms to scavenge
T. gondii in mice has shown that one of the several host lipids or lipidic precursors (Sonda
nitrogen-containing bisphosphonates, risedro- and Hehl, 2006; Goodman and McFadden, 2007;
nate, significantly increases the survival of Zhang et al., 2010; Ramakrishnan et al., 2013;
mice infected by T. gondii (Yardley et al., Coppens, 2013,2014; Amiar et al., 2016). The
2002). All these results indicate that bispho- parasite shows amazingly diverse features in
sphonates are promising candidate drugs to lipid metabolic pathways, with some of sharing
treat infections caused by T. gondii as well as close similarities to mammalian pathways,
some other protozoan parasites. whereas others are more evolutionary related to
bacteria and plant pathways. From a cell biolog-
ical viewpoint, no doubt exists that the lipid
8.8.3 Isoprenoid salvage by Toxoplasma metabolism and great plasticity of T. gondii will
likely reveal many more metabolic surprises in
Alternatively, the parasite can salvage isopre-
the future. One of the most striking feature of
noids such as labeled trans, trans-farnesol and
the parasite lipid synthetic pathways is the
labeled trans,trans,cis geranylgeraniol, from the
parasite’s ability to mix fatty acids that are scav-
medium to produce its prenylated proteins
enged and de novo synthesized to generate
(Ibrahim et al., 2001) and farnesyl diphosphate
most major phospholipid classes, forming mem-
and geranylgeranyl diphosphate from the host
brane made of “patchwork lipids.” T. gondii
cell (Li et al., 2013) (Fig. 8.5). This implies the
uses the two sources of fatty acids concomi-
presence of functional protein farnesyltransfer-
tantly resulting in complex trafficking and lipid
ase and geranylgeranyl transferase in T. gondii.
remodeling pathways. Characterization of more
Indeed, a protein farnesyltransferase activity
lipid-based target molecules and knowledge
has been detected in the parasite, responsible
about mechanisms promoting host lipid deliv-
for the catalysis of isoprene lipid modifications.
ery to the PV, lipid processing and trafficking
The survival of parasites lacking TgFPPS
pathways hold considerable potential for toxo-
depends on isoprenoids salvaged from the host
plasmosis chemotherapy. To this end, rationally
cells. Inhibition of the host mevalonate pathway
designed lipid synthesis/uptake inhibitors
with statin enhances the requirement for para-
would represent exciting prospects for the next
site isoprenoid synthesis (Li et al., 2013). Dual
generation of anti-Toxoplasma agents.
blockade of the parasite isoprenoid pathway
with fosmidomycin/bisphosphonates and the
host mevalonate pathway with atorvastatin Acknowledgments
results in parasite lethality in vitro and mice
The authors are grateful to the members of their laborato-
survival, emphasizing this double-hit strategy ries for helpful discussions on lipid metabolism in
combining inhibitors as a promising therapeutic Toxoplasma, in particular, Sheena Dassa for critical reading
approach (Li et al., 2017). of the manuscript and thoughtful comments.
References
8.9 Concluding remarks
Alonso, A.M., Coceres, V.M., De Napoli, M.G., Nieto, Guil,
A.F., Angel, S.O., et al., 2012. Protein palmitoylation
In this Section covering lipid metabolism, we inhibition by 2-bromopalmitate alters gliding, host cell
illustrate that T. gondii has sophisticated lipid invasion and parasite morphology in Toxoplasma gondii.
homeostatic pathways. It has retained the Mol. Biochem. Parasitol. 184, 39 43.
Toxoplasma Gondii
References 391
Amiar, S., MacRae, J.I., Callahan, D.L., Dubois, D., van Bullen, H.E., Jia, Y., Yamaryo-Botté, Y., Bisio, H., Zhang,
Dooren, G.G., Shears, M.J., et al., 2016. Apicoplast- O., Jemelin, N.K., et al., 2016. Phosphatidic acid-
localized lysophosphatidic acid precursor assembly is mediated signaling regulates microneme secretion in
required for bulk phospholipid synthesis in Toxoplasma toxoplasma. Cell Host Microbe 19, 349 360.
gondii and relies on an algal/plant-like glycerol 3- Cao, J., Zhou, Y., Peng, H., Huang, X., Stahler, S., Suri, V.,
phosphate acyltransferase. PLoS Pathog. 12, e1005765. et al., 2011. Targeting acyl-CoA:diacylglycerol acyltrans-
Anderson, R.G., Jacobson, K., 2002. A role for lipid shells in ferase 1 (DGAT1) with small molecule inhibitors for the
targeting proteins to caveolae, rafts, and other lipid treatment of metabolic diseases. J. Biol. Chem. 286,
domains. Science 296, 1821 1825. 41838 41851.
Arroyo-Olarte, R.D., Brouwers, J.F., Kuchipudi, A., Helms, Carman, G.M., Zeimetz, G.M., 1996. Regulation of phos-
J.B., Biswas, A., Dunay, I.R., et al., 2015. pholipid biosynthesis in the yeast Saccharomyces cerevi-
Phosphatidylthreonine and lipid-mediated control of siae. J. Biol. Chem. 271, 13293 13296.
parasite virulence. PLoS Biol. 13, e1002288. Charron, A.J., Sibley, L.D., 2002. Host cells: mobilizable
Azzouz, N., Rauscher, B., Gerold, P., Cesbron-Delauw, M. lipid resources for the intracellular parasite Toxoplasma
F., Dubremetz, J.F., Schwarz, R.T., 2002. Evidence for de gondii. J. Cell Sci. 115, 3049 3059.
novo sphingolipid biosynthesis in Toxoplasma gondii. Chen, A.L., Moon, A.S., Bell, H.N., Huang, A.S., Vashisht,
Int. J. Parasitol. 32, 677 6784. A.A., Toh, J.Y., et al., 2017. Novel insights into the com-
Baldock, C., Rafferty, J.B., Sedelnikova, S.E., Baker, P.J., position and function of the Toxoplasma IMC sutures.
Stuitje, A.R., Slabas, A.R., et al., 1996. A mechanism of Cell. Microbiol. 19. Available from: https://doi.org/
drug action revealed by structural studies of enoyl 10.1111.
reductase. Science 274, 2107 2110. Clayton, R.B., 1964. The utilization of sterols by insects. J.
Beck, J.R., Fung, C., Straub, K.W., Coppens, I., Vashisht, A. Lipid Res. 15, 3 19.
A., Wohlschlegel, J.A., et al., 2013. A Toxoplasma palmi- Coleman, R.A., Lee, D.P., 2004. Enzymes of triacylglycerol
toyl acyl transferase and the palmitoylated armadillo synthesis and their regulation. Prog. Lipid Res. 43,
repeat protein TgARO govern apical rhoptry tethering 134 176.
and reveal a critical role for the rhoptries in host cell Coppens, I., 2013. Targeting lipid biosynthesis and salvage
invasion but not egress. PLoS Pathog. 9, e1003162. in apicomplexan parasites for improved chemothera-
Bisanz, C., Bastien, O., Grando, D., Jouhet, J., Marechal, E., pies. Nat. Rev. Microbiol. 11, 823 835.
Cesbron-Delauw, M.F., 2006. Toxoplasma gondii acyl- Coppens, I., 2014. Exploitation of auxotrophies and meta-
lipid metabolism: de novo synthesis from apicoplast bolic defects in Toxoplasma as therapeutic approaches.
generated fatty acids versus scavenging of host cell pre- Int. J. Parasitol. 44, 109 120.
cursors. Biochem. J. 394, 197 205. Coppens, I., Joiner, K.A., 2003. Host but not parasite choles-
Bisio, H., Lunghi, M., Brochet, M., Soldati-Favre, D., 2019. terol controls Toxoplasma cell entry by modulating
Phosphatidic acid governs natural egress in Toxoplasma organelle discharge. Mol. Biol. Cell 14, 3804 3820.
gondii via a guanylate cyclase receptor platform. Nat. Coppens, I., Sinai, A.P., Joiner, K.A., 2000. Toxoplasma gondii
Microbiol. 4, 420 428. exploits host low-density lipoprotein receptor-mediated
van Blitterswijk, W.J., Houssa, B., 1999. Diacylglycerol endocytosis for cholesterol acquisition. J. Cell Biol. 149,
kinases in signal transduction. Chem. Phys. Lipids 98, 167 180.
95 108. Coppens, I., Dunn, J.D., Romano, J.D., Pypaert, M., Zhang,
Botté, C., Saı̈dani, N., Mondragon, R., Mondragón, M., H., Boothroyd, J.C., et al., 2006. Toxoplasma gondii
Isaac, G., Mui, E., et al., 2008. Subcellular localization sequesters lysosomes from mammalian hosts in the vac-
and dynamics of a digalactolipid-like epitope in uolar space. Cell 125, 261 274.
Toxoplasma gondii. J. Lipid Res. 49, 746 762. Dantas-Leite, L., Urbina, J.A., de Souza, W., Vommaro, R.
Botté, C.Y., Dubar, F., McFadden, G.I., Maréchal, E., Biot, C., 2004. Selective anti-Toxoplasma gondii activities of
C., 2012. Plasmodium falciparum apicoplast drugs: targets azasterols. Int. J. Antimicrob. Agents 23, 620 626.
or off-targets? Chem. Rev. 112, 1269 1283. Dubois, D., Fernandes, S., Amiar, S., Dass, S., Katris, N.J.,
Bottova, I., Hehl, A.B., Stefanić, S., Fabriàs, G., Casas, J., Botté, C.Y., et al., 2018. Toxoplasma gondii acetyl-CoA
Schraner, E., et al., 2009. Host cell P-glycoprotein is synthetase is involved in fatty acid elongation (of long
essential for cholesterol uptake and replication of fatty acid chains) during tachyzoite life stages. J. Lipid
Toxoplasma gondii. J. Biol. Chem. 284, 17438 17448. Res. 59, 994 1004.
Bretscher, M.S., Munro, S., 1993. Cholesterol and the Golgi Ehrenman, K., Sehgal, A., Lige, B., Stedman, T.T., Joiner, K.
apparatus. Science 261, 1280 1281. A., Coppens, I., 2010. Novel roles for ATP-binding
Toxoplasma Gondii
cassette G transporters in lipid redistribution in the parasite mitochondrion is required for efficient
Toxoplasma. Mol. Microbiol. 76, 1232 1249. growth but dispensable for survival of Toxoplasma gon-
Fadok, V.A., Bratton, D.L., Henson, P.M., 2001. Phagocyte dii. J. Biol. Chem. 289, 6809 6824.
receptors for apoptotic cells: recognition, uptake, and Heath, R.J., White, S.W., Rock, C.O., 2001. Lipid biosynthe-
consequences. J. Clin. Invest. 108, 957 962. sis as a target for antibacterial agents. Prog. Lipid Res.
Fadok, V.A., Bratton, D.L., Frasch, S.C., Warner, M.L., 40, 467 497.
Henson, P.M., 1998. The role of phosphatidylserine in Hu, X., Binns, D., Reese, M.L., 2017. The coccidian parasites
recognition of apoptotic cells by phagocytes. Cell Death Toxoplasma and Neospora dysregulate mammalian lipid
Differ 5, 551 562. droplet biogenesis. J. Biol. Chem. 292, 11009 11020.
Ferguson, M.A., Williams, A.F., 1988. Cell-surface anchor- Ibrahim, M., Azzouz, N., Gerold, P., Schwarz, R.T., 2001.
ing of proteins via glycosyl-phosphatidylinositol struc- Identification and characterisation of Toxoplasma gondii
tures. Annu. Rev. Biochem. 57, 285 320. protein farnesyltransferase. Int. J. Parasitol. 31,
Foe, I.T., Child, M.A., Majmudar, J.D., Krishnamurthy, S., 1489 1497.
van der Linden, W.A., Ward, G.E., et al., 2015. Global Ihara, F., Nishikawa, Y., 2014. Starvation of low-density
analysis of palmitoylated proteins in Toxoplasma gondii. lipoprotein-derived cholesterol induces bradyzoite con-
Cell Host Microbe 18, 501 511. version in Toxoplasma gondii. Parasites Vectors 7, 248.
Fu, Y., Cui, X., Fan, S., Liu, J., Zhang, X., Wu, Y., et al., Jakobsson, A., Westerberg, R., Jacobsson, A., 2006. Fatty
2018. Comprehensive characterization of Toxoplasma acid elongases in mammals: their regulation and roles
acyl coenzyme A-binding protein TgACBP2 and its crit- in metabolism. Prog. Lipid Res. 45, 237 249.
ical role in parasite cardiolipin metabolism. mBio 9, Jelenska, J., Crawford, M.J., Harb, O.S., Zuther, E.,
e01597. Haselkorn, R., Roos, D.S., et al., 2001. Subcellular locali-
Fu, Y., Cui, X., Liu, J., Zhang, X., Zhang, H., Yang, C., et al., zation of acetyl-CoA carboxylase in the apicomplexan
2019. Synergistic roles of acyl-CoA binding protein parasite Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A.
(ACBP1) and sterol carrier protein 2 (SCP2) in 98, 2723 2728.
Toxoplasma lipid metabolism. Cell. Microbiol. 21, Jelenska, J., Sirikhachornkit, A., Haselkorn, R., Gornicki, P.,
e12970. 2002. The carboxyltransferase activity of the apicoplast
Gallegos, A.M., Atshaves, B.P., Storey, S.M., Starodub, O., acetyl-CoA carboxylase of Toxoplasma gondii is the target
Petrescu, A.D., Huang, H., et al., 2001. Gene structure, of aryloxyphenoxypropionate inhibitors. J. Biol. Chem.
intracellular localization, and functional roles of sterol 277, 23208 23215.
carrier protein-2. Prog. Lipid Res. 40, 498 563. Jomaa, H., Wiesner, J., Sanderbrand, S., Altincicek, B.,
Glomset, J.A., 1968. The plasma lecithins:cholesterol acyl- Weidemeyer, C., Hintz, M., et al., 1999. Inhibitors of the
transferase reaction. J. Lipid Res. 9, 155 167. nonmevalonate pathway of isoprenoid biosynthesis as
Goldstein, J.L., Brown, M.S., 1990. Regulation of the meva- antimalarial drugs. Science 285, 1573 1576.
lonate pathway. Nature 343, 425 430. Kong, P., Ufermann, C.M., Zimmermann, D.L.M., Yin, Q.,
Gomes, A.F., Magalhães, K.G., Rodrigues, R.M., de Suo, X., Helms, J.B., et al., 2017. Two phylogenetically
Carvalho, L., Molinaro, R., Bozza, P.T., et al., 2014. and compartmentally distinct CDP-diacylglycerol
Toxoplasma gondii-skeletal muscle cells interaction synthases cooperate for lipid biogenesis in Toxoplasma
increases lipid droplet biogenesis and positively modu- gondii. J. Biol. Chem. 292, 7145 7159.
lates the production of IL-12, IFN-γ and PGE2. Parasit. Lekutis, C., Ferguson, D.J., Grigg, M.E., Camps, M.,
Vectors 7, 47. Boothroyd, J.C., 2001. Surface antigens of Toxoplasma
Goodman, C.D., McFadden, G.I., 2007. Fatty acid biosyn- gondii: variations on a theme. Int. J. Parasitol. 31,
thesis as a drug target in apicomplexan parasites. Curr. 1285 1292.
Drug Targets 8, 15 30. Lévêque, M.F., Berry, L., Yamaryo-Botté, Y., Nguyen, H.M.,
Gupta, N., Zahn, M.M., Coppens, I., Joiner, K.A., Voelker, Galera, M., Botté, C.Y., et al., 2017. TgPL2, a patatin-like
D.R., 2005. Selective disruption of phosphatidylcholine phospholipase domain-containing protein, is involved
metabolism of the intracellular parasite Toxoplasma gon- in the maintenance of apicoplast lipids homeostasis in
dii arrests its growth. J. Biol. Chem. 280, 16345 16353. Toxoplasma. Mol. Microbiol. 105, 158 174.
Gupta, N., Hartmann, A., Lucius, R., Voelker, D.R., 2012. Li, Z.H., Ramakrishnan, S., Striepen, B., Moreno, S.N.J.,
The obligate intracellular parasite Toxoplasma gondii 2013. Toxoplasma gondii relies on both host and parasite
secretes a soluble phosphatidylserine decarboxylase. J. isoprenoids and can be rendered sensitive to atorvasta-
Biol. Chem. 287, 22938 22947. tin. PLoS Pathog. 9, e1003665.
Hartmann, A., Hellmund, M., Lucius, R., Voelker, D.R., Li, Z.H., Li, C., Szajnman, S.H., Rodriguez, J.B., Moreno, S.
Gupta, N., 2014. Phosphatidylethanolamine synthesis in N.J., 2017. Synergistic activity between statins and
Toxoplasma Gondii
References 393
bisphosphonates against acute experimental toxoplas- Mazumdar, J., Wilson, E.H., Masek, K., Hunter, C.A.,
mosis. Antimicrob. Agents Chemother. 61, e02628. Striepen, B., 2006. Apicoplast fatty acid synthesis is
Lige, B., Romano, J.D., Bandaru, V.V., Ehrenman, K., essential for organelle biogenesis and parasite survival
Levitskaya, J., Sampels, V., et al., 2011. Deficiency of a in Toxoplasma gondii. Proc. Natl. Acad. Sci. U.S.A. 103,
Niemann-Pick, type C1-related protein in Toxoplasma 13192 13197.
is associated with multiple lipidoses and increased McFadden, G.I., 1999. Plastids and protein targeting. J.
pathogenicity. PLoS Pathog. 7, e1002410. Eukaryot. Microbiol. 46, 339 346.
Lige, B., Sampels, V., Coppens, I., 2013. Characterization of McLeod, R., Muench, S.P., Rafferty, J.B., Kyle, D.E., Mui, E.J.,
a second sterol-esterifying enzyme in Toxoplasma high- Kirisits, M.J., et al., 2001. Triclosan inhibits the growth of
lights the importance of cholesterol storage pathways Plasmodium falciparum and Toxoplasma gondii by inhibition
for the parasite. Mol. Microbiol. 87, 951 967. of apicomplexan Fab I. Int. J. Parasitol. 31, 109 113.
Lim, L., Kalanon, M., McFadden, G.I., 2009. New proteins de Melo, E.J., de Souza, W., 1996. Pathway of C6-NBD-
in the apicoplast membranes: time to rethink apicoplast Ceramide on the host cell infected with Toxoplasma gon-
protein targeting. Trends Parasitol. 25, 97 200. dii. Cell Struct. Funct. 21, 47 52.
Ling, Y., Sahota, G., Odeh, S., Chan, J.M., Araujo, F.G., Milovanović, I., Busarčević, M., Trbovich, A., Ivović, V.,
Moreno, S.N., et al., 2005. Bisphosphonate inhibitors of Uzelac, A., Djurković-Djaković, O., 2017. Evidence for
Toxoplasma gondii growth: In vitro, QSAR, and in vivo host genetic regulation of altered lipid metabolism in
investigations. J. Med. Chem. 48, 3130 3140. experimental toxoplasmosis supported with gene data
Ling, Y., Li, Z.H., Miranda, K., Oldfield, E., Moreno, S.N., mining results. PLoS One 12, e0176700.
2007. The farnesyl-diphosphate/geranylgeranyl-diphos- Mina, J.G., Thye, J.K., Alqaisi, A.Q.I., Bird, L.E., Dods, R.H.,
phate synthase of Toxoplasma gondii is a bifunctional Grøftehauge, M.K., et al., 2017. Functional and phyloge-
enzyme and a molecular target of bisphosphonates. J. netic evidence of a bacterial origin for the first enzyme
Biol. Chem. 282, 30804 30816. in sphingolipid biosynthesis in a phylum of eukaryotic
Liscum, L., Underwood, K.W., 1995. Intracellular choles- protozoan parasites. J. Biol. Chem. 292, 12208 12219.
terol transport and compartmentation. J. Biol. Chem. Mordue, D.G., Scott-Weathers, C.F., Tobin, C.M., Knoll, L.
270, 15443 15446. J., 2007. A patatin-like protein protects Toxoplasma gondii
Lourido, S., Moreno, S.N.J., 2015. The calcium signaling from degradation in activated macrophages. Mol.
toolkit of the apicomplexan parasites Toxoplasma gondii Microbiol. 63, 482 496.
and Plasmodium spp. Cell Calcium 57, 186 193. Mota, L.A., Roberto, Neto, J., Monteiro, V.G., Lobato, C.S.,
de Macedo, C.S., Shams-Eldin, H., Smith, T.K., Schwarz, R. Oliveira, M.A., et al., 2014. Culture of mouse peritoneal
T., Azzouz, N., 2003. Inhibitors of glycosyl- macrophages with mouse serum induces lipid bodies
phosphatidylinositol anchor biosynthesis. Biochimie 85, that associate with the parasitophorous vacuole and
465 472. decrease their microbicidal capacity against Toxoplasma
Marechal, E., Azzouz, N., de Macedo, C.S., Block, M.A., gondii. Mem. Inst, Oswaldo Cruz 109, 767 774.
Feagin, J.E., Schwarz, R.T., et al., 2002. Synthesis of chlo- Nishikawa, Y., Quittnat, F., Stedman, T.T., Voelker, D.R.,
roplast galactolipids in apicomplexan parasites. Choi, J.Y., Zahn, M., et al., 2005. Host cell lipids control
Eukaryot. Cell 1, 653 656. cholesteryl ester synthesis and storage in intracellular
Martin, M.B., Grimley, J.S., Lewis, J.C., Heath 3rd, H.T., Bailey, Toxoplasma. Cell. Microbiol. 7, 849 867.
B.N., et al., 2001. Bisphosphonates inhibit the growth of Nolan, S.J., Romano, J.D., Coppens, I., 2017. Host lipid dro-
Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, plets: an important source of lipids salvaged by the
Toxoplasma gondii, and Plasmodium falciparum: a potential intracellular parasite Toxoplasma gondii. PLoS Pathog.
route to chemotherapy. J. Med. Chem. 44, 909 916. 13, e1006362.
Martins-Duarte, E.S., Jones, S.M., Gilbert, I.H., Atella, G.C., Nolan, S.J., Romano, J.D., Kline, J.T., Coppens, I., 2018.
de Souza, W., Vommaro, R.C., 2009. Thiolactomycin Novel approaches to kill Toxoplasma gondii by exploiting
analogues as potential anti-Toxoplasma gondii agents. the uncontrolled uptake of unsaturated fatty acids and
Parasitol. Int. 58, 411 415. vulnerability to lipid storage inhibition of the parasite.
Martins-Duarte, É.S., Carias, M., Vommaro, R., Surolia, N., Antimicrob. Agents Chemother. 62, e00347.
de Souza, W., 2016. Apicoplast fatty acid synthesis is Oishi, H., Noto, T., Sasaki, H., Suzuki, K., Hayashi, T.,
essential for pellicle formation at the end of cytokinesis Okazaki, H., et al., 1982. Thiolactomycin, a new antibi-
in Toxoplasma gondii. J. Cell Sci. 129, 3320 3331. otic. I. Taxonomy of the producing organism, fermenta-
Mazumdar, J., Striepen, B., 2007. Make it or take it: fatty tion and biological properties. J. Antibiot. 35, 391 395.
acid metabolism of apicomplexan parasites. Eukaryot. Oppenheim, R.D., Creek, D.J., Macrae, J.I., Modrzynska, K.
Cell 6, 1727 1735. K., Pino, P., Limenitakis, J., et al., 2014. BCKDH: the
Toxoplasma Gondii
missing link in apicomplexan mitochondrial metabo- association between T. gondii seropositivity and obesity.
lism is required for full virulence of Toxoplasma gondii Front. Public Health 1, 73.
and Plasmodium berghei. PLoS Pathog. 1, e1004263. Roberts, C.W., McLeod, R., Rice, D.W., Ginger, M., Chance,
Pernas, L., Bean, C., Boothroyd, J.C., Scorrano, L., 2018. M.L., Goad, L.J., 2003. Fatty acid and sterol metabolism:
Mitochondria restrict growth of the intracellular para- potential antimicrobial targets in apicomplexan and try-
site Toxoplasma gondii by limiting its uptake of fatty panosomatid parasitic protozoa. Mol. Biochem.
acids. Cell Metab. 27, 886 897. Parasitol. 126, 129 142.
Polonais, V., Soldati-Favre, D., 2010. Versatility in the Romano, J.D., Sonda, S., Bergbower, E., Smith, M.E.,
acquisition of energy and carbon sources by the Coppens, I., 2013. Toxoplasma gondii salvages sphingoli-
Apicomplexa. Biol. Cell 102, 435 445. pids from the host Golgi through the rerouting of
Portugal, L.R., Fernandes, L.R., Cesar, G.C., Santiago, H.C., selected Rab vesicles to the parasitophorous vacuole.
Oliveira, D.R., Silva, N.M., et al., 2004. Infection with Mol. Biol. Cell 24, 1974 1995.
Toxoplasma gondii increases atherosclerotic lesion in Romano, J.D., Nolan, S.J., Porter, C., Ehrenman, K.,
ApoE-deficient mice. Infect. Immun. 72, 3571 3576. Hartman, E.J., Hsia, R.C., et al., 2017. The parasite
Portugal, L.R., Fernandes, L.R., Alvarez-Leite, J.I., 2009. Toxoplasma sequesters diverse Rab host vesicles within
Host cholesterol and inflammation as common key reg- an intravacuolar network. J. Cell Biol. 216, 4235 4254.
ulators of toxoplasmosis and atherosclerosis develop- Sampels, V., Hartmann, A., Dietrich, I., Coppens, I.,
ment. Expert. Rev. Anti Infect. Ther. 7, 807 819. Sheiner, L., Striepen, B., et al., 2012. Conditional muta-
Pratt, S., Wansadhipathi-Kannangara, N.K., Bruce, C.R., genesis of a novel choline kinase demonstrates plasticity
Mina, J.G., Shams-Eldin, H., Casas, J., et al., 2013. of phosphatidylcholine biogenesis and gene expression
Sphingolipid synthesis and scavenging in the intracellu- in Toxoplasma gondii. J. Biol. Chem. 287, 16289 16299.
lar apicomplexan parasite, Toxoplasma gondii. Mol. Santos, T.A., Portes Jde, A., Damasceno-Sá, J.C., Caldas, L.
Biochem. Parasitol. 187, 43 51. A., de Souza, W., Damatta, R.A., et al., 2011.
Pszenny, V., Ehrenman, K., Romano, J.D., Kennard, A., Phosphatidylserine exposure by Toxoplasma gondii is
Schultz, A., Roos, D.S., et al., 2016. A lipolytic lecithin: fundamental to balance the immune response granting
cholesterol acyltransferase secreted by toxoplasma facil- survival of the parasite and of the host. PLoS One 6,
itates parasite replication and egress. J. Biol. Chem. 291, e27867.
3725 3746. Schultz, A.J., Carruthers, V.B., 2018. Toxoplasma gondii
Qian, Y., Sebti, S.M., Hamilton, A.D., 1997. LCAT primarily contributes to tachyzoite egress.
Farnesyltransferase as a target for anticancer drug mSphere 3, e00073.
design. Biopolymers 43, 25 41. Seeber, F., 2003. Biosynthetic pathways of plastid-derived
Quittnat, F., Nishikawa, Y., Stedman, T.T., Voelker, D.R., organelles as potential drug targets against parasitic
Choi, J.Y., Zahn, M.M., et al., 2004. On the biogenesis of apicomplexa. Curr. Drug Targets Immune Endocr.
lipid bodies in ancient eukaryotes: synthesis of triacyl- Metabol. Disord. 3, 99 109.
glycerols by a Toxoplasma DGAT1-related enzyme. Mol. Sehgal, A., Bettiol, S., Wenk, M.R., Pypaert, M., Kaasch, A.,
Biochem. Parasitol. 138, 107 122. Blader, I., et al., 2005. Peculiarities of host cholesterol
Ramakrishnan, S., Docampo, M.D., Macrae, J.I., Pujol, F.M., transport to the unique intracellular compartment con-
Brooks, C.F., van Dooren, G.G., et al., 2012. Apicoplast taining Toxoplasma gondii. Traffic 6, 1 17.
and endoplasmic reticulum cooperate in fatty acid bio- Sharma, K., Shi, Y., 1999. The yins and yangs of ceramide.
synthesis in apicomplexan parasite Toxoplasma gondii. J. Cell Res. 9, 1 10.
Biol. Chem. 287, 4957 4971. Sibley, L.D., Niesman, I.R., Parmley, S.F., Cesbron-Delauw,
Ramakrishnan, S., Serricchio, M., Striepen, B., Bütikofer, P., M.F., 1995. Regulated secretion of multi-lamellar vesi-
2013. Lipid synthesis in protozoan parasites: a compari- cles leads to formation of a tubulo-vesicular network in
son between kinetoplastids and apicomplexans. Prog. host-cell vacuoles occupied by Toxoplasma gondii. J. Cell
Lipid. Res. 52, 488 512. Sci. 108, 1669 1677.
Ramakrishnan, S., Docampo, M.D., MacRae, J.I., Ralton, J.E., Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang,
Rupasinghe, T., McConville, M.J., et al., 2015. The intracel- T., Nasamu, A.S., et al., 2016. A genome-wide CRISPR
lular parasite Toxoplasma gondii depends on the synthesis screen in Toxoplasma identifies essential apicomplexan
of long-chain and very long-chain unsaturated fatty acids genes. Cell 166, 1423 1435.
not supplied by the host cell. Mol. Microbiol. 97, 64 76. Sleat, D.E., Wiseman, J.A., El-Banna, M., Price, S.M., Verot,
Reeves, G.M., Mazaheri, S., Snitker, S., Langenberg, P., L., Shen, M.M., et al., 2004. Genetic evidence for nonre-
Giegling, I., Hartmann, A.M., et al., 2013. A positive dundant functional cooperativity between NPC1 and
Toxoplasma Gondii
Further reading 395
NPC2 in lipid transport. Proc. Natl. Acad. Sci. U.S.A. https://doi.org/10.1371/journal.pcbi.1004261. eCollection
101, 5886 5891. 2015 May. PubMed PMID: 26001086; PubMed Central
Smith, S., Witkowski, A., Joshi, A.K., 2003. Structural and PMCID: PMC4441489.
functional organization of the animal fatty acid Uttaro, A.D., 2006. Biosynthesis of polyunsaturated fatty
synthase. Prog. Lipid Res. 42, 289 317. acids in lower eukaryotes. IUBMB Life 58, 563 5671.
Sonda, S., Hehl, A.B., 2006. Lipid biology of Apicomplexa: Vance, J.E., Vance, D.E., 2004. Phospholipid biosynthesis in
perspectives for new drug targets, particularly for mammalian cells. Biochem. Cell Biol. 82, 113 128.
Toxoplasma gondii. Trends Parasitol. 22, 41 47. Waller, R.F., Keeling, P.J., Donald, R.G., Striepen, B.,
Sonda, S., Ting, L.M., Novak, S., Kim, K., Maher, J.J., Handman, E., Lang-Unnasch, N., et al., 1998. Nuclear-
Farese, R.V., et al., 2001. Cholesterol esterification by encoded proteins target to the plastid in Toxoplasma gon-
host and parasite is essential for optimal proliferation of dii and Plasmodium falciparum. Proc. Natl. Acad. Sci. U.S.
Toxoplasma gondii. J. Biol. Chem. 276, 34434 34440. A. 95, 12352 12357.
Sonda, S., Sala, G., Ghidoni, R., Hemphill, A., Pieters, J., Welti, R., Mui, E., Sparks, A., Wernimont, S., Isaac, G.,
2005. Inhibitory effect of aureobasidin A on Toxoplasma Kirisits, M., et al., 2007. Lipidomic analysis of
gondii. Antimicrob. Agents Chemother 49, 1794 1801. Toxoplasma gondii reveals unusual polar lipids.
Striepen, B., Zinecker, C.F., Damm, J.B., Melgers, P.A., Biochemistry 46, 13882 13890.
Gerwig, G.J., Koolen, M., et al., 1997. Molecular struc- White, S.W., Zheng, J., Zhang, Y.M., Rock, C.O., 2005. The
ture of the “low molecular weight antigen” of structural biology of type II fatty acid biosynthesis.
Toxoplasma gondii: a glucose alpha 1-4 N-acetylgalacto- Annu. Rev. Biochem. 74, 791 831.
samine makes free glycosyl-phosphatidylinositols Wichroski, M.J., Ward, G.E., 2003. Biosynthesis of glycosyl-
highly immunogenic. J. Mol. Biol. 266, 797 813. phosphatidylinositol is essential to the survival of the
Swiezewskaa, E., Danikiewiczb, W., 2005. Polyisoprenoids: protozoan parasite Toxoplasma gondii. Eukaryot. Cell 2,
structure, biosynthesis and function. Prog. Lipid Res. 1132 1136.
44, 235 258. Yardley, V., Khan, A.A., Martin, M.B., Slifer, T.R., Araujo,
Tawk, L., Dubremetz, J.-F., Montcourrier, P., Chicanne, G., F.G., Moreno, S.N., et al., 2002. In vivo activities of far-
Merezegue, F., Richard, V., et al., 2011. nesyl pyrophosphate synthase inhibitors against
Phosphatidylinositol 3-Monophosphate Is Involved in Leishmania donovani and Toxoplasma gondii. Antimicrob.
Toxoplasma Apicoplast Biogenesis. PLoS Pathog. 7, Agents Chemother. 46, 929 931.
e1001286. Zhang, K., Bangs, J.D., Beverley, S.M.I., 2010. Sphingolipids
Tobin, C.M., Knoll, L.J., 2012. A patatin-like protein pro- in parasitic protozoa. Adv. Exp. Med. Biol. 688, 238 248.
tects Toxoplasma gondii from degradation in a nitric Zinecker, C.F., Striepen, B., Geyer, H., Geyer, R.,
oxide-dependent manner. Infect. Immun. 80, 55 61. Dubremetz, J.F., Schwarz, R.T., 2001. Two glycoforms
Tomavo, S., Schwarz, R.T., Dubremetz, J.F., 1989. Evidence for are present in the GPI-membrane anchor of the surface
glycosyl-phosphatidylinositol anchoring of Toxoplasma antigen 1 (P30) of Toxoplasma gondii. Mol. Biochem.
gondii major surface antigens. Mol. Cell Biol. 9, 4576 4580. Parasitol. 116, 127 135.
Tomavo, S., Dubremetz, J.F., Schwarz, R.T., 1992.
Biosynthesis of glycolipid precursors for glycosylpho-
sphatidylinositol membrane anchors in a Toxoplasma Further reading
gondii cell-free system. J. Biol. Chem. 267, 21446 21458.
Tomavo, C., Humbel, B.M., Geerts, W.J., Entzeroth, R., Caffaro, C.E., Boothroyd, J.C., 2011. Evidence for host cells
Holthuis, J.C., Verkleij, A.J., 2009. Membrane contact as the major contributor of lipids in the intravacuolar
sites between apicoplast and ER in Toxoplasma gondii network of Toxoplasma-infected cells. Eukaryot. Cell 10,
revealed by electron tomography. Traffic 10, 1471 1480. 1095 1099.
Tymoshenko, S., Oppenheim, R.D., Agren, R., Nielsen, J., Hoffmann, P.R., de Cathelineau, A.M., Ogden, C.A.,
Soldati-Favre, D., Hatzimanikatis, V., 2015. Metabolic Leverrier, Y., Bratton, D.L., Daleke, D.L., et al., 2001.
Needs and Capabilities of Toxoplasma gondii through Phosphatidylserine (PS) induces PS receptor-mediated
Combined Computational and Experimental Analysis. macropinocytosis and promotes clearance of apoptotic
PLoS Comput Biol. 11 (5), e1004261. Available from: cells. J. Cell Biol. 155, 649 659.
Toxoplasma Gondii

BAB VIII - Biochemistry and Metabolism of Toxoplasma Gondii Lipid Synthesis and Uptake

Uploaded by

Copyright:

Available Formats

BAB VIII - Biochemistry and Metabolism of Toxoplasma Gondii Lipid Synthesis and Uptake

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

BAB VIII - Biochemistry and Metabolism of Toxoplasma Gondii Lipid Synthesis and Uptake

Uploaded by

Copyright:

Available Formats

C H A P T E R

8.1 Introduction fatty acids and phosphatidic acid, are impor-

further be modified in the ER of Toxoplasma hydroxyacyl-CoA dehydratase and enoyl-CoA

growth of T. gondii consecutive to ablation of role of host mitochondria-PV association is still

phylogenetically divergent CDS enzymes: the analyses on the TgCDS1-deficient parasites

Ethanolamine Choline DME

Phosphoethanolamine Phosphocholine PhosphoDME

CDP-ethanolamine CDP-choline CDP-DME

parasite staining is located to the plasma lipoproteins stimulates parasite replication

LD. Blockade of cholesteryl ester synthesis is 8.7.2 Sphingolipid synthesis in

8.8.2 Isoprenoid synthesis in Toxoplasma

(Ling et al., 2007). Intracellular TgFPPS-deficient for isoprenylation. Peptidomimetics act as

You might also like