BBA - Molecular Cell Research 1865 (2018) 1846–1856

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BBA - Molecular Cell Research

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Calcium signaling and the lytic cycle of the Apicomplexan parasite T

Toxoplasma gondii☆
Miryam Andrea Hortua Trianaa,1, Karla M. Márquez-Noguerasa,1, Stephen A. Vellaa,1,
⁎
Silvia N.J. Morenoa,b,
a
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA
b
Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Toxoplasma gondii has a complex life cycle involving different hosts and is dependent on fast responses, as the
Calcium signaling parasite reacts to changing environmental conditions. T. gondii causes disease by lysing the host cells that it
Toxoplasma gondii infects and it does this by reiterating its lytic cycle, which consists of host cell invasion, replication inside the
Lytic cycle host cell, and egress causing host cell lysis. Calcium ion (Ca2+) signaling triggers activation of molecules in-
Pathogenicity
volved in the stimulation and enhancement of each step of the parasite lytic cycle. Ca2+ signaling is essential for
Calcium dependent protein kinases
the cellular and developmental changes that support T. gondii parasitism.
Calcium binding proteins
The characterization of the molecular players and pathways directly activated by Ca2+ signaling in
Toxoplasma is sketchy and incomplete. The evolutionary distance between Toxoplasma and other eukaryotic
model systems makes the comparison sometimes not informative. The advent of new genomic information and
new genetic tools applicable for studying Toxoplasma biology is rapidly changing this scenario. The Toxoplasma
genome reveals the presence of many genes potentially involved in Ca2+ signaling, even though the role of most
of them is not known. The use of Genetically Encoded Calcium Indicators (GECIs) has allowed studies on the role
of novel calcium-related proteins on egress, an essential step for the virulence and dissemination of Toxoplasma.
In addition, the discovery of new Ca2+ players is generating novel targets for drugs, vaccines, and diagnostic
tools and a better understanding of the biology of these parasites.

1. Calcium and the Toxoplasma lytic cycle
Toxoplasma gondii belongs to the phylum Apicomplexa, which in-
cludes a number of unicellular eukaryotes that infect humans and an-
imals and cause diseases, such as malaria (Plasmodium spp.), babesiosis
(Babesia spp.), toxoplasmosis (Toxoplasma gondii), cryptosporidiosis
(Cryptosporidium parvum), and cyclosporiasis (Cyclospora cayetanensis),
among others. Apicomplexan are highly divergent from mammals and
more closely related to ciliates and dinoflagellates [1]. The name of the
phylum Apicomplexa denotes the presence of a unique set of secretory
organelles at the apical complex of zoites. These specialized organelles
secrete unique effectors, in a highly regulated manner, critical for en-
tering into their host cell [2].
Toxoplasma gondii is an obligate intracellular parasite that infects
approximately one third of the world population [3,4]. Toxoplasma can
be transmitted through contaminated food or water via oocyts released
with the feces of infected cats, the definitive host [3,5]. Intermediate
hosts, such as animals and humans, can be infected via mature oocysts
containing sporozoites or via contaminated meat with tissue cysts
containing bradyzoites. Both sporozoites and bradyzoites transform
into tachyzoites, the fast replicating form, which change back into slow
replicating bradyzoites within tissue cysts. These are characteristic of
the chronic infection and are usually found in the brain, eye, and stri-
ated muscle tissues [6]. Disseminated toxoplasmosis can cause severe
complications in immunocompromised patients including HIV-infected
individuals, fetuses, and organ transplant recipients [7,8]. Some of the
most devastating effects of toxoplasmosis are the result of the parasite
lytic cycle (Fig. 1), which starts when the tachyzoite attaches and in-
vades host cells, replicates inside a parasitophorous vacuole and
egresses to find another host cell to invade and re-start the cycle [9,10].
Invasion and egress are dynamic processes that are highly regulated
and essential for the propagation of the infection. Toxoplasma and other

☆
This article is part of a Special Issue entitled: Calcium signaling in health, disease and therapy edited by Geert Bultynck and Jan Parys.
⁎
Corresponding author at: Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602, USA.
E-mail address: smoreno@uga.edu (S.N.J. Moreno).
1
Equal Contribution, Authors' names are alphabetical.

https://doi.org/10.1016/j.bbamcr.2018.08.004
Received 15 April 2018; Received in revised form 6 August 2018; Accepted 7 August 2018
Available online 10 August 2018
0167-4889/ © 2018 Elsevier B.V. All rights reserved.
M.A. Hortua Triana et al.
Fig. 1. The lytic cycle of Toxoplasma gondii. Toxoplasma tachyzoites attach and invade host cells and form a parasitophorous vacuole (PV) where they reside and
replicate by endodyogeny. The PV is surrounded by a PV membrane (PVM) that allows small molecules to sieve through. After several rounds of replication, they
rupture the PVM and the host cell membrane and egress followed by a short extracellular phase and invasion of another host cell. Christina Moore designed the Lytic
Cycle.
Apicomplexan parasites like Plasmodium rely on gliding motility for
host-cell invasion, egress and dissemination in the infected host.
Gliding motility, vital to a successful lytic cycle, is a peculiar mode of
substrate dependent motility, unique to Apicomplexan parasites, that
facilitates entry into host cells and is powered by a macromolecular
complex termed the glideosome [11].
During invasion, the parasite secretes proteins from specific apical
organelles [12], and their contents mediate attachment to host cells and
formation of the specialized parasitophorous vacuole (PV) occupied by
the parasite [13]. Each step during the lytic cycle has to be precise, fast
and effective and the parasite possesses specific molecules that become
activated at the right instant. A strong body of evidence supports that
intracellular calcium oscillations in the parasite precede the activation
or stimulation of the specific steps of the lytic cycle [14]. Both extra-
cellular and intracellular Ca2+ pools contribute to cytosolic Ca2+ in-
creases resulting in downstream signaling pathways that are decoded
into critical biological functions of the parasite lytic cycle.
Genomic analysis suggests the presence of a variety of calcium
channels, which could potentially play a role in Ca2+ entry or release
from intracellular stores [15]. There is evidence for Ca2+ release sti-
mulated by IP3 and cADP ribose [16,17] but no receptors for these
second messengers have been identified for any Apicomplexan parasite
[18]. A number of Ca2+-binding proteins including calmodulins, cal-
modulin-like proteins, and an array of Ca2+-dependent protein kinases
are predicted to be present in these parasites [19,20]. A large number of
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BBA - Molecular Cell Research 1865 (2018) 1846–1856
recent studies have provided evidence of their function in the lytic cycle
of Toxoplasma.
2. The Toxoplasma calcium signaling toolkit
The information on the elements that form part of the calcium
signaling toolkit of Toxoplasma and other Apicomplexan parasites is
fragmented. The main challenge is the evolutionary distance of the
Apicomplexa from mammalian model systems for which most of the
information is available, and making direct comparisons is not always
informative. However, the characterization of the molecular pathways
involved in calcium signaling in Toxoplasma and other pathogens is
highly relevant for several reasons. First, it is known that calcium
fluctuations control vital cellular processes essential for parasitic life.
Second, it is very likely that new molecules with unique characteristics
may result in the discovery of druggable targets. Lastly, as an early
branching eukaryote, Toxoplasma occupies a unique phylogenetic po-
sition providing insights into the early origin of complex signaling
pathways.
Toxoplasma possesses characteristic calcium compartments such as
the endoplasmic reticulum (ER), Golgi apparatus and mitochondrium,
and additional compartments that could contribute to calcium signaling
like acidocalcisomes [21–23] and endosome-like compartments like the
plant-like vacuole [24].
The concentration of Ca2+ in the cytosol of Toxoplasma tachyzoites
M.A. Hortua Triana et al.
is around 50–100 nM [25], similar to the cytosolic concentration of
other eukaryotic cells. Ca2+ signaling starts when the cytoplasmic le-
vels of Ca2+ are raised due to Ca2+ entry or Ca2+ release from in-
tracellular stores. Ca2+ entry has an important role in refilling in-
tracellular organelles that participate in signaling pathways that
respond to elevated cytosolic Ca2+ [26]. T. gondii does not appear to
express the molecular elements of Store Operated Calcium Entry
(SOCE). There is no genomic evidence for the presence of store-oper-
ated channels (ORAI), or for the ER sensor protein stromal interaction
molecule (STIM). It appears that ligand-operated channels [26], or
second messenger-operated channels are also missing in T. gondii. Gene
sequences with similarity to voltage dependent Ca2+ channels are
present in its genome [15,19]. The role of the encoded proteins as
functional Ca2+ channels has not been demonstrated although evidence
has been presented for the presence of a nifedipine-sensitive Ca2+ entry
pathway that supports the function of a voltage-gated Ca2+ channel
[27]. Sequences with similarity to transient receptor potential (TRP)
channels were also found in T. gondii [15] but have not been char-
acterized. TRP channels are a superfamily of channels with diverse
cellular functions [28] that can localize at the plasma membrane and
allow Ca2+ influx or to intracellular stores and allow Ca2+ release into
the cell cytosol.
In vertebrate cells binding of a ligand to a surface receptor like a G-
protein linked receptor (GPCR) may result in the stimulation of the
phosphoinositide-signaling pathway via the activation of a phosphati-
dylinositol phospholipase C (PI-PLC), which hydrolyzes phosphatidyli-
nositol 4,5-bisphosphate (PIP2) to form inositol 1,4,5-trisphosphate
(IP3) and diacylglycerol (DAG). Binding of IP3 to an IP3 receptor usually
present in the membrane of the ER, will result in its opening allowing
Ca2+ to be released into the cytosol [29]. A diversity of cell signaling
pathways can be generated through this mechanism [29]. Cyclic ADP
ribose (cADPR) is also able to release intracellular Ca2+ acting through
ryanodine receptors usually located in the ER [30]. Some of the ele-
ments of this signaling pathway have been found and characterized in
Toxoplasma but a large number of them remains to be identified. The T.
gondii phosphoinositide phospholipase C (TgPIPLC) has been studied
[31] and characterization of conditional mutants of the Tgpiplc gene
highlighted its importance for parasite survival and its participation in
a vast and currently incompletely understood signaling cascade [32]. It
is not clear yet, what is the target of the IP3 generated by TgPIPLC
because there is no genomic support for the presence of IP3 receptors in
any Apicomplexan parasite. However, IP3 stimulates Ca2+ release from
isolated microsomes of T. gondii [17] and cyclic ADP ribose does as
well, presumably by stimulating a ryanodine-type receptor [17]. Ca2+
release by IP3 is inhibited by the IP3 receptor inhibitor xestospongin C
[17], which also inhibited secretion of micronemes, secretory proteins
involved in host invasion [16]. T. gondii possesses ADP ribosyl cyclase
and hydrolase [17] but there is no molecular evidence of a ryanodine
receptor.
The ER plays critical roles in calcium signaling in eukaryotic cells.
Ca2+ influx into the ER is driven by a SERCA-type Ca2+ ATPases, which
has been characterized in T. gondii [33]. Inhibition of the T. gondii
SERCA-type Ca2+-ATPase by thapsigargin [34] results in increase of
cytosolic Ca2+ in T. gondii [25], due to Ca2+ leakage from the ER
through an unknown pathway. Ca2+ leakage is the passive calcium
efflux from the ER that is thought to prevent ER calcium overload and
thus allow cytosolic Ca2+ signaling [35]. In mammalian cells it has
been proposed that leak channels in the membrane of the ER play a role
in the steady state concentration of the ER luminal Ca2+ [36]. Several
types of membrane proteins have been proposed to be involved in the
ER calcium leak pathway, Bcl-2, pannexin 1, presenilins and TRPC1.
The Toxoplasma genome contains ortholog genes for a presenilin
(TGME49_204040) and two TRP channels (TGME49_247370 and
TGME49_310560) [15]. These Toxoplasma molecules have not been
characterized except for a recent study using high-affinity tags, which
localized TGME49_247370 to the ER [37]. An active ER Ca2+ release
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BBA - Molecular Cell Research 1865 (2018) 1846–1856
channel, termed transmembrane and coiled-coil domains 1 (TMCO1) or
Ca2+ load-activated Ca2+ (CLAC) channel has been shown to assemble
as a homotetramer upon ER Ca2+ overload [38]. The main function of
the channel is to prevent ER Ca2+ overload. A small portion of TMCO1
exists as homotetramer even under normal levels of ER Ca2+, which
could also cause passive leak of Ca2+ from the ER. The genome of
Toxoplasma shows evidence for the presence of a TMCO1 ortholog
(TGME49_310870), predicted to have a signal peptide at its N-terminus.
This Toxoplasma gene has not been characterized.
Acidocalcisomes are acidic organelles that store large amounts of
Ca2+, which is mostly bound to polymers of phosphate [39]. Acid-
ocalcisomes of T. gondii possess a plasma membrane-type Ca2+-ATPase
for Ca2+ uptake named TgA1 [40]. Purified acidocalcisome fractions
from T. gondii tachyzoites show vanadate-sensitive Ca2+ uptake sup-
porting the presence of this enzyme [23].
A vacuolar compartment with lysosomal characteristics (Plant-Like
Vacuole or PLV) that expresses several orthologs of pumps and trans-
porters usually found in the plant vacuole has been characterized in
Toxoplasma and shown to store Ca2+ [24]. An increase in cytosolic
Ca2+ in response to glycyl-L-phenylalanine-naphthylamide (GPN) in-
dicated the presence of Ca2+ in the PLV where lytic activities are also
present. GPN has been used to study lysosomal Ca2+ because it causes
swelling of these compartments leading to leakage of Ca2+ into the
cytosol. In Toxoplasma, GPN-dependent Ca2+ release was independent
from release from other Ca2+ stores, such as the ER [24].
The mitochondrion of T. gondii maintains a membrane potential and
carries out energy-linked functions like respiration coupled to oxidative
phosphorylation [41]. However, it was not possible to demonstrate
Ca2+-uptake reliant on its membrane potential (unpublished results). In
addition, there is no genomic evidence for the presence of a mi-
tochondrial Ca2+ uniporter (MCU) in the inner membrane of any api-
complexan parasite [42]. A Ca2+/H+ antiporter (CAX) was localized to
the mitochondria of P. falciparum [43] but the Toxoplasma ortholog did
not localize to the mitochondrion [44]. The potential role of the Tox-
oplasma mitochondria in cytoplasmic Ca2+ homeostasis or even the role
of mitochondrial Ca2+ on the regulation of mitochondrial enzymes is
unknown [45].
Downstream to Ca2+, the signaling pathways that control essential
and specific biological functions of Toxoplasma are not clearly defined.
However, the information available is growing and several important
players are emerging (Fig. 2). A role for a cyclic GMP (cGMP)-activated
Protein Kinase G (PKG) in the activation of Toxoplasma egress, motility
and differentiation, has been reported [46,47]. Plant like Ca2+ de-
pendent proteins kinases (CDPKs) are expanded across Apicomplexa
and play pivotal roles in Ca2+ signaling throughout the lytic cycle [20].
CDPKs have been implicated in a range of processes in Toxoplasma, like
invasion [48], egress [49,50,51] and microneme secretion [20,48]. A
recent study linked the central regulating role of PKG in the Ca2+
signals that precede egress to the activity of Protein kinase A. It was
proposed that PKA down-regulates PKG-dependent signaling leading to
egress by potentially activating a phosphodiesterase that would hy-
drolyze cGMP [52]. In a second study on Toxoplasma PKA the authors
proposed that PKA is in the pathway that leads to suppression of cy-
tosolic Ca2+ right after invasion promoting a decrease in motility and
the beginning of intracellular replication [53]. It is clear that Ca2+, PKG
and PKA signaling pathways crosstalk but we still do not know all the
elements involved and the order in which they interact (Fig. 2). How-
ever, the expanse of information during the last three years about sig-
naling pathways involved in the regulation of Toxoplasma parasitism is
encouraging and it is very likely that we will soon know how those
elements interact.
3. Calcium binding proteins in T. gondii
Intracellular Ca2+ increase activates a variety of cellular processes,
including the mechanisms that return cytosolic Ca2+ concentration to
M.A. Hortua Triana et al. BBA - Molecular Cell Research 1865 (2018) 1846–1856

Fig. 2. Ca2+ signaling in T. gondii tachyzoites. Ca2+ enters the parasite likely through a Ca2+ channel that is sensitive to nifedipine. Inside the cell, Ca2+ is pumped
either outside by a plasma membrane type Ca2+ ATPase or into organelles like the SERCA-Ca2+-ATPases localized to the endoplasmic reticulum (ER). The plasma
membrane type Ca2+-ATPase in Toxoplasma also localizes to acidic stores (AS) like acidocalcisome or PLV. A signaling cascade that starts by an increase in cGMP
level would lead to activation of a cGMP-protein kinase G that would result in cytosolic Ca2+ fluctuations that would activate biological responses like microneme
(MIC) secretion. In this hypothetical scenario PKA could phosphorylate an unidentified phosphodiesterase (PDE) resulting in its inhibition and leading to an increase
in cGMP levels. Downstream to PKG the pathway is hypothetical because the evidence is incomplete but CDPK3 could be part of it together with PI-PLC. The
hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP2) by PI-PLC forms two second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3
would act on an unknown release channel in the ER allowing Ca2+ to be released into the cytosol, which could activate CDPK1 stimulating microneme secretion.
Changes in DAG levels would activate DAGK1 (DAG kinase 1), which converts DAG into phosphatidic acid (PA), which is sensed by the micronemal protein APH
(acylated pleckstrin homology domain-containing protein) stimulating secretion. A Ca2+/H+ exchanger that was characterized in the PLV could use the proton
gradient to take-up Ca2+ into the acidic store [140]. How Ca2+ is released into the cytoplasm is not known. The bar at the upper right represents a Ca2+
concentration scale by blue color gradient (light to dark). Known pathways are shown by continuous lines, while hypothetical ones are represented by dashed lines.

its steady-state level, such as the membrane transporters that sequester
Ca2+ into intracellular organelles or actively pump it out of the cell.
Most signaling responses, however, involve Ca2+-binding proteins
(CaBPs). One of the most common motifs that binds Ca2+ and trans-
duces these signals is the helix-loop-helix motif known as EF-hand. EF-
hands frequently occur in pairs (also called EF-hand domains) that
allow for the cooperative binding of two Ca2+ ions per domain. CaBPs
with single or odd number of EF hands sometimes function via dimer-
ization mechanisms. Analysis of the T. gondii genome has revealed ap-
proximately 68 EF-hand domain-containing proteins encoded by its
genome (ToxoDB, http://toxodb.org/toxo/). The majority of these
genes have not been characterized.
EF-hand containing proteins can be grouped into the calmodulin
(CaM) family, the calcineurin (CN) family, and the Ca2+-dependent
protein kinase (CDPK) family. Both CaM and CN family members lack
effector domains and are thought to act by regulating other proteins.
The CaM group includes classical CaMs (with > 75% sequence identity
to human CaM), calmodulin-like (CML) proteins, which include myosin
light chains (sequence identity with human CaM < 75%), and centrins
or calcactrins. Calmodulins comprise two globular domains (each with
a pair of EF hands) linked by a flexible helical region [54,55]. The T.
gondii genome encodes a single prototypical CaM and a variable number
of CML proteins. The T. gondii prototypical CaM binds Ca2+ in vitro
[56] and was localized to the apical domain [57], and more recently to
the conoid where it may interact with calcineurin [58]. No studies have
addressed directly the role of CaM in the specific steps of the parasite
lytic cycle.
Toxoplasma contains plant-like features because of its early
branching prior to the animal-plant separation [1] and also because of
the acquisition of an endosymbiont derived from an algal cell [59].
Because of this, signaling pathways and molecules present in Tox-
oplasma will likely be very unique. For example, as in plants,
Toxoplasma possesses a large number of CML proteins with low se-
quence identity and with degenerate EF-hand motifs [60]. CML proteins
can have diverse subcellular localizations broadening the potential
substrates for their interaction [61]. Three of the CaM-like proteins,
named CaM1, CaM2, and CaM3, are important for motility and invasion
and localize to the conoid where they overlap with the motor protein
myosin H (MyoH). These proteins were shown to interact with MyoH
and its light chain MLC7 [62,63]. CaM1 has two conserved EF hands,
while CaM2 has one conserved and one degenerate EF hand. Analysis of
mutants unable to bind Ca2+ was consistent with Ca2+ binding to the
conserved EF hands for the regulation of function of CaM1 and CaM2
[62]. CaM3 is essential for growth and it was proposed that it may
replace CaM1 and CaM2 in the activation of MyoH [64]. In agreement
with the function of CaMs in invasion, the CaM inhibitors calmidazo-
lium and trifluoperazine were shown to inhibit T. gondii host cell entry
[65].
Centrins are Ca2+ binding proteins usually present with centro-
somes. Apicomplexan centrins have been shown to associate with var-
ious cytoskeletal components including the centriole and conoid
[66,67], but none have been demonstrated to be Ca2+ regulated. In-
terestingly, a recent finding showed that TgCentrin2 co-localized with
the TgPIPLC [37]. This localization was secondary to the plasma
membrane localization of the TgPIPLC and it was only possible to ob-
serve with high-affinity tags. The significance of this finding is intri-
guing and deserves further investigation.
Myosin light chains (MLCs) are calmodulin (CaM)-related proteins
containing four EF-hand motifs and are important for the stability of the
myosin motor complex [68–70]. It is unlikely that these CaM-like
proteins will bind Ca2+ given that most of their EF-hands are degen-
erate. Two myosin regulatory light chain proteins, essential light chains
1 and 2 (ELC1, ELC2), which form part of the glideosome, have been
studied and ELC1 has been shown to bind Ca2+ as estimated by

1849
M.A. Hortua Triana et al.
modeling studies and thermal shift assays [71,72]. The affinity for Ca2+
of this protein is low (37 ± 9 μM) suggesting that for this interaction to
be physiologically relevant it would have to be in a high Ca2+ con-
centration microdomain. It is interesting that four CML proteins localize
to the conoid. Considering what occurs with plant CML proteins and
CaMs, which can activate a variety of target proteins that regulate
metabolism, transcription and ion transport, it is very likely that the
Toxoplasma CML proteins that co-localize could have more than one
target. The redundancy of these proteins, suggests that they play more
than one role in Ca2+ signaling.
The CN family members have been identified only in some protist
genomes. Calcineurin is a Ca2+ regulated-phosphatase that consists of a
catalytic subunit (CnA) and a Ca2+-binding regulatory subunit (CnB).
Calcineurin phosphatase activity is usually regulated by changes in
intracellular Ca2+ [73]. The role of calcineurin in T. gondii was studied
by analyzing conditional CnA mutants and found to be important for
host cell attachment, invasion and stage-specific development of the
parasite [58]. The next challenge will be to determine which are the
substrates for T. gondii calcineurin.
A less prominent Ca2+-binding motif is the C2 domain, which was
originally identified as the domain responsible for the Ca2+-dependent
phospholipid binding of protein kinase C (PKC). Although no PKC can
be found in apicomplexan genomes, various C2-domain containing
proteins have been identified. Among them, DOC2 has been shown to
be required for the Ca2+-regulated secretion of micronemes in T. gondii
[74]. Apicomplexan PIPLC, which is thought to mediate intracellular
Ca2+ store release through the production of IP3, is also known to
contain a C2 domain, in addition to an EF-hand, although it is unknown
how Ca2+ impacts its regulation [31].
In plants there is a unique group of Ca2+ sensors and regulators
known as Calcium Dependent Protein Kinases (CDPKs). Canonically,
CDPKs are composed of a kinase domain followed by a CaM-like do-
main, also known as the CDPK activation domain (CAD) [75,76]. The
activity of these kinases is directly regulated by Ca2+ binding to the
CAD. The important role of CDPKs in apicomplexans is reflected by
their overrepresentation in the parasite genome. T. gondii CDPKs reg-
ulate a variety of events in the parasites like egress, replication, motility
and protein secretion.
4. Ca2+-dependent protein kinases
The classical calmodulin-dependent kinases (CaMK) typical of an-
imal cells [77] are absent in Apicomplexan. Instead, they contain cal-
cium-dependent protein kinases (CDPKs), a family of serine/threonine
kinases that is present in plants and protists, including ciliates [20,78].
In plants, CDPKs regulate a diversity of pathways like cell cycle pro-
gression, stress response, stomatal closure, and root-nodule formation
[75]. In Apicomplexan parasites CDPKs represent the best characterized
link between Ca2+ signaling and parasite biological functions like in-
vasion, motility, egress and differentiation [20].
The typical structure of CDPKs comprises an N-terminal serine/
threonine kinase domain (KD) connected by a junction region to a
calcium-binding regulatory domain with four EF-hands forming a cal-
modulin-like domain. The regulatory domain is termed the CDPK-ac-
tivation domain (CAD) [76]. The structures of T. gondii CDPK1 in the
presence and absence of Ca2+ revealed the molecular details of their
activation [76,79]. In the absence of Ca2+, the junction region lodges in
the substrate-binding region, blocking the catalytic pocket of the en-
zyme keeping it inactive. Upon binding of Ca2+ to the EF-hands a
dramatic structural rearrangement leads to a segmentation of the au-
toinhibitory helix around the calmodulin like-domain altering the in-
teraction and relieving the inhibition [76]. A study with CDPK1 showed
that upon binding calcium the CAD domain rotates with respect to the
kinase domain and in addition to relieving autoinhibition stabilizes the
active form of the enzyme [80]. The Toxoplasma CDPK1 is essential and
conditional knockdown mutants of TgCDPK1 are defective in
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BBA - Molecular Cell Research 1865 (2018) 1846–1856
microneme secretion, motility, host-cell invasion and egress [48]. These
studies highlighted the role of CDPKs in transducing Ca2+ signals.
A remarkable characteristic of TgCDPK1 is the presence of a small
glycine gatekeeper residue in the ATP binding pocket making it sensi-
tive to ATP-competitive inhibitors with bulky substituents. Because of
this, it is possible to inhibit TgCDPK1 with bulky ATP-analogues de-
signed for kinases with extended ATP-binding pockets [48,79,81], as
well as with similar compounds designed for their improved anti-
parasitic properties [82–84].
TgCDPK3 has been shown to have a more specific function in
parasite egress, which was shown by characterizing a direct knockout of
the gene as well as with a chemical-genetic approach [49–51]. CDPKs
represent attractive drug targets because of their absence from the
mammalian genome [20]. Several recent screens have targeted CDPKs
for the development of new antiparasitic compounds [85–87].
T. gondii also expresses a number of noncanonical CDPKs with dif-
ferent numbers of EF-hands, N-terminal EF hands, or with additional
domains. Much less is known about the function of these noncanonical
CDPKs with the exception of TgCDPK7, which contains the kinase do-
main preceded by a pleckstrin homology (pH) domain and two or more
incomplete EF-hands. Conditional knockout of TgCDPK7 showed a
defect in cell division [88]. Another study developed seven individual
mutants of noncanonical CDPKs, plus double and triple mutants [89].
Only one of these mutants, ΔCDPK6, showed a mild growth phenotype.
There is not a clear link between Ca2+ signaling and the potential
function of these noncanonical CDPKs.
The challenge has been to characterize the biological targets of
CDPKs. Several studies have addressed this question by searching for
widespread changes in the phosphorylation state of diverse proteins
following an increase in intracellular Ca2+ [70,90]. These studies have
uncovered interesting molecules but it is still not clear what are the
specific effectors that are controlled by Ca2+. A comparison of the
phosphoproteomes of wild type and TgCDPK3-defficient parasites un-
covered hundreds of changes in diverse cellular pathways including
proteins associated with gliding motility [90]. Several components of
the motor complex are heavily phosphorylated and this has been
documented [91]. Mutation of the identified phosphorylation sites was
undertaken to identify which one was relevant for motor function [91].
The central player of the glideosome, a multisubunit motor complex, is
T. gondii myosin A (TgMyoA), an unconventional myosin that is es-
sential for motility, invasion and egress [69,92]. An increase in cyto-
plasmic Ca2+ resulted in increased MyoA phosphorylation and muta-
tion of the major sites for TgMyoA phosphorylation altered the parasite
response to cytoplasmic Ca2+ elevation by ionophores and other drugs
[93]. The expression of MyoA bearing phosphoro-mimetic mutations
rescued the response to ionophores [93]. A more recent study using the
proximity-based protein interaction trap BioID identified 13 targets of
CDPK3, among them TgMyoA [94]. In this study, a peptide array ap-
proach identified serines 21 and 743 as the targets for CDPK3 phos-
phorylation. CDPK3 phosphorylation of MyoA was important for in-
itiation of motility and egress providing a mechanistic link between
CDPK3 regulation and the lytic cycle [94]. A study on egress and the
role of CDPKs on Ca2+ signaling showed that CDPK1 and CDPK3 play a
role in the Ca2+ signal recovery prior to egress [95]. A forward-genetic
screen to isolate gain-of-function CDPK3 mutants [96] followed this
study, which uncovered a new component that was characterized as
suppressor of Ca2+-dependent cell egress [96].
It is very likely that the essential phenotypes attributed to CDPKs
are the result of their important regulatory functions but it is clear that
more work is needed to clarify the signaling network that is triggered
upon cytoplasmic Ca2+ increase and its downstream stimulation of
kinases, phosphatases or other targets. Deciphering the specific targets
of CDPKs will be an essential task to understand the signaling pathways
that lead to the biological functions that are essential for Toxoplasma
parasitism.
M.A. Hortua Triana et al.
5. Microneme secretion, motility, host cell invasion and egress
Toxoplasma is an obligate intracellular parasite that multiplies in-
side host cells. The process of invasion is actively driven by the parasite
and is fast, precise, and involves gliding motility, conoid extrusion, and
microneme secretion [97–99]. An increase in cytosolic Ca2+ artificially
elevated with ionophores, like ionomycin or A23187, stimulated conoid
extrusion of T. gondii tachyzoites, an effect that was prevented if the
parasites were pre-loaded with BAPTA-AM [98]. Conoid extrusion also
responded to other cytosolic Ca2+ triggers like ethanol [100] and ex-
tracellular Ca2+ enhanced and extended the extrusion time [27].
BAPTA-AM also prevented gliding motility [97] and host cell invasion
[101] by T. gondii, supporting a role for Ca2+ in these essential parasite
functions. Conditional downregulation of the expression of a conoid
protein hub 1 (CPH1) caused the conoid to collapse and become shorter
[64]. In this work, the stimulation of conoid extrusion or microneme
secretion by ionophore was not affected even though CPH1 was es-
sential for invasion and conoid structure [64]. The specifics about how
Ca2+ increase leads to extension of the conoid are not clearly defined.
Several Ca2+ binding proteins have been localized to the structure [62].
A recently described conoid ring protein, RNG2, was shown to change
its orientation in response to stimulation of conoid extrusion with Ca2+
ionophores [102]. Based on this result, a role for RNG2 in secretion was
proposed.
Toxoplasma gondii contains micronemes, specialized secretory or-
ganelles important for gliding motility and host cell invasion [103,104].
Micronemes are small apical organelles with an elongated shape and
electron-dense structure [104]. Micronemes store adhesins that upon
secretion participate in the interactions with the host-cell surface and
participate in the early stages of host-cell invasion. Secretion of mi-
cronemes can be triggered by elevating cytosolic Ca2+ with ionophores
and can be blocked by chelating intracellular Ca2+ with BAPTA-AM
[103,105]. A number of studies have shown that multiple external
stimuli can trigger microneme secretion such as a drop in extracellular
K+ [106], an increase in H+ [107], serum albumin [108] and extra-
cellular Ca2+ [27]. The precise signaling pathway that leads to mi-
croneme exocytosis, and especially how Ca2+ stimulates it, is not well
defined, but some participating molecules have been identified. One
study on the characterization of Ca2+-ATPase (TgA1) [40] knockout
mutants, which showed altered levels of intracellular Ca2+, examined
the role of Ca2+ in the lytic cycle of Toxoplasma. These mutant parasites
are deficient in microneme secretion, invasion, and showed reduced
virulence in vivo [109].
A study looking at the mechanism of action of the antimalarial drug
artemisinin, showed that thapsigargin and artemisinin triggered Ca2+-
dependent secretion of micronemes. Artemisinin treatment altered
Toxoplasma intracellular Ca2+ and increased the periodicity of Ca2+
oscillations as imaged in parasites loaded with the Ca2+ fluorescent
indicator Fluo4 [33]. Previous reports had shown in Plasmodium, that
the SERCA orthologue PfATPase6 could be the target of artemisinin
[110] and considering that artemisinin altered Ca2+ signaling in Tox-
oplasma [111] it was proposed that the drug could inhibit TgSERCA,
which was further supported by the sensitivity of yeast expressing
TgSERCA to artemisinin. Presently, the mechanism (s) of action of ar-
temisinin, still remains unclear because Toxoplasma artemisinin-re-
sistant mutants did not show apparent mutations or alterations in the
expression levels of TgSERCA or other Ca2+-ATPase genes [111].
CDPK1 conditional knockdowns highlighted the role of the Ca2+-
stimulated kinase in attachment and secretion of micronemes [48]. The
authors characterized the inhibition of TgCDPK1 by 3-methylbenzyl-
PP1 (3-MB-PP1), an ATP analog that inhibited the enzyme due to the
presence of the amino acid glycine in the gate-keeper position allowing
the inhibitor to block the ATP binding site. The PP1 inhibitor blocked
microneme secretion and related functions. Mutation of glycine to a
methionine (G for M) made the enzyme insensitive to the inhibitor [48].
A chemical genetic study with the inhibitor WRR-086, identified an
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BBA - Molecular Cell Research 1865 (2018) 1846–1856
homologue of the human redox chaperone protein, T. gondii DJ-1, with
specific function in parasite motility and microneme secretion [112],
even when stimulating secretion with ionophore or ethanol. This sug-
gested that the role of TgDJ-1 is downstream of Ca2+ signaling [112] as
was also shown for TgCDPK1. It was proposed that the regulation of
microneme secretion by CDPK1 was in part due to its interaction with
TgDJ-1 in a Ca2+ and H2O2-dependent manner [113].
The role of the cGMP-dependent protein kinase (PKG) in microneme
exocytosis was first demonstrated by its inhibition with compound 1
(Cpd1), a trisubstituted pyrrole derivative with in vivo activity against
Toxoplasma [114]. The validation of PKG as target of Cpd1 was done
with transgenic parasites complemented with mutated versions of
TgPKG (T761Q), which are insensitive to the inhibition of microneme
secretion by Cpd1 [46]. Increase of cytosolic Ca2+ with ionophores did
not reverse the inhibition of microneme secretion by Cpd1 suggesting a
downstream role for PKG in this process [46]. Toxoplasma possesses a
single gene for PKG, which is essential, but expresses two isoforms with
identical regulatory and catalytic domains, PKGI is membrane bound
and PKGII is cytosolic [115]. Recent work on PKG differentiated the
contribution of each PKG isoform to Toxoplasma biology using the
auxin-inducible degron (AID) tagging system for conditional depletion
of PKG protein followed by complementation with mutated versions of
PKG [116]. PKGI, the membrane associated isoform is essential and
sufficient for microneme secretion and parasite survival. PKGII, the
cytosolic version is dispensable but its function became relevant when
the gene was modified so the protein localized to the plasma membrane
[116]. These results support a role for PKG downstream or in-
dependently of the events that lead to intracellular Ca2+ increase
[46,116]. However, studies with parasites loaded with the Ca2+ in-
dicator Fura2-AM showed that accumulation of cGMP by inhibiting its
phosphodiesterases (PDE) with zaprinast, resulted in an increase in
cytosolic Ca2+, which is dependent on PKG activity because this
Ca2+increase was almost 80–90% inhibited by Cpd1 [117].
It is interesting that serum albumin acts synergistically with ethanol
to increase cytosolic Ca2+ and microneme secretion [108]. The sti-
mulation of microneme secretion was proposed to occur through a
Ca2+-independent pathway on the basis of Ca2+ measurements using
the genetic indicator GCaMP6f. However, it remains to be determined if
the Ca2+ increase was below the GCaMP6 sensitivity as this indicator
exhibits different kinetics and affinities for Ca2+ than Fura2 [118].
Early studies evaluating microneme secretion of Toxoplasma were per-
formed in the presence of bovine serum, which has been shown to
stimulate microneme secretion in related apicomplexan
[16,99,105,108]. More recently it was determined that serum albumin,
the major component of serum, was sufficient to induce microneme
secretion, while γ-globulins, the second major component of serum had
no effect [108]. It is well characterized that Ca2+ ionophores such as
ionomycin or A23187 induce microneme secretion, however in a study
characterizing the role of extracellular Ca2+, microneme secretion was
shown to correlate with the concentration of extracellular Ca2+ sup-
porting an enhancing role for Ca2+ influx [27]. This study did not use
ionophores or other triggers, or serum and parasites were kept in low
Ca2+ buffer until the microneme assay was performed. It is possible
that Ca2+ influx leads to an increase of Ca2+ at specific peripheral
domains of the parasite and bypasses the intracellular signaling trig-
gered by PKG. Additional studies are needed to clarify the link between
Ca2+ and PKG signaling in T. gondii.
Most evidence linking Ca2+ signaling to parasite replication is in-
direct. Conditional knock-downs of CDPK7, a potentially Ca2+ regu-
lated kinase, results in parasites with replication phenotypes [88].
Knockdown of CDPK7 alters centrosome duplication and positioning.
CDPK7 appears to play a role in the positioning of secretory organelles
like rhoptries and micronemes. The miss-positioning of these organelles
in the mutants implicated CDPK7 in the trafficking of these organelles
to daughter cells [88]. During intracellular growth, GRA1, a dense
granule protein secreted within the PV was shown to be important for
M.A. Hortua Triana et al.
the formation of the tubulovesicular network [119,120]. GRA1 pos-
sesses two EF-hand domains, which were shown to bind Ca2+ and it
was proposed that it would act as a Ca2+ buffer within the PV [119].
The role of host Ca2+, specifically Ca2+ entry, was shown with the
inhibitor L-651,582, which apparently blocks Ca2+ entry in the host
cell affecting parasite replication [121]. L-651,582 inhibited Tox-
oplasma and Eimeria growth in a variety of mammalian host cells [121].
This result suggests that during its intracellular replication phase the
parasite might need to take up Ca2+ from the host to keep its stores
replenished. It is also possible that a number of Ca2+ regulated proteins
are involved in the replication of the parasite. There is evidence for the
presence of a higher concentration of Ca2+ within the PV [122] and
host Ca2+ oscillation do cause oscillations in the parasite [123]. It is
still not clear how host cytosolic Ca2+ impacts the biological functions
of the parasite. Parasites mutants on a dense granule protein secreted to
the PV, GRA41, showed alteration in their cytosolic Ca2+ levels and
egress [124].
Active egress of Toxoplasma from host cells requires rupture of the
parasitophorous vacuole membrane (PVM) and the host cell membrane
[125]. Egress is essential for the dissemination of the infection and it
has been known for several years that Ca2+ ionophores can trigger
egress [126]. However, it was the use of Genetically Encoded Ca2+
Indicators (GECIs) that provided the final and conclusive evidence that
there was a cytosolic Ca2+ increase right before egress [123]. The use
of these indicators has vastly impacted the studies of Ca2+ in in-
tracellular parasites [95,117,123,127]. Secretion from the micronemes
of the perforin-like protein TgPLP1, assists in the permeabilization of
the PVM and host cell membrane [128]. Initiation of parasite motility
results in mechanical pressure and final rupture of the host cell allowing
the release of the parasites. Both, secretion of the micronemal protein
TgPLP1 and initiation of motility during egress are stimulated by an
increase in cytosolic Ca2+. A role for a change in extracellular po-
tassium concentration was proposed to stimulate Ca2+ signaling
leading to egress [106]. The presence of extracellular Ca2+ also affected
the rate of egress, which was blocked with the Ca2+-channel blocker
nifedipine [123]. A plant-like pathway involving the phytohormone
abscisic acid (ABA) was proposed to be the pathway leading to the
production of the natural signal for Toxoplasma egress [129]. The initial
trigger that starts the Ca2+ signaling leading to egress is still a mystery.
The isolation of parasite mutants resistant to ionophore-induced
egress (or delayed response) identified mutants defective in Ca2+ sig-
naling. A mutant defective in a Na+/H+ exchanger (TgNHE1), was
isolated in this way and found to have elevated intracellular Ca2+ levels
and reduced pathogenicity [130]. A similar screen identified TgCDPK3
as a mediator of ionophore-induced egress [50]. TgCDPK3 likely con-
trols rapid exit from the host by phosphorylating proteins involved in
the activation of motility. Another CDPK3 substrate that plays a role in
egress is the Suppressor of Ca2+ Egress 1 or SCE1, and its phosphor-
ylation relieves the suppression of Ca2+-dependent host cell egress
[96].
Extracellular tachyzoites exhibit three types of motility (Fig. 3):
circular (counter-clockwise circular motion), helical (forward cork-
screw motion), and twirling (attachment to posterior end and spinning
vertically) [131], though within a 3D matrix, motility is one continuous
irregular corkscrew motion [132]. Helical gliding is considered the
most effective and essential for invasion [131]. Ca2+ oscillations were
first observed in actively motile parasites loaded with Fluo-4 that pre-
ceded periods of microneme secretion and bursts of speed [97,133].
Cytosolic Ca2+ fluctuations were further characterized with parasites
expressing GECIs and a direct correlation between distance traveled
and amplitude of the oscillations was exposed [95,123]. Cytosolic Ca2+
levels are reduced at the moment of invasion and stay low right after
invasion. The oscillation pattern of cytosolic Ca2+ in motile parasites
suggests that Ca2+ influx channels and reuptake mechanisms are highly
active in gliding parasites and necessary for effective lytic cycle events
[95,123,133]. Similar perturbations of Ca2+ signaling also affect
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BBA - Molecular Cell Research 1865 (2018) 1846–1856
gliding motility and microneme secretion [134], which has been used
to deduce our current understanding of this form of parasite movement.
For example, increasing the frequency and lowering the amplitude of
Ca2+ oscillations with calmidazolium enhanced microneme secretion
and gliding motility [97]. Calmidazolium stimulated gliding and this
was proposed to depend on MIC2 secretion. The increase of cytosolic
Ca2+ levels was shown to occur through release from intracellular
stores and entry from the extracellular milieu. The calmidazolium study
highlighted the importance of Ca2+ oscillations vs. constant Ca2+ in-
crease for efficient gliding of T. gondii [97]. It is possible that Ca2+
oscillations enable the parasite to traverse longer distances over a
longer period of time, via coordinating the release of micronemes only
when necessary instead of constitutively [95,123]. Whether Ca2+ reg-
ulates aspects of gliding motility beyond secretion of adhesins remains
to be determined.
Release of intracellular Ca2+ stores was proposed to be sufficient to
initiate gliding motility and host-cell invasion by use of ionophores or
intracellular Ca2+ chelators like BAPTA [16]. However, extracellular
Ca2+ influx impacted and enhanced invasion-linked traits like motility,
conoid extrusion, microneme secretion and invasion [27]. T. gondii
replicates through endodyogeny, and its lytic cycle is complex and
parasites commonly invade and exit with little or no replication
[135,136]. This complex behavior, all of which depends on Ca2+, likely
requires replenishing intracellular Ca2+ stores with extracellular Ca2+.
It would be advantageous for the parasite to utilize the high con-
centration of extracellular Ca2+ to enhance invasion-related processes
and to resupply intracellular stores for use during subsequent rounds of
invasion and egress. Experimental evidence supports a general me-
chanism of Ca2+ entry, which may not be sensitive to the filling state of
the ER, as is the case in mammalian cells, but which could work in
parallel with intracellular store release to elevate intracellular Ca2+,
and activate downstream events like invasion and motility [27].
The glideosome is the molecular machinery that generates the me-
chanical force needed for completion of the lytic cycle [137]. Inhibition
(using pharmacological agents) or disruption (using knockouts or con-
ditional depletion of core components) of the glideosome has been
known to adversely affect the lytic cycle events of egress, extracellular
motility, and invasion thus highlighting its significance across the lytic
cycle [10,125]. Egress results from the initiation of the glideosome and
parasites will continue to be actively motile until invasion of new host
cells suggesting that extracellular motility serves as the bridge con-
necting egress and invasion [10]. As an intracellular parasite, T. gondii
must replicate inside host cells; and as such, glideosome activation must
be tightly regulated across time and space [91]. Several studies have
demonstrated that glideosome activation occurs in a Ca2+-dependent
manner, through multiple, interconnected and cross-talking mechanism
(s) [51,94,125] (Fig. 4A). Furthermore, a recent study identified that
the force and direction of gliding motility occurs in a Ca2+-dependent
manner. At basal levels the net mechanical force of the parasite is
random and disorganized. Post increases in cytosolic Ca2+, the net
mechanical force of the parasite becomes polarized and oriented across
the long axis of the parasite thus sufficient for productive motility
[138].
The glideosome is a multi-subunit actomyosin motor complex that
resides at the periphery of the parasite; confined to the space below the
plasma membrane (PM) and above an underlying structure termed the
inner membrane complex (IMC) (Fig. 4A) [11,125]. The complex
(Fig. 4B) is anchored to the IMC via the scaffolding complex of gli-
deosome-associated proteins (GAP's), and members of the GAPM family
[11]. TgGAP45 serves as a molecular tether that links the IMC to the PM
through lipid modification links [11,70]. TgMyoA, an important com-
ponent of the glideosome [70,92], is an unconventional myosin of the
class XIVa that interacts with its associated light chain, TgMLC1 and
either the essential light chain 1 or 2 (ELC1 or ELC2) [70,72]. Though
MyoA serves as the primary source for the generation of force needed
for motility, it is the interaction between the MyoA neck with ELC1/2
M.A. Hortua Triana et al. BBA - Molecular Cell Research 1865 (2018) 1846–1856

Fig. 3. T. gondii Motility Types. (From left to right)

Representative stills of Circular (counter clockwise
circular motion), Helical (forward corkscrew mo-
tion), and Twirling (attachment on posterior end with
windmill-like motion) motility post extracellular
Ca2+ addition. T. gondii tachyzoites are expressing
cytosolic GCaMP6f [123] and exposure to extra-
cellular Ca2+ stimulates motility and fluorescence of
the indicator. Size bar: 5 μM.

that supports force transduction throughout the motor complex and
more broadly throughout the body of the parasite as GAP proteins are
anchored to the cytoskeleton [11,72,139]. Within the glideosome
MLC1, ELC1 and ELC2, interact either directly with Ca2+ or via Ca2+
dependent phosphorylation to stimulate motility and microneme se-
cretion [14,51,70] (Fig. 4B).
Though several Ca2+-dependent phosphorylation sites of the gli-
deosome are dispensable, mutations of S20, S21, and S29 of MyoA have
been determined to be important for MyoA activity, whereas S21 ap-
pears to be the primary site for phosphorylation [70,91,93]. A recent
study determined that disruption of the phosphorylation state(s) of
MyoA by expressing mutated versions of MyoA serine residues, altered
the motility behavior of gliding parasites as compared to wild type cells
[93]. These results suggest that the summation of Ca2+ oscillations may
coordinate the dynamic cycles of phosphorylation/dephosphorylation
of MyoA, needed for effective gliding motility/invasion [51,93,94].
An in vitro assay utilized to monitor actin displacement of purified
MyoA, MLC1, and ELC1 identified that when ELC1 was added to
complexed MyoA-MLC1, MyoA moved actin filaments at more than
twice the speed (and more in line with motor complexes isolate from
parasites) as compared to MyoA-MLC1 alone [139]. Later it was de-
termined that in addition to enhancing the thermostability of ELC1 in
the presence of Ca2+, ELC1 had an enhanced affinity for MyoA and is
recruited by MLC1 [70,72]. Given the low affinity (μM) of ELC1 (and
presumably ELC2) for Ca2+, it is reasonable to hypothesize that ELC1(/
2) may have evolved to optimally bind Ca2+ at high concentrations
and/or rapidly release Ca2+ in continuous succession [72]. Perhaps
through rapidly binding and releasing Ca2+, during the Ca2+

Fig. 4. A) Signaling and activation of Motility. Activation of Ca2+ signaling will translate into the activation of CDPK3 which will phosphorylate proteins from the
molecular machinery and activating motility. The glideosome is the main molecular motility machinery and is anchored to the underlying cytoskeleton termed the
inner membrane complex (IMC). Adapted from [14], with permission. (B) The glideosome of T. gondii. MLC1 and/or ELC1 or ELC2 interact with Ca2+ directly or via
Ca2+ dependent phosphorylation events to stimulate motility. The glideosome was adapted from [71].

1853
M.A. Hortua Triana et al.
oscillations that govern parasite motility, ELC1(/2) may serve as a
“molecular throttle” to enhance MyoA activity, thus highlighting its
potential role in decoding the Ca2+ signals that govern parasite moti-
lity.
Outlook
In the last 3 years since our last review of the Ca2+ research lit-
erature [14] we have witnessed how new genetic tools have advanced
our knowledge on Ca2+ signaling in Toxoplasma. Some of these ad-
vances include the use of CRISPR-Cas9 for genome editing increasing
the efficiency for the characterization of Toxoplasma genes, the ex-
pression of genetically encoded Ca2+ indicators in Toxoplasma tachy-
zoites, the use of a biotin ligase fusion protein to identify proximal and
interacting proteins, and the adaptation of tools to down-regulate
protein levels allowing studies of more immediate phenotypic differ-
ences without previous adaptation. The field is moving leap wise and it
is expected that we will see more and more discoveries of new elements
and pathways components.
During these last three years we have finally been able to see Ca2+
oscillations prior to exit of the parasite from its host cells. This was
years after Endo [126] showed that ionophores trigger egress and as-
sumed that it was because of a Ca2+ response. These studies were
possible thanks to the use of GECIs that allowed to specifically detect
Ca2+ changes in live intracellular parasites. We still need to know how
intracellular vs extracellular Ca2+ crosstalk to deliver the threshold
needed for increase in motility and egress.
We have also seen evidence of crosstalk between second messengers
like Ca2+, cGMP and cAMP. The picture is still not very clear, especially
about which one is upstream of which, but there are no doubts that
information is flowing and we will see this mystery uncovered in the
near future.
There is no doubt about the significance of Ca2+ for gliding moti-
lity, microneme secretion, host cell invasion and egress of T. gondii.
How Ca2+ regulates aspects of gliding motility beyond secretion of
adhesins remains to be determined. The same argument can be ex-
tended to our understanding of parasite invasion and egress from host
cells, although certain cellular components may affect the different
phenotypes to different extents. Ca2+ signaling is controlled by its
uptake and release from different cellular compartments and there are
important differences with the processes that control Ca2+ homeostasis
in other eukaryotic cells, providing opportunities for finding novel drug
targets. It is also apparent that the more we learn about Ca2+ signaling
in Toxoplasma the more we know about the basic mechanisms that
regulate how Toxoplasma, and other related pathogens, cause disease.
Transparency document
The Transparency document associated with this article can be
found, in online version.
Acknowledgments
S.N.J.M. is supported by grants from the U.S. National Institutes of
Health (AI-096836, and AI128356). KMN was supported in part by a
fellowship from the T32 training grant to the Center for Tropical and
Emerging Global Diseases (AI060546). SAV was partially supported by
a fellowship from the UGA Office of the Vice-President for Research
(OVPR). We thank Christina Moore for help with the drawings.
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