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REVIEW ARTICLE

Mechanisms of Toxoplasma gondii persistence and latency


William J. Sullivan Jr1,2 & Victoria Jeffers1
1

Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, IN, USA; and 2Department of Microbiology
and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA

Correspondence: William J. Sullivan Jr,


Department of Pharmacology and Toxicology,
Indiana University School of Medicine, 635
Barnhill Drive, MS A-525, Indianapolis, IN
46202, USA. Tel./fax: 317 274 1573/7714;
e-mail: wjsulliv@iupui.edu
Received 27 May 2011; revised 22 August
2011; accepted 25 August 2011.
Final version published online 4 October
2011.
DOI: 10.1111/j.1574-6976.2011.00305.x
Editor: Colin Berry

Abstract
Toxoplasma gondii is an obligate intracellular protozoan parasite that causes
opportunistic disease, particularly in immunocompromised individuals. Central
to its transmission and pathogenesis is the ability of the proliferative stage
(tachyzoite) to convert into latent tissue cysts (bradyzoites). Encystment allows
Toxoplasma to persist in the host and affords the parasite a unique opportunity
to spread to new hosts without proceeding through its sexual stage, which is
restricted to felids. Bradyzoite tissue cysts can cause reactivated toxoplasmosis
if host immunity becomes impaired. A greater understanding of the molecular
mechanisms orchestrating bradyzoite development is needed to better manage
the disease. Here, we will review key studies that have contributed to our
knowledge about this persistent form of the parasite and how to study it, with
a focus on how cellular stress can signal for the reprogramming of gene expression needed during bradyzoite development.

MICROBIOLOGY REVIEWS

Keywords
parasite; Apicomplexa; differentiation;
eukaryotic pathogen; microbial persistence;
stress response.

Introduction
Toxoplasma gondii is one of the most successful parasites
on Earth. Despite its obligate intracellular lifestyle, this
protozoan parasite has remarkable transmissibility and
has permanently infected most species of mammals and
birds around the world. As we will discuss, the ability of
Toxoplasma to encyst inside the cells of host tissues and
reconvert back into its proliferative stage was an evolutionary paradigm shift, circumventing the need for the
parasite to undergo its sexual stage to be transmitted to a
new host. This unusual property affords Toxoplasma a
second major route of transmission: the capacity to disseminate clonally through intermediate hosts.
Toxoplasma belongs to phylum Apicomplexa, which
contains many other protozoan pathogens of human and
veterinary importance, such as Plasmodium spp. (malaria),
Cryptosporidium spp. (cryptosporidiosis), and Eimeria spp.
(poultry coccidiosis). Toxoplasmosis is a notorious opportunistic disease in patients with AIDS and other immunocompromised individuals which most commonly results
from reactivation of a previous infection (Luft et al.,
1984). Ocular infection by Toxoplasma is a major cause
of retinochoroiditis in both immunocompromised and
FEMS Microbiol Rev 36 (2012) 717733

immunocompetent individuals (Wallace & Stanford,


2008). Additionally, Toxoplasma can cross the placental
barrier and cause abortion or congenital birth defects if the
mother becomes infected for the first time shortly before
or during pregnancy (Jones et al., 2003). In utero infection
is also associated with an elevated risk of ocular toxoplasmosis owing to spontaneous reactivation of disease
(Wallace & Stanford, 2008).
Toxoplasma has a complex life cycle consisting of multiple stages that fluctuate between proliferative and latent
stages (Fig. 1). The proliferative stage, known as the tachyzoite, replicates exponentially by endodyogeny (asexual
division whereby two daughter cells form within a single
mother cell) inside of host cells with a doubling time of
c. 7 h (Radke & White, 1998). The intracellular tachyzoites, housed in a membrane-bound compartment called
the parasitophorous vacuole (PV), can convert into virtually quiescent forms known as bradyzoites, which transform the parasitophorous vacuole membrane (PVM) into
a cyst wall in the process. Bradyzoite cysts remain infectious and can form in skeletal, heart, and CNS tissues,
granting Toxoplasma the ability to spread to a new host
following predation of its former host. Felines serve as
the definitive host for Toxoplasma, whose intestinal milieu
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W.J. Sullivan Jr & V. Jeffers

is the only known environment suitable for the sexual


stage of the parasite life cycle. Owing to factors that have
yet to be identified, the cat gut induces bradyzoites to differentiate into macrogametes and flagellated microgametes that fuse and lead to the formation of oocysts.
Dissemination of the stable, highly infectious oocysts into
the environment provides another major route of transmission through the food and water chain (Fig. 1). While
it is not possible to distinguish infection from meat vs.
oocysts, it has been proposed that the surge of infection
in teenagers and low prevalence in younger children indicate that transmission through cysts in undercooked meat
is important (Dubey & Jones, 2008). As a consequence of
these multiple routes of transmission, Toxoplasma has
made itself at home in at least one in three people. Seroprevalence varies widely between geographical regions,
but estimates suggest that between 30% and 65% of
humans worldwide are infected (Tenter et al., 2000).
The ability to differentiate into bradyzoites and form
these impenetrable cysts makes it currently impossible to
eradicate Toxoplasma from the host. While several drugs
are available which control acute toxoplasmosis, such as
pyrimethamine plus sulfadiazine, no short-term treatment

exists which can eliminate the cysts, which also appear


impervious to the immune response. The presence of
latent bradyzoite cysts makes Toxoplasma a chronic infection, and the ability of bradyzoites to reconvert into rapidly growing tachyzoites explains the high frequencies of
acute toxoplasmosis often observed in immunocompromised individuals (Wong & Remington, 1993; Montoya
& Liesenfeld, 2004). Toxoplasmic encephalitis, characterized by intracerebral lesions, is a significant CNS complication in patients with AIDS (Fig. 2).
Microbial persistence and latency are conserved strategies that numerous pathogens use to their advantage. The
ability of Toxoplasma to form latent cysts is an evolutionary trade-off that reduces virulence while increasing transmission a winwin situation for the parasite. In terms
of human and veterinary health, understanding bradyzoite
conversion is the key to minimizing transmission and
managing chronic infection. In this review, we will discuss bradyzoite development and physiology, with a focus
on the molecular mechanisms orchestrating differentiation. We will also highlight the methods used to study
bradyzoite cysts in vitro and in vivo and present important questions that require further investigation.

Fig. 1. Life cycle of Toxoplasma gondii. The definitive host of


Toxoplasma is the cat (members of the Felidae family), the only
organism capable of supporting the sexual stage of the parasite.
Infected cats excrete stable, infectious oocysts into the environment
that transmit the parasite to other warm-blooded animals, which
serve as intermediate hosts. Within intermediate hosts, Toxoplasma
persists as infectious bradyzoites within tissue cysts, providing another
route of transmission via carnivorism. Infection of humans may occur
via two routes: direct exposure to oocysts in the environment or from
contaminated food or water and ingestion of bradyzoite tissue cysts
in undercooked meat. Primary infection of the fetus can occur during
pregnancy if tachyzoites cross the placental barrier, leading to
congenital birth defects or spontaneous abortion.

Fig. 2. Reactivated Toxoplasma infection. Brain CT scan with contrast


shows reactivation of disease in an HIV-positive patient (male Hispanic
age 28 with CD4 count of 48). The ring-enhancing lesion has
surrounding edema. Image courtesy of Dr L.M. Weiss Albert Einstein
College of Medicine.

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Toxoplasma bradyzoite latency

Biology of the bradyzoite cyst


Morphological features of bradyzoites and
cysts

Bradyzoite tissue cysts develop and remain inside nucleated cells, exhibiting tropism similar to tachyzoites, but
are more frequently found in brain, eye, skeletal muscle,
and cardiac tissue. Within the brain, bradyzoite cysts
appear to form in a wide variety of regions, although a
subtle tropism for the medial and basolateral amygdala
has been reported (Vyas et al., 2007). Mature tissue cysts
can be detected within 710 days postinfection, but hallmarks of bradyzoite differentiation can be detected within
3 days postinduction in vitro. Recent studies using bioluminescence imaging technology also show that conversion
to bradyzoites begins rapidly in vivo, as early as 1 day
postinfection (Di Cristina et al., 2008). Tissue cysts vary
in size and shape. Young cysts can be as small as 5 lm in
diameter with as few as two bradyzoites, while mature
cysts can be up to 70 lm with c. 1000 bradyzoites (Dubey et al., 1998) (Fig. 3a). Individual bradyzoites (7 9
1.5 lm) are slender relative to tachyzoites (6 9 2 lm),
with a more pronounced bow or crescent shape (Mehlhorn & Frenkel, 1980). Also different from tachyzoites,
the nucleus in mature bradyzoites resides at the parasites
posterior, rhoptry organelles are electron dense, and there
is a marked increase in micronemes and amylopectin
(glucose storage) granules (Dubey et al., 1998) (Fig. 3b).
The bradyzoite PVM/cyst wall is < 0.5 lm thick and
elastic to accommodate expansion. The cyst wall is built
from chitin and glycoproteins secreted by the parasites
(Boothroyd et al., 1997; Zhang et al., 2001). The sugars
in the cyst wall are capable of binding lectins concanavalin A, wheat germ agglutinin, soy bean agglutinin, and
Dolichos biflorus agglutinin (Sethi et al., 1977), the latter

(a)

of which is commonly used as a diagnostic tool for cyst


wall staining (Fig. 4). In mature cysts, a matrix material
fills the PV space between bradyzoites (Ferguson &
Hutchison, 1987). The number of cyst wall/matrix proteins that have been characterized to date is limited.
CST1 is a 116-kDa cyst wall glycoprotein that is the
major binding constituent of Dolichos lectin (Zhang et al.,
2001). MAG1 is a 65-kDa protein that localizes to the
cyst wall and matrix (Parmley et al., 1994). At least one
dense granule protein, GRA5, has been reported to be
highly concentrated in the cyst wall; less intense staining
of GRA1, GRA3, and GRA6 can also be observed (Lane
et al., 1996; Ferguson, 2004). Upon ingestion by a host,
the cyst wall is degraded by acid-pepsin digestion, releasing the bradyzoites. Unlike tachyzoites, bradyzoites are
resistant to proteolytic enzymes (Jacobs et al., 1960), a
key feature that assures survival in the host stomach. The
bradyzoites proceed to invade intestinal epithelial cells to
begin infection and dissemination throughout the host.
Studies have demonstrated that mature bradyzoites isolated from the brains of mice infected with Toxoplasma
oocysts represent a growth-arrested stage with parasites
suspended in G0 with uniform 1N DNA content (Radke
et al., 2003). To provide insights into the dynamics of
early stage conversion, an in vitro system using type II
(Prugniaud) parasites subjected to CO2 deprivation was
developed to maintain bradyzoites for up to 2 weeks.
Using this system, it was observed that immature bradyzoites replicate slowly (requiring > 12 h compared to
the c. 7 h for tachyzoites) and asynchronously, using a
combination of endodyogeny and endopolygeny (asexual
replication whereby more than two daughter cells form
within the mother cell) (Dzierszinski et al., 2004). Interestingly, within the context of this system, 1020%
of developing bradyzoite vacuoles contained individual

(b)

Fig. 3. Bradyzoite cyst and morphology. (a) Phase image of a brain cyst harboring hundreds of bradyzoites isolated from a chronically infected
mouse. Image courtesy of Dr L.M. Weiss, Albert Einstein College of Medicine. (b) Electron micrograph shows details of bradyzoites within an
intracellular tissue cyst. Note the posterior location of the parasite nucleus and abundance of amylopectin granules (white) and micronemes
(black). Image courtesy of Dr David Ferguson, University of Oxford.

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720

Fig. 4. Dolichos lectin stains bradyzoite cyst wall. Type II strain ME49
tachyzoites were grown in human foreskin fibroblasts and induced to
form bradyzoite tissue cysts by alkaline stress (pH 8.2) for 7 days.
Following methanol fixation, tissue cysts were detected using FITCconjugated Dolichos biflorus lectin. 4,6-diamidino-2-phenylindole
(DAPI) was used as a costain of DNA. Image courtesy of Dr Christian
Konrad (Sullivan laboratory).

parasites lacking an apicoplast, a nonphotosynthetic plastid-like organelle acquired from an algal endosymbiont
(Dzierszinski et al., 2004; McFadden, 2010). Despite their
reputation for quiescence, the developing bradyzoites generated under these conditions were highly motile and
capable of exiting a host cell without destroying it to
invade a neighboring cell; bradyzoites within mature cysts
purified from mouse brains also exhibit intravacuolar
motility (Dzierszinski et al., 2004). Bradyzoite motility
within and between host cells may explain why increases
in cyst burden are observed in chronically infected mice
despite the low frequency of cyst rupture.

Metabolism in the latent stage

Changes in parasite metabolism accompany the switch to


a latent lifestyle. A number of metabolic enzymes have
tachyzoite- and bradyzoite-specific isoforms (e.g. ENO2/
ENO1 and LDH1/LDH2), suggesting a fine tuning of
metabolism between these two life cycle stages. The abundance of polysaccharide in the form of amylopectin granules in the bradyzoites reflects a major shift in
carbohydrate metabolism. This idea is supported by biochemical analyses which concluded that bradyzoites lack a
functional TCA cycle and respiratory chain, suggesting a
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W.J. Sullivan Jr & V. Jeffers

predominant role for anaerobic glycolysis during this


stage (Denton et al., 1996). In contrast, tachyzoites likely
use both mitochondrial oxidative phosphorylation and
glycolysis to generate ATP. Pyruvate kinase and lactate
dehydrogenase activities are markedly higher in bradyzoites, suggesting that lactate production is particularly
important during latency (Denton et al., 1996). A bradyzoite-specific isoform of lactate dehydrogenase (LDH2)
has been identified which is likely to be specialized for
the latent stage (Yang & Parmley, 1997). LDH2 is resistant to acidic pH and would continue to function during
the acidification that would occur during the catabolism
of amylopectin to lactate in bradyzoites. Knockdown of
LDH2 resulted in parasites that were unable to establish
significant cyst burdens in the brains of infected mice
(Al-Anouti et al., 2004).
Two other bradyzoite-specific isoforms of key glycolytic
enzymes have been identified: glucose-6-phosphate isomerase (G6-PI) and enolase-1 (ENO1) (Dzierszinski et al.,
1999). The enolase isoforms display distinct enzymatic
properties and differ in their stability. The tachyzoitespecific enolase (ENO2) exhibits a similar Km value for the
2-phosphoglycerate substrate but has a threefold higher
specific activity at Vmax compared to ENO1 (Dzierszinski
et al., 2001). Considered together, these studies are
consistent with the idea that stage-specific enzymes exist
which are designed to tune glycolysis to accommodate
proliferation or dormancy. Alternatively, or in addition to
tailoring glycolysis, the enolases may play a role in transcriptional regulation. ENO1 and ENO2 have been found
not only in the cytoplasm but also in the parasite nucleus
of bradyzoites and tachyzoites (Ferguson et al., 2002). An
alternatively translated form of human ENO1 also localizes
to the nucleus and can repress transcription by binding to
the c-myc promoter (Feo et al., 2000).
A number of enzymes with roles in the metabolism of
oxygen radicals appear to be upregulated in bradyzoites,
suggesting that the encysted forms are equipped to deal
with long-term exposure to reactive metabolites (Manger
et al., 1998). Consistent with this idea, it has been
reported that mRNAs encoding various DNA repair
enzymes increase in bradyzoites (Manger et al., 1998;
Yahiaoui et al., 1999).

Triggers of bradyzoite formation


Strain differences

Three predominating strains of Toxoplasma (designated


type I, II, and III) have been documented in North
America and Europe which differ in growth rate, virulence in mice, and ability to form cysts (Howe & Sibley,
1995). Genetic polymorphism analyses indicate that these
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clonal lineages emerged after a single genetic cross, and


their swift expansion resulted from the acquisition of
direct oral infectivity (Su et al., 2003). It is estimated that
this genetic cross occurred c. 10 000 years ago, coincident
with the establishment of agriculture and domestication
of animals such as the cat in the Fertile Crescent. Oral
infectivity is a critical factor linked with the ability of
some strains to efficiently differentiate into bradyzoites
(Fux et al., 2007).
Type I strains grow rapidly and are hypervirulent in
mice (LD50 c. 1 parasite). The commonly used type I
laboratory strain is designated RH, after the initials of the
boy from which it was isolated in 1939 (Sabin, 1941). RH
strain is generally considered to have lost the ability to
develop into mature cysts at high frequency, perhaps as a
consequence of prolonged propagation in vitro. To what
degree RH strain has lost this developmental competency
is a matter of debate and seems to vary between individual laboratory strains. During stress, type I strains including RH will activate gene expression of some bradyzoite
marker genes, and in some cases, bradyzoite surface antigens and cyst wall proteins have been detected (Soete
et al., 1994; Bohne & Roos, 1997; Lescault et al., 2010).
Isolated reports claim RH parasites can form cysts in
mice, via treatment with atovaquone and pyrrolidine
dithiocarbamate (Djurkovic-Djakovic et al., 2005) or
prior vaccination with soluble tachyzoite antigen and
IL-12, although the bradyzoites formed by RH differ
ultrastructurally and in their sensitivity to pepsinHCl
(Yap et al., 1998). In addition, RH strain parasites attenuated in virulence owing to disruption of dense granule
gene GRA2 are capable of establishing chronic infection
in mice (Mercier et al., 1998). Other type I strains that
have not been subject to extensive in vitro culture, such
as GT1, are able to form normal cyst walls in vitro during
stress (Khan et al., 2009). Interestingly, it has been suggested that the phenotypic differences between type I
strains are unlikely to be owing to sequence variation
(Khan et al., 2009). Indeed, both epigenetic and translational control mechanisms have been linked with stage
conversion.
Type II (e.g. Prugniuad or Pru, ME49) and type III
strains (e.g. VEG) have lower replication rates and readily
form cysts in vitro and in vivo; consequently, they are
hypovirulent in mice (LD50 c. 104). The most commonly isolated strain from clinical toxoplasmosis is type
II (Howe et al., 1997), although type I strains are typically responsible for acute outbreaks. The genomes of
representatives from all three lineages have been
sequenced (data are available at ToxoDB.org), but which
genes account for the differences reported in these strains
have only begun to be resolved.

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In terms of studying bradyzoite differentiation, strain


choice is a crucial factor. While type I strains, particularly
RH, grow faster and are easier to genetically manipulate,
they are limiting in the study of cyst formation. However,
several groups have made important discoveries regarding
bradyzoite development using type I RH strain. There are
also more transgenic parasites made in the RH background that facilitate gene tagging/disruption, or conditional gene expression (Meissner et al., 2007; Fox et al.,
2009; Huynh & Carruthers, 2009). Type II and III strains
form mature cysts readily but grow slowly and are more
difficult to manipulate. Consequently, there are presently
fewer tools available to study parasite physiology in the
hypovirulent strains.
In vitro stresses induce stage conversion

It is well documented that conversion to the latent


stage is a stress-mediated response, coupled with a
slowing of the parasite cell cycle. One of the most
commonly used in vitro methods to prompt bradyzoite
differentiation is alkaline pH 8.08.2 (Soete et al.,
1994). A wide variety of other stress agents have since
been reported, including sodium nitroprusside, which
acts as a source of exogenous nitric oxide (NO) and
also inhibits proteins involved in the parasite mitochondrial respiratory chain (Bohne et al., 1994). Similarly,
drugs that interfere with the parasite mitochondria also
induce differentiation to bradyzoites (Bohne et al., 1994;
Tomavo & Boothroyd, 1995). Heat shock and treatment
with sodium arsenite also trigger the expression of
bradyzoite antigens (Soete et al., 1994). Nutrient deprivation is a potent inducer of bradyzoite formation and
can be achieved through arginine starvation (Fox et al.,
2004), axenic incubation (Yahiaoui et al., 1999), or
pyrimidine depletion in uracil phosphoribosyltransferase
(UPRT)-deficient parasites subjected to ambient (0.03%)
CO2 (Bohne & Roos, 1997; Dzierszinski et al., 2004).
More recently, insults that cause endoplasmic reticulum
(ER) stress or that interfere with calcium-induced egress
have also been found to induce bradyzoite cyst formation (Nagamune et al., 2008; Narasimhan et al., 2008).
Treatment with interferon gamma (IFN-c) does not
induce conversion to bradyzoites cultured in human
fibroblasts (Soete et al., 1994) but will do so in murine
macrophages, presumably owing to the stimulated
releases of NO (Bohne et al., 1993). Long-term culturing of cysts was accomplished in vitro using murine
astrocytes and intermittent inclusion of IFN-c in the
culture media (Jones et al., 1986). Methods used to
convert tachyzoites to bradyzoites in vitro are summarized in Box 1.

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W.J. Sullivan Jr & V. Jeffers

Box 1. Induction of bradyzoites in vitro

Acidic or alkaline pH
Sodium nitroprusside
Atovaquone
Heat shock (43 C)
Sodium arsenite
Arginine starvation
Axenic incubation
Tunicamycin
Pyrimidine deprivation (UPRT parasites, 0.03% CO )
Fluridone (disruption of ABA-mediated calcium signaling)
2

Whether these stress treatments act on the parasite


directly (while they are extracellular), and/or if they act
indirectly on intracellular parasites by stressing the host
cell, is unclear. Weiss et al. (1998) have shown that when
extracellular parasites are exposed to alkaline stress for
1 h and allowed to reinfect host cells, bradyzoite differentiation is observed, albeit at a lower frequency than
stressed intracellular parasites. Additionally, extracellular
tachyzoites deprived of host cells for 12 h converted to
bradyzoites upon reinfection of host cells (Yahiaoui et al.,
1999). These studies suggest that extracellular parasites
not only sense their environment but are marked in some
way to initiate the bradyzoite development program upon
reentry into a host cell. Epigenetic-mediated gene regulation offers a mechanism by which this form of short
term memory of the parasites environment could occur.
Conversion to bradyzoite cysts may be driven by physiological factors other than exogenous stresses. Tachyzoites
can spontaneously convert into bradyzoites, and low
MOIs and/or frequent removal of egressed tachyzoites
from cultures of infected cells enriches for cysts (McHugh
et al., 1993). The proclivity toward spontaneous differentiation is influenced by the type of parasite strain and host
cell background (Ferreira da Silva et al., 2008), and cysts
are more frequently detected in differentiated host cells
that are long-lived (Dubey et al., 1998). Further evidence
that the host cell environment is a determinant in parasite
differentiation comes from studies of a trisubstituted pyrrole known as Compound 1 (Donald et al., 2002). Compound 1 was shown to act directly on human host cells to
slow tachyzoite replication and induce bradyzoite-specific
gene expression in type II and III strain parasites (Radke
et al., 2006). In these studies, human cell division autoantigen-1 (CDA1) was associated with promoting parasite
differentiation, as tachyzoites infecting host cells overexpressing CDA1 underwent bradyzoite conversion (Radke
et al., 2006). Prestressing human foreskin fibroblast host
cells prior to infection can also stimulate bradyzoite formation (Radke et al., 2006). Collectively, these studies
argue that tachyzoites are capable of assessing not only the
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type of host cell they invade, but also whether those host
cells are under strain. These studies also suggest that the
trigger(s) to differentiate are complex and multifactorial,
consisting of both endogenous and exogenous factors.

In vivo factors relevant to the


maintenance of bradyzoite cysts
The primary host response controlling Toxoplasma infection is mediated by CD8+ T cells with synergistic action
from CD4+ T cells (Gazzinelli et al., 1991, 1992). While
CD8+ T cells have been shown to be cytotoxic to Toxoplasma-infected peritoneal macrophages (Kasper et al.,
1992), their production of IFN-c likely has a greater impact
on controlling infection. IFN-c is well documented as a key
cytokine in the host immune response to Toxoplasma infection that limits intracellular replication of the parasite.
Infected mice that are immunodepleted of IFN-c succumb
to toxoplasmosis instead of developing chronic infection
(Suzuki et al., 1988). Similarly, administration of IFN-c
protects against lethal infection (McCabe et al., 1984).
With regard to the mechanism of IFN-c, it has been
reported that in human brain microvascular endothelial
cells, IFN-c induces the tryptophan-degrading enzyme
indoleamine 2,3-dioxygenase (Daubener et al., 2001),
which in turn would starve parasites of tryptophan (Pfefferkorn et al., 1986). Starvation for key amino acids like
tryptophan is likely to slow parasite growth and trigger cyst
formation. Further support for this idea comes from studies demonstrating that arginine starvation induces bradyzoite differentiation (Fox et al., 2004). IFN-c also induces an
oxidative burst and the production of NO, which may act
as a direct trigger for bradyzoite differentiation (Bohne
et al., 1994). IFN-c-inducible immunity-related GTPases
(IRG proteins/p47 GTPases), such as IGTP and LRG-47,
have also been linked to reduced parasite viability in activated macrophages (Butcher et al., 2005). Most recently,
the IRG protein Irga6 (IIGP1), which participates in the
disruption of the PVM, is phosphorylated and inactivated
by the type I ROP18 kinase as an immune evasion strategy
that promotes virulence (Fentress et al., 2010; Steinfeldt
et al., 2010). In short, numerous mechanisms exist which
account for how IFN-c controls tachyzoite proliferation
in vivo, but whether IFN-c acts directly on parasites to
induce or maintain bradyzoites in cyst forms is less clear.
Other cytokines have been linked to promoting bradyzoite differentiation. In murine macrophages, tumor
necrosis factor (TNF) a synergizes with IFN-c to facilitate
conversion to bradyzoites (Bohne et al., 1994). Another
study reported that the proinflammatory interleukin IL-6
favors the formation of bradyzoites in vitro in human
fibroblasts (Weiss et al., 1995). Given that heat shock is a
potent in vitro trigger for bradyzoite differentiation, it is
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conceivable that fever could contribute to stage conversion during acute toxoplasmosis in vivo.
As discussed in the preceding section, host cell background appears to be a key factor influencing bradyzoite
formation. Bradyzoite cysts have a clear predilection to
form in brain (specifically in neurons, astrocytes, and microglia) and muscle tissue rather than lungs, liver, spleen,
or kidneys (Dubey et al., 1998; Luder et al., 1999). Cysts
have been found in several regions of the brain, including
the cerebral cortex, superior and inferior colliculus, cerebellum, olfactory bulbs, and medulla oblongata, and have
also been found in the spinal cord (Di Cristina et al.,
2008). It remains to be determined whether there is something special about the intracellular environments of these
host cells that favor spontaneous conversion to latent cysts
in addition to being immunologically privileged sites.
The pathogenesis of Toxoplasma infection also relies in
part on host genetics. Studies in humans and mice have
revealed varying abilities to control infection and cyst burden (Brown et al., 1995; Suzuki et al., 1996; Mack et al.,
1999; Suzuki, 2002). For example, BALB/c mice are genetically resistant to Toxoplasma infection, harboring fewer
brain cysts compared to other infected mouse strains. It
was recently determined that BALB/c CD8+ T cells can
eliminate brain cysts through their perforin-mediated
activity (Suzuki et al., 2010). This study provides evidence
that certain cytotoxic T cells harbor a capacity to recognize and destroy intracellular brain cysts, which have been
previously believed to be impervious to host immunity.

sone has also been used to reactivate toxoplasmosis in


chronically infected mice (Nicoll et al., 1997; DjurkovicDjakovic & Milenkovic, 2001). It is not currently known
whether host immunity directly prevents bradyzoites from
reactivating and/or functions to quickly kill or trigger the
differentiation of reactivated tachyzoites escaping cysts.

Role of stress signaling in bradyzoite


development and maintenance
As described earlier, virtually all types of stress can induce
cyst development in vitro. Common denominators
between the stresses that induce conversion to bradyzoites
include the slowing of parasite proliferation, the induction of heat shock proteins, and translational control
mediated by eIF2a phosphorylation (Fig. 5).

Reactivation of acute infection


In vitro generated bradyzoites will quickly revert to proliferative tachyzoites upon removal of the stress agent used
to differentiate the parasites. These studies support the
idea that cellular stress is a key factor not only in
prompting the development of bradyzoites, but also in
maintaining the encysted form. In immunocompromised
patients, reactivation of Toxoplasma infection is typically
seen after the CD4+ T-cell count drops below 100
200 cells per mm3 (Pereira-Chioccola et al., 2009)
(Fig. 2). Ferguson et al. have reported that on rare occasion, bradyzoite cysts will rupture in immunocompetent
mice, leading to rapid recruitment of inflammatory cells
to the site (Ferguson et al., 1989). In the absence of a
normal immune response to induce the newly released
tachyzoites to convert into bradyzoites, the tachyzoites
will continue to replicate and disseminate throughout the
host, leading to serious complications and possibly death.
Mouse models for reactivated toxoplasmosis have been
developed which further underscore the relevance of
IFN-c in controlling latency (Suzuki & Joh, 1994; Dunay
et al., 2009). The steroid immunosuppressant dexamethaFEMS Microbiol Rev 36 (2012) 717733

Fig. 5. Stage differentiation of Toxoplasma. Replicating tachyzoites


convert to latent bradyzoites in response to a wide variety of stresses
(see text and Box 1), and bradyzoites can reconvert into proliferating
tachyzoites if stress is removed or immunity is impaired. The transition
from tachyzoite to bradyzoite is associated with morphological
changes that include narrowing of the parasite body, movement of
the nucleus toward the parasite posterior, an increase in the number
of micronemes (blue) and amylopectin granules (white), and more
electron-dense rhoptries (maroon). One stress response pathway
linked to stage conversion involves the phosphorylation of the alpha
subunit of eukaryotic initiation factor-2 (TgIF2a), which is mediated by
stress-activated TgIF2a kinases (TgIF2Ks). Phosphorylated TgIF2a
represses global translation, allowing preferential translation of a
subset of mRNAs that encode stress-responsive factors. Bradyzoites
maintain high levels of phosphorylated TgIF2a relative to proliferating
tachyzoites. In addition to translational control, transcription and
epigenetic factors have also been implicated in stage conversion.
Histone acetylation in particular has been correlated with stagespecific gene expression: in tachyzoites, promoters of tachyzoitespecific genes (TZ) contain acetylated nucleosomes, while promoters
of bradyzoite-specific genes (BZ) do not (active genes are denoted in
green, repressed genes in red). Histone acetylation contributes to the
restructuring of chromatin to create a local environment more
conducive to gene expression.

2011 Federation of European Microbiological Societies


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724

Bradyzoite development and the parasite cell


cycle

Cell cycle blocks do not trigger bradyzoite differentiation,


demonstrating that cell cycle progression is required for
cyst formation (Gubbels et al., 2008). Virtually all of the
stress conditions that promote bradyzoite differentiation
mentioned above reduce the proliferation of tachyzoites.
Slowing of the parasite cell cycle has been linked to the
initiation of the bradyzoite developmental program from
a late-S/G2 subpopulation containing 1.82N DNA content (Jerome et al., 1998; Radke et al., 2003). During
bradyzoite differentiation, these parasites proceed through
M phase and then arrest in G1/G0 with uniform 1N DNA
content. The unique late-S/G2 stage represents a premitotic cell cycle checkpoint for the commitment to bradyzoite
formation and growth arrest following mitosis. The identification of cyclic expression of several bradyzoite-specific
mRNAs, which exhibit peak expression in the late mitotic
period, lends support to this model (Behnke et al., 2010).
Curiously, one of the mRNAs with peak expression in
tachyzoite cytokinesis is also significantly increased during
bradyzoite differentiation; this mRNA encodes AP2VIIa-1,
a likely transcription factor. It is enticing to speculate that
AP2VIIa-1 is a master regulator that coordinates changes
in the transcriptome that lead to bradyzoite conversion.
The nature of the signaling mechanisms that accompany
slowed growth and ultimately lead to bradyzoite differentiation has yet to be fully elucidated; the following sections
detail what has been determined to date.
Heat shock proteins and signaling pathways

One of the earliest described bradyzoite-specific genes is


Hsp30/BAG1, related to the small heat shock proteins
found in plants (Bohne et al., 1995; Parmley et al., 1995).
BAG1 appears c. 23 days after bradyzoite induction, but
its relevance in differentiation was clouded when disruption of the gene in a type II strain failed to block tissue
cyst formation (Bohne et al., 1998). A similar study by
Zhang et al. (1999) confirmed that BAG1 knockouts still
form cysts in mice but do so at a significantly lower frequency. Weiss et al. (1998) have demonstrated that
Hsp70 is induced during alkaline-mediated bradyzoite
differentiation, and that an inhibitor of Hsp90, Hsp70,
and Hsp27 synthesis can suppress bradyzoite development
in vitro. Another study also implicated a role of Hsp70
during the reactivation of chronic toxoplasmosis in vivo
(Silva et al., 1998). Hsp90, in complex with p23 co-chaperone, has also been implicated in bradyzoite development (Echeverria et al., 2010) and exhibits different
localization patterns, being found in both the nucleus and
cytoplasm in bradyzoites rather than just the cytoplasm
2011 Federation of European Microbiological Societies
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W.J. Sullivan Jr & V. Jeffers

(Echeverria et al., 2005). Consistent with these data, serial


analysis of gene expression (SAGE) libraries indicate that
Hsp90 is detected early in bradyzoite differentiation (Radke et al., 2005). Intriguingly, geldanamycin, an antibiotic
that perturbs the normal function of Hsp90, blocks the
conversion of tachyzoites to bradyzoites as well as the
reversion of bradyzoites to tachyzoites (Echeverria et al.,
2005). The mitochondrial chaperone Hsp60 exists as two
alternatively spliced transcripts, both of which are upregulated in bradyzoites (Toursel et al., 2000). Upon bradyzoite induction, TgHsp60 localizes to two unknown
vesicular bodies distinct from the parasite mitochondrion.
Another heat shock protein, DnaK-tetratricopeptide
repeat (DnaK-TPR), which also interacts with p23, was
recently found to be expressed predominantly in bradyzoites (Ueno et al., 2011). Three Hsp40/DnaJ family
members were also found to be upregulated in alkalinestressed tachyzoites: TGME49_115690, TGME49_010430,
and TGME49_023950 (S. O. Angel and W. J. Sullivan Jr.,
unpublished data).
Other conventional stress response signaling pathways
well characterized in higher eukaryotes may also function
in Toxoplasma and possibly contribute to bradyzoite
development. Homologues of mitogen-activated protein
kinases (MAPKs), which regulate diverse biological processes including cellular stress responses, have been identified in Toxoplasma (Lacey et al., 2007). During in vitro
differentiation to bradyzoites, mRNA levels for TgMAPK1 increase, suggesting a possible role in stage conversion
(Brumlik et al., 2004). Increasing levels of cGMP and
cAMP, as well as inhibiting downstream cGMP- and
cAMP-dependent kinases, have also been linked to bradyzoite differentiation (Kirkman et al., 2001; Eaton et al.,
2006). The precise roles of Toxoplasma HSPs and signaling pathways during stage conversion warrant more
detailed investigation.
It was recently discovered that stress conditions also
induce the synthesis of the phytohormone abscisic acid
(ABA) by the apicoplast (Nagamune et al., 2008). ABA
leads to the production of cyclic ADP ribose (cADPR),
which controls the release of intracellular calcium stores
in Toxoplasma and induces egress. Pharmacological inhibition of ABA synthesis with fluridone blocked egress and
triggered bradyzoite differentiation, suggesting that ABAmediated calcium signaling is an important factor in
whether the parasite is lytic or latent (Nagamune et al.,
2008).
Translational control

Translational repression appears to operate in both tachyzoites and bradyzoites. Appreciable mRNAs encoding
the bradyzoite-specific proteins G6-PI and MAG1 are
FEMS Microbiol Rev 36 (2012) 717733

725

Toxoplasma bradyzoite latency

readily detected in tachyzoites as well, suggesting that


such messages are translationally repressed during the
tachyzoite stage (Dzierszinski et al., 1999; Weiss & Kim,
2000). Similarly, LDH1 protein is only expressed in
tachyzoites, yet LDH1 mRNAs are equally detectable in
both stages, suggesting that LDH1 is translationally
repressed in bradyzoites (Yang & Parmley, 1997). The
Toxoplasma eukaryotic translation initiation factor 4A
(eIF4A), which facilitates the binding of capped mRNA to
the 40S ribosomal subunit, is downregulated in bradyzoites, possibly reflective of the global reduction in protein
synthesis during latency (Gastens & Fischer, 2002).
Recent studies have expanded and clarified the role of
translational control in the parasite stress response and
differentiation. Cellular stresses are well-known inducers
of protein translation control through phosphorylation of
the alpha subunit of eukaryotic translation initiation factor-2 (eIF2a). When phosphorylated, eIF2a dampens global translation initiation, thereby favoring the preferential
translation of a subset of mRNAs that encode proteins
geared toward alleviating the stress (Wek et al., 2006). As
in other species, Toxoplasma phosphorylates its eIF2a
orthologue (TgIF2a) in response to numerous stresses,
including those that trigger bradyzoite differentiation
(Sullivan et al., 2004). Alkaline pH, heat shock, sodium
nitroprusside, sodium arsenite, and tunicamycin have all
been shown to cause phosphorylation of TgIF2a (Sullivan
et al., 2004; Narasimhan et al., 2008). Moreover, TgIF2a
remains phosphorylated in mature bradyzoites generated
in vitro (Narasimhan et al., 2008). We have also found
that salubrinal, a specific inhibitor of eIF2a dephosphorylation (Boyce et al., 2005), induces bradyzoite development in vitro (Narasimhan et al., 2008). Collectively,
these studies suggest a major role of TgIF2a phosphorylation (TgIF2a~P) in the development and maintenance of
bradyzoite cysts. This idea has been supported in studies
performed in the related apicomplexan parasite Plasmodium. Zhang et al. demonstrated that a Plasmodium eIF2
kinase designated IK2 controls the latency of sporozoites
in the mosquito salivary gland (Zhang et al., 2010).
TgIF2a~P may also facilitate Toxoplasma dissemination
to immune-privileged sites where bradyzoites tend to
form. A greater capacity to survive outside host cells may
help explain the increased ability of type I strains to disseminate throughout the host organism, particularly to
the CNS. Two prevailing models describing how tachyzoites traverse biological barriers include the Trojan horse
mechanism and paracellular transmigration (Elsheikha &
Khan, 2010). Type I parasites are not as efficient as type
II and III strains in inducing migratory phenotypes in
infected dendritic cells (Lambert et al., 2009), suggesting
that type I strains may rely more heavily on dissemination as extracellular parasites (Barragan & Sibley, 2002).
FEMS Microbiol Rev 36 (2012) 717733

Being ill-equipped to respond robustly to the stress of an


extracellular environment, type II and III strains must
remain confined to the intracellular environment, a limitation that is likely to contribute to their decreased virulence. Through the generation of a nonphosphorylatable
mutant, we have found that TgIF2a~P promotes survival
of extracellular tachyzoites and does so in a strain-dependent manner (Joyce et al., 2010). Type I RH strain shows
rapid and robust TgIF2a~P within hours after egress,
whereas the type II strain is slower to phosphorylate
TgIF2a (Joyce et al., 2010). It is tempting to speculate
that the translational control induced by TgIF2a reprograms gene expression to protect extracellular parasites,
and the increased viability of RH strain may stem from
its increased capacity to phosphorylate TgIF2a for cytoprotection.
The relevance of eIF2a~P in Toxoplasma and Plasmodium latent forms underscores the need to characterize the
parasite family of eIF2a kinases. Higher eukaryotes possess
four eIF2a kinases, each appearing to respond to specific
stress arrangements: GCN2 is activated by nutrient deprivation, protein kinase RNA-activated (PKR) is activated by
viral infection, PEK/Perk is activated by ER stress, and HRI
is activated by heme deficiency and heat shock (Wek et al.,
2006). Toxoplasma also possesses four eIF2a kinases
(TgIF2K-A through D), some of which contain motifs
suggestive of functional equivalency to mammalian
enzymes. For example, based on the presence of a transmembrane domain, we hypothesized that TgIF2K-A may
be equivalent to PEK/Perk. Indeed, TgIF2K-A localizes to
the parasite ER and associates with the ER-resident chaperone BiP/GRP in a stress-dependent fashion (Narasimhan
et al., 2008). TgIF2K-D is most similar to GCN2, possessing a histidyl-tRNA synthetase (HisRS)-related domain
that stimulates kinase activity by binding to uncharged
tRNAs that accumulate during nutrient starvation. We
have recently demonstrated that TgIF2K-D enhances the
survival of extracellular tachyzoites deprived of host cell
resources (Sullivan, unpublished data). The roles of the
other parasite eIF2 kinases, and how they become activated
during different stress conditions, are important areas for
future study.

Reprogramming the genome for


bradyzoite development
In other species, stress-induced eIF2a~P reduces global
protein synthesis in favor of a subset of mRNAs encoding
factors needed to respond to the cellular insult or signal.
Master regulator transcription factors such as GCN4/
ATF4 are preferentially translated during stress, which
subsequently reprogram the genome for stress remedy.
Using polyribosome profiling and [35S]-Met/Cys labeling,
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726

we have verified that TgIF2a~P significantly curtails protein production (Narasimhan et al., 2008). These data
align with an earlier study showing that eIF4A, a DEADbox RNA helicase that facilitates binding of capped
mRNA to the 40S ribosomal subunit, is strongly downregulated in bradyzoites (Gastens & Fischer, 2002).
Recently, we have used Toxoplasma microarrays to identify mRNAs that are preferentially translated in the polyribosome fraction during ER stress (Sullivan, unpublished
data). Among these genes were several so-called AP2 proteins, which contain plant-like DNA-binding domains
and represent a major lineage of transcription factors in
Apicomplexa (Painter et al., 2011). In the absence of
GCN4/ATF4 homologues in Apicomplexa, it is plausible
to speculate that AP2 proteins may be functional equivalents of these well-conserved master regulators.
Transcriptional regulation clearly plays a major role in
bradyzoite development as evidenced by numerous studies showing stage-specific gene expression (Manger et al.,
1998; Cleary et al., 2002; Radke et al., 2005; Sullivan
et al., 2009). A seminal study generated multiple SAGE
libraries to construct progressive snapshots of the developmental transition from sporozoite to tachyzoite to
bradyzoite in different Toxoplasma strains (Radke et al.,
2005). Results from this study showed that distinct gene
expression cascades occur through developmental transitions, underscoring the importance of transcriptional regulation throughout these events. Promoters that regulate
BAG1 and a bradyzoite-specific NTPase during bradyzoite
development were fine-mapped to a 6- to 8-bp resolution, and these minimal cis-elements were capable of converting a constitutive promoter to one that is induced by
bradyzoite conditions (Behnke et al., 2008). Together,
these studies reveal that conventional eukaryotic promoter mechanisms are fundamentally at work to coordinate gene expression driving stage differentiation, but the
usage of AP2 proteins as transcriptional regulators is different than higher eukaryotic counterparts. Details on the
roles of AP2 factors, 11 of which are induced in bradyzoites, are emerging in ongoing studies.
Recently, the bradyzoite-specific promoter ENO1 was
used as bait in an affinity-based strategy to isolate DNAassociating factors that may repress this gene in tachyzoites. Thirty-nine putative nuclear factors were found and
divided into three categories: (i) 11 proteins with significant similarity to known nuclear factors, including a protein with a RNA-specific DEAD/DEAH box helicase
domain, a pinin domain, and an FK506BP homologue,
(ii) 7 proteins corresponding to kinases, phosphatases,
and HSPs, and (iii) 21 hypothetical proteins, two of which
have significant homology to Alba proteins, which are
chromatin-associated silencing factors best characterized
in Archaea. The FK506BP homologue (TgNF3) was exten 2011 Federation of European Microbiological Societies
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W.J. Sullivan Jr & V. Jeffers

sively characterized. While not a direct binder of DNA,


TgNF3 physically interacts with free core and nucleosomeassociated histones to exert a gene silencing function at
ENO1 and 18S ribosomal RNA genes, consistent with the
yeast homologue known to be a histone chaperone that
regulates rDNA silencing. Interestingly, TgNF3 is in the
nucleus (enriched in the nucleolus) of tachyzoites but in
the cytoplasm of bradyzoites (Olguin-Lamas et al., 2011),
suggesting that differential compartmentalization may
influence activation of the bradyzoite ENO1 gene.
Another important factor implicated in contributing to
bradyzoite gene expression is histone modification and
chromatin remodeling (Bougdour et al., 2010; Dixon
et al., 2010). The first indication that histone modifications play a role in Toxoplasma differentiation emerged
when it was reported that histones at promoter regions of
bradyzoite genes are hypoacetylated in tachyzoites and
become acetylated after bradyzoite induction. Conversely,
tachyzoite-specific genes that are hyperacetylated in the
tachyzoite stage display decreased acetylation levels upon
induction of differentiation (Saksouk et al., 2005). The
lysine (K) acetyltransferase (KAT) TgGCN5-A was localized to promoters of active developmentally regulated
genes, while the lysine deacetylase TgHDAC3 was situated
at the promoters of inactive genes (Saksouk et al., 2005).
Further study of a TgGCN5-A knockout parasite has
revealed a vital role for this KAT in the expression of bradyzoite-specific genes during differentiation. Although
TgGCN5-A appears dispensable in type I tachyzoites during normal culture conditions, the TgGCN5-A knockout
parasites fail to upregulate bradyzoite marker genes upon
induction of differentiation by alkaline stress (Naguleswaran et al., 2010). The importance of histone acetylation for
the control of differentiation is underscored by the finding
that chemical inhibition of TgHDAC3 with low doses of
the compound FR235222 caused conversion to bradyzoites. The conversion was accompanied by hyperacetylation
of the upstream regions of > 350 genes, one-third of which
are specific to bradyzoites (Bougdour et al., 2009).
While acetylation of histone lysine residues leads to
gene activation, methylation of histone residues can result
in either gene activation or repression. Monomethylation
of lysine 20 of histone H4 (H4K20) by the methyltransferase TgSET8 promotes heterochromatin formation and
subsequent gene silencing (Sautel et al., 2007). Concurrent with the global downregulation of gene expression in
the quiescent bradyzoite, monomethylation of H4K20 is
significantly enriched (Sautel et al., 2007), suggesting that
TgSET8 is involved in the maintenance of this transcriptionally suppressed state. In contrast, arginine methylation of histones has been linked to gene activation.
Chemical inhibition of the arginine methyltransferase
TgCARM1 triggers bradyzoite differentiation (Saksouk
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Toxoplasma bradyzoite latency

et al., 2005). Consistent with this observation, TgCARM1


associates with the promoter regions of tachyzoite-specific
genes in tachyzoites but is enriched at bradyzoite-specific
genes upon the induction of differentiation with alkaline
pH (Saksouk et al., 2005).
The Toxoplasma genome contains 17 predicted members of the SWI/SNF family of ATP-dependent nucleosome remodeling complexes (Dixon et al., 2010), a
number of which may be involved in stage conversion.
An EST analysis of bradyzoites formed in vivo by Manger
et al. identified a SNF2-like protein (TGME49_073870)
that is upregulated during differentiation (Manger et al.,
1998). Message levels for another SNF2-like homologue,
TgSRCAP, were shown to be upregulated during in vitro
bradyzoite differentiation (Sullivan et al., 2003).
Finally, alterations in nucleosome composition are
another likely method of gene expression control in bradyzoite differentiation. Toxoplasma possesses novel H2A
and H2B histone variants, substitutions of which can
modulate gene expression (Sullivan et al., 2006). The
H2AZ and H2Bv variants are enriched at active genes;
however, the H2AX variant present at inactive genes is
upregulated in the bradyzoite stage (Dalmasso et al.,
2009). Toxoplasma expresses many more chromatin remodelers that have yet to be fully characterized which are
candidates contributing to the reprogramming of the genome during stage conversion (Dixon et al., 2010).

Bradyzoite mutants
The haploid nature of tachyzoite/bradyzoite stages facilitates the generation of gene knockouts and disruptions
for mutational analysis. The power of Toxoplasma as a
molecular genetics system has been exploited to determine genes involved in the stage conversion. A number
of genes have been linked to having a role in bradyzoite
differentiation using this approach, including the aforementioned BAG1 gene, the P-type H+-ATPase PMA1
(Holpert et al., 2006), and the bradyzoite surface antigen
SAG1-related sequence SRS9 (Kim et al., 2007). Recently,
knockouts of dense granule proteins GRA4 and GRA6
were shown to have dramatically reduced cyst burdens at
3 weeks postinfection in C57BL/6 mice, especially as a
dual knockout (Fox et al., 2011). A significant defect in
cyst burden was also reported at 5 weeks postinfection
for mutants lacking the entire 14-kb ROP4/7 locus (Fox
et al., 2011). It should be mentioned that mutations
resulting in decreased cyst numbers in vivo may not
directly correlate to effects on tachyzoite to bradyzoite
conversion, as it is difficult to rule out the effects on
tachyzoite viability and dissemination in vivo.
To discover novel genes involved in bradyzoite development, various groups have generated developmental
FEMS Microbiol Rev 36 (2012) 717733

727

mutants. Singh et al. (2002) developed a transgenic parasite clone in type II Pru strain that contained GFP fused
to the bradyzoite-specific promoter LDH2. After exposing
these parasites to a chemical mutagen, variants defective
in bradyzoite conversion (GFP negative) could be isolated
using FACS following culture in bradyzoite-inducing conditions. Microarray analysis of the mutants following various stresses revealed a hierarchy of gene activation,
supporting the idea that multiple induction conditions
lead to a common pathway that reprograms for bradyzoite gene expression. It was noted that a 14-3-3 homologue, PITSLRE kinase, and a vacuolar ATPase exhibited
decreased expression levels in most of the bradyzoite
differentiation mutants (Singh et al., 2002). A similar
approach was followed to develop insertional mutants
impaired in bradyzoite gene activation, leading to the discovery that a nucleolar CCHC zinc finger protein (ZFP1)
and a pseudouridine synthase homologue (PUS1) are
involved in bradyzoite differentiation (Vanchinathan
et al., 2005; Anderson et al., 2009).
Matrajt et al. (2002) applied insertional mutagenesis on
type I RH parasites lacking UPRT, which facilitates differentiation to bradyzoites following CO2 starvation. Using a
selectable marker driven by a bradyzoite-specific promoter,
insertional mutants were isolated which have deficiencies
in BAG1 expression and cyst wall formation. Like the
mutants generated in the Singh study, these mutants replicated well under bradyzoite differentiation conditions, and
microarray analysis revealed a gene expression profile
more aligned with that seen for tachyzoites. Among the
genes rescued and implicated as having a role in the differentiation process include a splicing factor, an oocyst wall
protein, and AP2XII-6 (Lescault et al., 2010). AP2XII-6 is
particularly intriguing as AP2 domain proteins are potential transcription factors. One mutant contained an insert
just upstream of a gene prediction for a DNA replication
factor, but the mapped disrupted locus may be a noncoding RNA (Matrajt, 2010). Moreover, state modeling was
used to capture hidden variation between parasite lines to
reveal additional genes likely to be involved in bradyzoite
differentiation, including transcription elongation factor
Spt5, DNA primase subunit, TBC domain protein, Homologous to the E6-AP carboxyl terminus (HECT) type
ubiquitin ligase, and six hypothetical genes of unknown
function (Lescault et al., 2010).
The Knoll laboratory has generated and screened over
8000 insertional mutants by immunofluorescence microscopy for defects in bradyzoite cyst formation. Nine mutants
were identified as defective in both cyst wall formation and
expression of BAG1. One of these mutants contained an
insertion in a gene encoding a serine/proline-rich proteophosphoglycan. This proteophosphoglycan is upregulated
in bradyzoites and enhances cyst wall component
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728

expression and assembly through an unknown mechanism


(Craver et al., 2010). Another insertional mutant indicates
that TgRSC8, which has homology to the catalytic component of the SWI/SNF and Remodel the Structure of Chromatin (RSC) complexes found in Saccharomyces cerevisiae,
is important for the upregulation of a subset of bradyzoite
genes during in vitro differentiation (Rooney et al., 2011).
The latter further highlights the importance of chromatin
remodeling during stage conversion.
How these gene products identified from the mutants
contribute to bradyzoite conversion awaits further investigation. It is important to note that in each of these studies, the bradyzoite-deficient mutants are leaky, suggesting
that control of bradyzoite differentiation is a complex
process with redundancies, and no single gene appears to
be able to completely ablate bradyzoite formation.

Summary and future outlook


A schematic highlighting recent key discoveries in the
mechanisms of stage conversion is presented in Fig. 5.
Despite our advances, there are still many fundamental
questions concerning bradyzoite differentiation to address,
such as the composition of the cyst wall and matrix, how
multiple signals are integrated into a common differentiation response, and which AP2 factors (or other transcription factors) operate as master regulators to coordinate
the bradyzoite gene expression program.
As the molecular toolbox for parasite investigation
expands, so will our understanding of bradyzoite differentiation. A major impediment to interrogating bradyzoite
development is the lack of tools developed specifically for
type II strains. The recent generation of Ku80 knockouts
in type II Pru strain will greatly facilitate the study of
genes linked to bradyzoite conversion (Fox et al., 2011).
Conditional knockout systems also need to be developed
in type II backgrounds for the study of essential genes.
Like EST and SAGE library predecessors, Toxoplasma
microarrays (ToxoGeneChiP) continue to be utilized to
resolve the bradyzoite transcriptome further, and ChIPchip approaches can be applied to elucidate the bradyzoite epigenome; high-throughput sequencing promises to
increase the resolution of these datasets even further.
Methods that need improvement include techniques to
purify tissue cysts from host cells, which would provide a
clearer picture of the bradyzoite proteome and metabolome. Another important advance is the application of bioluminescence imaging to study the course of Toxoplasma
infection, including the reactivation of chronic infection,
in real time in living mice (Saeij et al., 2005).
Future studies must also begin to focus on targeting
bradyzoite formation and/or eliminating tissue cysts. Is it
possible to develop avirulent, nonencysting Toxoplasma
2011 Federation of European Microbiological Societies
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W.J. Sullivan Jr & V. Jeffers

mutants for potential vaccines, particularly in livestock to


reduce zoonotic transmission? The study of cytokines relevant to cyst formation and stability, and the observation
that certain CD8+ T cells can directly target cysts (Suzuki
et al., 2010), opens the door for immunomodulation
approaches to eliminate cysts. More traditional pharmacological methods may still be useful in eradicating cysts if
the drugs are conjugated to arginine oligomers, which have
been shown to penetrate cyst walls (Samuel et al., 2003).
D-luciferin, used during bioluminescent imaging of infection, was also noted as being able to access and be metabolically processed by bradyzoites within tissue cysts (Di
Cristina et al., 2008). The latest discoveries illuminating
the mechanics of how the bradyzoite gene program unfolds
offer a rich repository of potential novel drug targets.
Numerous recent studies suggest that the persistence of
Toxoplasma cysts in the brain may have a significant
impact on the health of immunocompetent hosts. It is
now well documented that infected rats undergo an
exquisite change in behavior that would work to enhance
transmission of the parasite back to its definitive host
(Webster, 2007). Remarkably, rodents with latent Toxoplasma infection converted their aversion to feline odors
into attraction, suggesting that they would be more easily
preyed upon in the field (Vyas et al., 2007). Whether
bradyzoite cysts within human brains affect behavior is
less clear, and we are far from understanding the mechanisms (Fekadu et al., 2010; Webster & McConkey, 2010).
A great deal of progress has been made since Toxoplasma tissue cysts were first observed in 1928, but much
work remains to be completed to fully understand the
molecular mechanisms driving latency and the impact of
chronic infection on host behavior. The significance for
continued study of latency in Toxoplasma is underscored
by parallels between bradyzoites and other eukaryotic
pathogens with dormant stages, exemplified by the discovery that translational control through eIF2a phosphorylation is a critical determinant in latent malarial
sporozoites as well as Toxoplasma bradyzoites.

Acknowledgements
Research in the Sullivan laboratory is supported by grants
from the National Institutes of Health (R01 AI077502,
R21 AI084031, and R21 AI083732). We thank Drs Louis
Weiss (Albert Einstein College of Medicine) and David
Ferguson (University of Oxford) for supplying images.
Illustrations in Figures 1 and 5 were completed by Christopher M. Brown (Office of Visual Media, Indiana University School of Medicine). We also thank Drs Louis
Weiss and Sherry Queener (Indiana University School of
Medicine) for critically reading the manuscript and providing helpful suggestions.
FEMS Microbiol Rev 36 (2012) 717733

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