Toxoplasma gondii Development of Its Replicative Niche: in Its HostCell and Beyond
Ira J. Blader,a Anita A. Koshyb
Departments of Microbiology and Immunology and Ophthalmology, University at Buffalo, Buffalo, New York, USAa; Departments of Neurology and Immunobiology, BIO5Institute, University of Arizona, Tucson, Arizona, USAb
Intracellular pathogens can replicate efficiently only after they manipulate and modify their host cells to create an environmentconducive to replication. While diverse cellular pathways are targeted by different pathogens, metabolism, membrane and cyto-skeletal architecture formation, and cell death are the three primary cellular processes that are modified by infections. Toxo-plasma gondii is an obligate intracellular protozoan that infects �30% of the world’s population and causes severe and life-threatening disease in developing fetuses, in immune-comprised patients, and in certain otherwise healthy individuals who areprimarily found in South America. The high prevalence of Toxoplasma in humans is in large part a result of its ability to modu-late these three host cell processes. Here, we highlight recent work defining the mechanisms by which Toxoplasma interacts withthese processes. In addition, we hypothesize why some processes are modified not only in the infected host cell but also in neigh-boring uninfected cells.
Toxoplasma gondii is a protozoan, obligate intracellular parasitethat is considered one of the world’s most successful pathogens
(1). Multiple factors contribute to this success, including a com-plex life cycle in which the parasite can be transmitted by bothvertical and horizontal means, efficient propagation within bothits primary (felines) and intermediate hosts, extensive mecha-nisms to evade and disarm host immunity, an ability to formchronic lifelong infections in intermediate hosts, and a wide hosttropism in which the parasite can infect most nucleated cells ofwarm-blooded animals (2). Central to most of these factors is thatToxoplasma has developed the means to replicate efficientlywithin the hostile intracellular environment of its host cell. In thisreview, we highlight recent data that have shed light on how par-asite growth is achieved by the parasite interacting with its host cellto manipulate host signaling cascades, transcription, cell survivalpathways, and membrane transport. In addition, we discuss howparasites interact with neighboring host cells and propose howthis may contribute to establishing a permissive microenviron-ment to improve its overall success. In particular, we focus onthose processes that are essential for the growth of all parasitestrains and we refer readers to recent reviews that highlight howpolymorphic parasite molecules contribute to Toxoplasma viru-lence (3–5).
NUTRIENT ACQUISITION
As an obligate intracellular parasite that resides within a nonfuso-genic vacuole, Toxoplasma must satisfy its nutritional needs byscavenging essential nutrients from its host cell. These nutrientsinclude carbon sources (glucose and glutamine) to fuel its energydemands, specific amino acids, lipids, and other nutrients. Below,we discuss each of these and highlight pathways and processes thatare unique to the parasite that could serve as novel drug targets(Fig. 1).
GLUCOSE AND GLUTAMINE POWER THE PARASITE
Toxoplasma expresses a full complement of glycolytic and tricar-boxylic acid (TCA) enzymes, and both metabolic pathways areactive in tachyzoites (6). Toxoplasma glycolytic genes function
both in glycolysis and in other parasite processes such as parasitemotility (7–9). These data led several groups to conclude thatglucose was the primary carbon source that was scavenged byToxoplasma from its host cell. In turn, this conclusion led to ques-tions such as how was the parasite scavenging glucose, what im-pact did siphoning this nutrient have on the host cell’s physiology,and what was the function of the parasite’s TCA cycle in growth?
Toxoplasma expresses a hexose transporter (TgGT1) on itsplasma membrane that shows the highest affinity for glucose. De-letion of the TgGT1 gene results in a significant defect in glucoseuptake and a defect in parasite motility and replication (10). Therequirement for glucose in parasite motility is linked to the obser-vation that during motility, glycolytic enzymes relocalize to theinner membrane complex (a membranous structure that lies di-rectly adjacent to the plasma membrane and serves as an anchorfor the actomyosin machinery to propel the parasite into the hostcell), suggesting that glucose provides the energy needed for inva-sion (8, 9). Surprisingly, loss of TgGT1 had no impact on virulence(10), suggesting that Toxoplasma uses other carbon sources togenerate ATP.
Identification of this other carbon source came from the ob-servation that motility of the TgGT1 knockout parasites could berestored by the addition of glutamine to the media (10). Together,these data suggested that parasites could generate ATP througheither glycolysis or glutaminolysis. This hypothesis was confirmedby isotope labeling and metabolite profiling that showed that Tox-oplasma uses host-derived glucose and glutamine to generate ATPvia cytosolic glycolysis and mitochondrial oxidative phosphoryla-tion. It is unknown how acetyl-coenzyme A (acetyl-CoA) is gen-erated for the TCA cycle, since the parasite lacks a mitochondrial
pyruvate dehydrogenase complex. Rather, this complex is local-ized within the apicoplast, where it generates acetyl-CoA that isused by the fatty acid II biosynthetic pathway (11, 12). See Fig. 1for a current model of glucose and glutamine uptake and use bythe parasite.
Whether the parasite’s TCA cycle is essential for growth is un-clear. Fleige and coworkers reported that reduced expression ofthe TCA enzyme succinyl-CoA synthase did not impact parasitegrowth, whereas MacRae et al. reported that chemical inhibitionof aconitase did (11, 13). These seemingly contradictory data werereconciled by the discovery that Toxoplasma synthesizes �-ami-nobutyric acid (GABA) from glutamine and that this compoundcan be shunted into the TCA cycle as succinate via succinic-semi-aldehyde dehydrogenase and thus bypasses succinyl-CoA syn-thase (11). However, neither the GABA shunt nor, by extension,the TCA pathway appears to be essential since deletion of the geneencoding glutamate decarboxylase (the enzyme that converts glu-tamate to GABA) has a relatively minor impact on parasite growthand virulence (11).
LIPIDS: WE ALL NEED FATCholesterol. Sterols are essential components of eukaryotic mem-branes, and cholesterol is the major sterol in mammalian cells.While parasite membranes contain cholesterol, Toxoplasma lackscholesterol biosynthetic enzymes and must scavenge it from its
host (14). Serum-derived cholesterol is the primary source forcholesterol since parasite growth is not reduced in cell lines unableto synthesize cholesterol (14). Parasites scavenge cholesterol fromlow-density lipoprotein (LDL) particles and do so by redirectingLDL receptor trafficking to the parasitophorous vacuole (PV) (14,15). An unexpected player in this process is the P-glycoproteinhost multidrug-resistance efflux pump that appears to be requiredfor parasite uptake of cholesterol at some point after cholesteroldelivery to the PV (16). But how cholesterol crosses the PV mem-brane (PVM) and the parasite plasma membrane remains to bedetermined.
Isoprenoids. The mevalonate and deoxy-D-xylulose-5-phos-phate (DOXP) pathways produce isopentyl pyrophosphate,which is the precursor for the biosynthesis of longer isoprenoids.Only the DOXP pathway is expressed by the parasite, and deletionof several genes encoding DOXP enzymes is lethal (17). Toxo-plasma expresses a bifunctional farnesyl/geranyl diphosphate syn-thase (TgFPPS) (18) whose activities are required for isoprenoidsto be incorporated into cholesterol, dolichols, or isopentyls. De-letion of the TgFPPS gene results in parasites with growth defectsin specific types of host cells (e.g., the knockout can grow in hu-man foreskin fibroblasts but cannot grow in macrophages) (17,18). TgFPPS knockouts also cannot survive as extracellular para-sites for extended periods of time because of a mitochondrial de-
FIG 1 Glucose and glutamine utilization by Toxoplasma gondii. Glucose (Glc) is scavenged from the host cell by the GT1 glucose transporter and metabolizedby either the apicoplast pathway or the cytosolic pathway. Glutamine (Gln) is scavenged by an unknown transporter, where it is converted to glutamate that canbe utilized by the mitochondrial Krebs cycle as either �-ketoglutarate (�-KG) or succinate (via the GABA shunt).
fect that is likely due to a loss of ubiquinone, which is an isopre-nylated cofactor of the mitochondrial respiratory chain (18).
Why is the DOXP pathway essential while TgFPPS is not?DOXP isoprenoid biosynthesis occurs within the apicoplast, andpresumably its products are transported from the apicoplast to themitochondria where TgFPPS is localized. TgFPPS, on the otherhand, is not essential because the parasite can salvage longer iso-prenoids (e.g., farnsyl diphosphate and geranylgeranyl diphos-phate) from the host cell. This requirement for host isoprenoidsrenders the parasites highly susceptible to statins, which inhibitHMG (3-hydroxy-3-methyl-glutaryl)-CoA reductase that is spe-cifically expressed by the host but not by the parasite. Importantly,statin treatment increases murine resistance to Toxoplasma, indi-cating that simultaneous inhibition of host and parasite iso-prenoid biosynthesis processes may be a novel approach to treat-ing Toxoplasma infections. It is also possible that statins impactparasite growth in vivo by reducing cholesterol levels in the host,although in vitro data suggest that statins primarily act by inhib-iting host isoprenoid synthesis (19). The finding that TgFPPSknockout growth is severely restricted in macrophages suggeststhat isoprenoid scavenging may be restricted in these cells. Thefact that the TgFPPS knockout grows in human foreskin fibro-blasts suggests either that there are differences in the basal rates ofisoprenoid biosynthesis between fibroblasts and macrophages orthat restricting isoprenoid availability is a novel innate immunemechanism in macrophages.
SPHINGOLIPIDS
Sphingolipids are a diverse group of lipids that have importantfunctions in cell structure, membrane trafficking, and cell signal-ing. Toxoplasma, which contains at least 20 different sphingolipidspecies (primarily sphingomyelin and ceramide), expresses sph-ingolipid biosynthetic enzymes and thus can synthesize these lip-ids. Many parasite sphingolipids contain saturated and unsatu-rated long-chain fatty acid moieties (C:20 to C:24), making themstructurally distinct from host sphingolipids whose fatty acids areprimarily C:16 and C:18.
Addition of fluorescently labeled ceramide to uninfected hostcells normally stains the Golgi apparatus as well as punctate cyto-plasmic structures. Infected host cells are similarly stained earlyafter labeled ceramide addition, but with time, the ceramide accu-mulates in intracellular parasites (20, 21). These data suggestedthat Toxoplasma not only synthesizes its own sphingolipids butalso scavenges host-derived ones; subsequent radiolabeling assaysconfirmed this model (22). Since infection induces a redistribu-tion of the host Golgi apparatus to the PV, it was possible thatparasites scavenged host sphingolipids through this rerouting ofhost Golgi membrane trafficking. Rabs are a family of �40 low-molecular-weight GTPases whose primary function is to regulateintracellular membrane trafficking. Consistent with Toxoplasmainducing a redistribution of the host Golgi apparatus, infectionaltered the localization of 3 Rab GTPases (Rab14, Rab30, andRab43) established as regulators of Golgi assembly and dynamics.Significantly, expression of dominant-negative Rab14 and Rab43,but not Rab30, significantly reduced host-derived sphingolipidaccumulation in the parasite (20).
AMINO ACIDSTryptophan. Tryptophan is an essential amino acid that Toxo-plasma scavenges from the host. The first evidence for this require-
ment came from studies showing that gamma interferon (IFN-�),the key cytokine for limiting parasite replication, upregulates in-doleamine-dioxygenase (IDO), a gene that encodes the first andrate-limiting enzyme in tryptophan catabolism. In addition, par-asite growth cannot be controlled in IFN-�-treated cells lackingIDO, and the repressive effect of IDO on parasite growth can bereversed by the addition of excess tryptophan to the growth me-dium (23). These data not only highlighted one manner by whichIFN-� controls Toxoplasma growth in human cells but also sug-gested that Toxoplasma is a tryptophan auxotroph that scavengesthe amino acid from its host cytosol. Definitive proof that Toxo-plasma is a tryptophan auxotroph came from the ability to growparasites in tryptophan-limited medium when they express theEscherichia coli trpB gene, which encodes tryptophan synthase(24). Moreover, in silico metabolic pathway reconstruction indi-cates that the parasite lacks tryptophan biosynthetic enzymes(http://www.genome.jp/kegg-bin/show_pathway?org_name�tgo&mapno�00400&mapscale�&show_description�hide).
More recent work, however, has suggested that IDO is not auniversally utilized anti-Toxoplasma control mechanism in hu-man cells. Niedelman et al. reported that while IDO could restrictparasite growth in IFN-�-treated HeLa cells, IDO was not in-volved in killing Toxoplasma in the human fibroblasts that theyused (25). Since the work by Pfefferkorn discussed above on thefunction of IDO during Toxoplasma infections also used humanfibroblasts (23), a likely explanation for the discrepancy betweenthese studies is that genetic and/or epigenetic differences betweenthe different fibroblasts dictate the antiparasitic mechanism usedby IFN-� to kill Toxoplasma.
Arginine. Arginine plays a unique role in Toxoplasma growthand virulence. First, it is an essential amino acid that the parasitescavenges from its host and a decrease in its availability triggersbradyzoite development (26). Besides being an essential aminoacid, arginine must also be metabolized by the host cell to generatepolyamines (e.g., spermine, spermadine, etc. . . .) that are thentransported into the parasite (27, 28). Like tryptophan and otheramino acids that the parasite scavenges from its host, arginine andpolyamine transporters remain to be identified.
Arginase 1 is a host cell enzyme that catabolizes arginine toornithine and urea. Its expression in macrophages is the hallmarkof alternatively activated macrophages, whereas its lack of expres-sion and the macrophage’s exposure to IFN-� produce classicallyactivated macrophages (29). Arginase 1 expression is upregulatedby Toxoplasma type I and III strains due to their expression of theROP16 polymorphic serine/threonine kinase that is secreted intothe host cell, where it phosphorylates and activates the STAT3 andSTAT 6 (STAT3/6) transcription factors (29–35). In contrast, typeII parasites trigger the development of classically activated macro-phages by limiting STAT3/6 activity, presumably by expressing aless efficient ROP16 allele as well as by activating other proteinssuch as the suppressor for cytokine signaling 3 (SOCS3) proteinsthat act to limit STAT3 activity (36) and also by expressingGRA15, which activates NF-�B, which controls the expression ofgenes that help skew the macrophages toward becoming classi-cally activated (34). Because arginine availability is rate limitingfor parasite growth, upregulation of arginase 1 would be predictedto limit parasite growth. Indeed, arginine limitation reducesgrowth of wild-type type 1 parasites but not ROP16 knockouts(35). But arginase 1 expression would also act to reduce polyaminelevels in the host cell, and how parasites handle decreased avail-
ability of these nutrients, which also must be scavenged, remainsto be determined (27).
THE HOST PLASMA MEMBRANE AS A KEY TARGET FORTOXOPLASMA TO REGULATE ITS HOST CELL
Being the initial site of contact, the host plasma membrane repre-sents a key interface between Toxoplasma and its host cell bothduring and after parasite invasion. Yet little is known about howthey interact. Before parasites begin to invade a host cell, unknownsignals induce rhoptry and microneme secretion. Among the se-creted rhoptry proteins, the RONs are a complex of proteins thatare injected into the host cell and localize to the host cell surface(37). The RONs then bind AMA1, which is a micronemal proteinthat is secreted onto the parasite surface. Once AMA1 and theRON complex engage, the parasite can then propel itself into thehost cell. Thus, the parasite places its own receptor on the surfaceof its host cell, which likely explains Toxoplasma’s diverse host celltropism. But this does not mean that the host cell plasma mem-brane plays a passive role during invasion. Other microneme pro-teins (e.g., MIC2) are adhesins that mediate parasite-host cell at-tachment by binding to as-yet-unidentified host cell surfacefactors (38).
A primary function of the plasma membrane is to activate cel-lular responses to extracellular cues by triggering intracellular sig-naling pathways. Interactions of micronemal and parasite surfaceantigens with the host plasma membrane suggested that the par-asite might regulate host cell signaling by engaging host plasmamembrane receptors. This hypothesis was supported by the find-ing that addition of parasite-secreted factors (mainly composed ofmicroneme-derived factors) to uninfected host cells led tochanges in gene expression (39, 40). The fact that many of thesehost genes encoded chemokines, cytokines, and other immune-response-associated proteins suggested a role for Toll-like recep-tor (TLR) and/or other pathogen detection receptors in this re-sponse. More-recent studies have, however, provided more directevidence for how Toxoplasma uses host cell plasma membranereceptors to help in establishing its replicative niche.
Concomitant with Toxoplasma penetrating into its host cell, itforms its PV, while avoiding the endolysosomal pathway, sincethat route would result in autophagy-mediated degradation of thePV and parasite (41–43). Parasite avoidance of the phagolysosomeis inhibited by treatment of cells with tyrosine kinase inhibitors,and the epidermal growth factor (EGF) receptor, which is a recep-tor tyrosine kinase, was identified as at least one target of theseinhibitors (44). Significantly, parasite growth was attenuated incells transfected with small interfering RNAs (siRNAs) targetingthe EGF receptor and the PVs in these cells appeared as if they wereundergoing autophagic destruction. AKT kinase activation ofphosphatidylinositol-3 kinase appears to be the critical down-stream target of the EGF receptor signaling. These data support amodel where EGF/AKT signaling either prevents the invadingparasite from entering the endolysosomal pathway or preventslysosomal recruitment to the PV. While the parasite ligand(s) thatactivates the EGF receptor is unknown, the receptor is not acti-vated by parasites with knockout mutations in the MIC1 andMIC3 micronemal proteins (44).
Besides allowing the PV to properly develop, host plasmamembrane signaling-dependent reprogramming of host gene ex-pression and intracellular signaling is likely also to be importantfor Toxoplasma to establish its replicative niche. As an example,
initial DNA microarray studies revealed that Toxoplasma infec-tion causes dramatic alterations to the host cell transcriptome (45,46). While modulation of many of these genes is most likely im-portant for either promoting host resistance or allowing the par-asite to evade host defenses, others likely act to modify the hostcell’s intracellular environment to make it hospitable for parasitegrowth. Host genes predicted to promote parasite growth wouldinclude those that prevent host cell death and those that functionin biosynthetic pathways that provide the parasite with a necessarynutrient.
One clade of upregulated host genes that fulfilled the criteriafor possibly being important for parasite replication includedthose that encoded vascular endothelial growth factor (VEGF),glucose transporter, and glycolytic transcripts (45). These genesare targets for the hypoxia-inducible factor-1 (HIF-1) transcrip-tion factor, which is a heterodimer composed of � and � subunitsthat regulates cellular responses to decreased oxygen availability(47, 48). Toxoplasma activates HIF-1 via a host- or parasite-de-rived soluble secreted factor that signals through a family ofplasma membrane-localized, serine/threonine kinase receptorsnamed the activin-like kinase receptors (ALK4,5,7) (49). Follow-ing infection, ALK4,5,7 signaling triggers HIF-1 activation by in-creasing the stability of the HIF-1� subunit, which is a proteinwith an inherently short half-life. The mechanism by whichHIF-1� is degraded is a well-studied pathway where, immediatelyafter the protein is synthesized, two proline residues are hydroxy-lated by a prolyl hydroxylase (PHD) (50). Prolyl-hydroxylatedHIF-1� is then recognized by and ubiquitylated by the Von Hip-pel-Lindau (VHL) ubiquitin ligase, which targets HIF-1� to theproteasome. Toxoplasma infection stabilizes HIF-1� by blockingits prolyl hydroxylation, and this correlates to a decreased abun-dance of PHD2, which is the PHD most critical for controllingHIF-1� protein levels (49). The importance of HIF-1 for parasitegrowth was demonstrated by the finding that parasite growth isreduced in HIF-1�-deficient cells (48). Interestingly, Toxoplasmadependence on HIF-1� is increasingly important at physiologicalO2 levels but it is unknown how HIF-1 promotes parasite growthunder this condition.
KEEPING THE HOST CELL ALIVE: APOPTOSIS AND THEINFLAMMASOME
Toxoplasma must ensure that its host cell remains alive longenough for the parasite to replicate and then egress and invade thenext host cell. Thus, the death of infected host cells is a key weaponin the metazoan host’s defense against infection (51). The twobest-characterized forms of host cell death during infection areapoptosis and pyroptosis, and both are modulated during infec-tion. Apoptosis, which is dependent on the activation of a signal-ing cascade that leads to caspase-3 activation, is typified by mem-brane blebbing, nuclear condensation, and DNA fragmentation.The membrane blebbing results in the formation of apoptoticbodies that are taken up by phagocytic cells. In contrast, pyropto-sis is dependent on the inflammasome activating caspase-1, whichleads to the release of interleukin-1 � (IL-1�) and IL-18 and mem-brane permeabilization following by cell swelling and osmotic ly-sis (51, 52).
APOPTOSIS
Apoptosis is a noninflammatory form of programmed celldeath that been implicated as an innate defense mechanism to
eliminate intracellular pathogens (51). There are two majorapoptotic pathways. The first, the intrinsic pathway, is trig-gered by cytotoxic stress and DNA damage (e.g., UV irradiationand chemotherapy) and involves increased expression and mi-tochondrial localization of proapoptotic Bcl-2 family mem-bers. This leads to release of cytochrome C, promoting assem-bly of the apoptosome, which is a multiprotein complex thatactivates caspase-9. The second is the extrinsic pathway that istriggered by death receptor activation (e.g., FasL and TRAIL)and involves activation of caspase-8 (53–55). Activatedcaspase-8 or -9 can then cleave caspase 3, which then allows itto trigger apoptosis by cleaving cellular targets.
Toxoplasma infection renders the host cells resistant to stimulithat activate either the intrinsic pathway or the extrinsic pathway(56–58). Consistent with these findings, Toxoplasma-infectedcells show decreased levels of cleaved caspase-3, as well as ofcaspase-8 (extrinsic pathway) and caspase-9 (intrinsic pathway)(57, 59). A variety of potential upstream changes have also beennoted, including increases in expression of inhibitor of apoptosisproteins (IAPs) (60) and of antiapoptotic members of the Bcl-2family (45), activation of PI 3-kinase signaling (61), an NF-�B-dependent degradation of proapoptotic Bcl-2 family members(BAX, BAD, and BID) (59, 62), and upregulation and expressionof STAT6-dependent specific serine protease inhibitors, includingSERPIN B3 and B4, which are known to protect against tumornecrosis factor alpha (TNF-�)-induced apoptosis (63).
Although Toxoplasma clearly impacts host cell apoptosis, a keyissue that remains to be addressed is whether this effect on the hostcell’s physiology provides an advantage for parasite growth and/orimmune evasion. Or are these effects on host cell apoptosis anoff-target effect of the parasite modulating other host cell path-ways? Directly testing these possibilities awaits identification ofthe parasite factor(s) that impacts host cell apoptosis.
THE INFLAMMASOME
The inflammasome is a multiprotein complex first described in2002 that assembles in the cytoplasm after a sensor proteinwithin the complex detects a microbial or environmental factordanger signal (52, 64, 65). Inflammasome activation requirestwo signals. The first is signal 1, which is often initiated by TLRbinding of PAMPs (pathogen-associated molecular patterns)or other receptors sensing DAMPs (danger-associated molec-ular patterns). This signal leads to an upregulation of pro-IL-1� through activation of the transcription factor NF-�B.Signal 2 then triggers the activation of an intracellular sensorthat leads to inflammasome assembly and caspase-1 activation,which then leads to the processing and secretion of IL-1� andIL-18. (See Fig. 2 for a schematic of inflammasome activation.)Several inflammasome sensors have been defined, but the onespertinent to this review belong to the nod-like receptor (NLR)family and are called NLRP1 (NALP1) and NLRP3 (NALP3).Unlike other intracellular sensors that respond only to foreignmolecules (e.g., RIG-I binding to double-stranded RNA[dsRNA]), inflammasome sensors respond to both PAMPs andDAMPs (52, 66). In some cells, such as macrophages, inflam-masome activation can trigger pyroptosis, a rapid, inflamma-tory cell death that is caspase-1 dependent (52, 66).
In humans, polymorphisms in the nlrp1 gene have been linkedto susceptibility in congenital toxoplasmosis (67). Moreover, par-asite growth was enhanced whereas host cell viability and IL-1�
and IL-18 expression were reduced in monocytes engineered toexpress decreased levels of NLRP1 protein (67, 68). The idea of alink between the inflammasome and human toxoplasmosis wasfurther supported by data showing that IL-1� expression in hu-man monocytes was dependent on caspase-1 and ASC, which is anadaptor protein that mediates caspase-1 binding to either theNLRP1/caspase-5 or the NLRP3/CARDB inflammasomes (64,68). IL-1� was significantly upregulated by type II strain parasites(i.e., the Pru strain), and this polymorphic effect was dependenton GRA15, which mostly likely upregulates expression of IL-1� byvirtue of its ability to activate NF-�B (68). Infection of humanmonocytes with type I, II, or III Toxoplasma tachyzoites did notresult in rapid cell death, as the assays in the studies cited abovewere carried out at 24 and 36 h (67, 68), suggesting that underthose conditions, inflammasome activation was not triggering py-roptosis. Thus, in human monocytes, activation of the inflam-masome restricts parasite growth either through IL-1�- and IL-18-dependent mechanisms or through another unknownmechanism. This manner of controlling Toxoplasma is distinctfrom that seen in macrophages from Toxoplasma-resistant rats, inwhich NLRP1 inflammasome activation led to rapid cell death(within 3 to 10 h) after infection with Toxoplasma, consistent withpyroptosis (69, 70).
It had previously been noted that diverse rat strains differed intheir susceptibility to Toxoplasma and that this difference waslinked to a specific 1.7-cM genetic locus named the toxo1 locus(71). The rat nlrp1 gene lies within this locus, and polymorphismsin this gene determine how macrophages from Toxoplasma-sus-ceptible and -resistant rats differentially respond to Toxoplasma.In susceptible rats, macrophages infected by Toxoplasma do notundergo pyroptosis or secrete IL-1�, while in resistant rats, themacrophages do secrete IL-1� and undergo pyroptosis (72, 73).This difference in triggering the inflammasome then leads to adifference in parasite expansion within macrophages (72). MostToxoplasma strain types are similarly detected by the resistant-ratinflammasome (72) regardless of whether or not the macrophageswere first primed with lipopolysaccharide (LPS) (69). In mice,both NLRP1 and NLRP3 contribute to inflammasome activationby Toxoplasma as evidenced by induction of IL-1� maturationand secretion from macrophages (73, 74). But unlike the resultsseen with rats and akin to those seen with humans, inflammasomeactivation did not trigger pyroptosis (73). Interestingly, in mice,few strain-specific differences in inflammasome activation werenoted when the macrophages were primed with a substance suchas LPS or Pam3CSK4 (73, 74). Finally, in mice, Toxoplasma doesnot activate NLRP1 by cleaving its N terminus, unlike the Bacillusanthracis lethal factor (69, 72, 73), the only previously knownmechanism for activation of NLRP1 (75). Thus, even though Tox-oplasma activates the inflammasome through NLRP1 (andNLRP3 in mice), this activation occurs through novel mecha-nisms.
Collectively, these studies clearly implicate inflammasome ac-tivation in playing an important role in innate defenses againstToxoplasma, but their contradictions also raise questions. Why dopriming murine macrophages eliminate the strain-specific initia-tion of the NLRP1 inflammasome seen in unprimed murine mac-rophages? In mouse macrophages, certain strains can clearly giveboth signal 1 and signal 2 (e.g., type II Pru- and GRA15-dependentNF-�B activation), but other strains may trigger only signal 2 (73),which means that they do not activate the inflammasome in vitro
unless the cell has been exogenously primed to activate signal 1(i.e., LPS or Pam3CSK4). This may also explain differences re-ported in human monocytic cells in two studies; the cells were notprimed in either study, but one study found that type I parasitestriggered the inflammasome by 36 h postinfection (hpi) (67) andthe other found strain-specific differences in inflammasome acti-vation at 24 hpi (68). Thus, without priming, type I (RH) strainsmay activate signal 1 and signal 2 of the inflammasome only by 36hpi whereas another strain type (type II) can do so more rapidly.Currently, it is unknown if prestimulation of the human macro-phages/monocytes with LPS or Pam3CSK4 would eliminate thesestrain differences (70, 73, 76–78).
While these are very exciting developments in the Toxoplas-ma-inflammasome story, there is much that remains to be un-derstood. Macrophages have been the major focus of the in-flammasome work, but it is possible (and likely) that theinflammasome is important in Toxoplasma resistance in other celltypes. In addition, definitive proof that macrophage (or other cell)inflammasome activation drives Toxoplasma susceptibility or re-sistance remains to be developed, possibly through studies usingbone marrow-chimeric rats and mice. In addition, understandinghow Toxoplasma is sensed by NLRP1 will offer insights into why
polymorphisms in this gene impact infection outcomes in hu-mans (congenital disease) and rodents. Clearly, the effect of Tox-oplasma on both apoptosis and pyroptosis/inflammasome activa-tion underscores the importance of host cell viability for parasitegrowth and survival.
TOXOPLASMA REGULATES HOST CELL NUCLEARFUNCTIONS
As described in the preceding sections, modulating host cell geneexpression is one important way for the parasite to develop itsreplicative niche (Fig. 3). This is achieved by the activation of hosttranscription factors such as STAT3/6, NF-�B, and HIF-1. In con-trast to factors that activate NF-�B (GRA15) and HIF-1 (identitycurrently unknown), STAT3/6 is activated by ROP16, which is apolymorphic protein that translocates to the nucleus after it isinjected into a host cell (32). This is reminiscent of another rhop-try-localized protein phosphatase 2C (PP2c) homolog that alsotranslocates to the host cell nucleus following invasion (75), al-though the function of this protein is unknown. Spurred by thesefindings, Bougdour and colleagues used an in silico approach toidentify parasite proteins that, like ROP16 and the rhoptry PP2chomolog, contained both a signal sequence (to facilitate export
FIG 2 Schematic of inflammasome activation by Toxoplasma gondii. Increased production of secreted IL-1� via the inflammasome requires two steps. Signal 1causes activation of NF-�B, leading to increased expression of pro-IL-1� (*, pro-IL-18 is constitutively expressed). In Toxoplasma studies, the triggering of signal1 is achieved either through natural parasite sensors or factors (e.g., type II GRA15 in human monocytes) or through priming of the cell by exogenous factors (e.g.,LPS and TLR-4). Signal 2 triggers the activation of an intracellular sensor that leads to assembly of the inflammasome, which causes activation of caspase-1, whichthen cleaves pro-IL-1� and pro-IL-18 and allows the secretion of processed IL-1� and IL-18. NLRP1 is an inflammasome sensor for human, rat, and mouseimmune cells. In mice, NLRP3 is also activated by Toxoplasma. How Toxoplasma triggers either sensor is currently unknown. NLRP3 and CARDB are italicizedto indicate that they interact with each other.
into the host cell) and a nuclear localization signal. This screen ledto the identification of GRA16 and GRA24, which are releasedfrom the dense granules and are constitutively secreting, apicom-plexan-specific organelles. GRA16 binds to a complex composedof a host deubiquitinase (HAUSP) and a host PP2A phosphatase(79). Together, the components of this complex act to maintainlevels of the host p53 protein, which may impact host cell growthand cycle progression and/or proinflammatory responses (79, 80).Importantly, deletion of GRA16 severely attenuates the virulenceof type II strain parasites. Thus, GRA16 is the first nonpolymor-phic factor to be identified that is secreted into host cells andimpacts parasite virulence.
GRA24 was the second protein identified by this screen, and itbinds to and promotes and maintains activation of the host p38mitogen-activated protein (MAP) kinase. GRA24-dependent ac-tivation of p38 MAP kinase results in the activation of severaltranscription factors, including the early-growth-response pro-teins (81). GRA24 deletion leads to a significant reduction in theexpression of chemokines, including those critically required forrecruitment of inflammatory monocytes (81). Since inflamma-tory monocytes are required for resistance to Toxoplasma infec-
tion (82, 83), it was surprising that loss of GRA24 had no discern-ible impact on virulence, and further experiments are required toestablish the basis for this (81).
Studies on immune evasion have largely revealed that many(but not all) of the known Toxoplasma virulence factors act bydisengaging the immune response from properly detecting andresponding to the infection. IFN-� is the key cytokine that medi-ates the host cell defense and does so by upregulating the expres-sion of IFN-� effectors that kill Toxoplasma by a number of dis-tinct mechanisms. These include limiting nutrient scavenging,degrading the parasitophorous vacuole, and increasing antigenpresentation in infected cells so that they can be recognized byToxoplasma-specific T cells. Recent work in both human and mu-rine cells demonstrated that virulent Toxoplasma strains can pre-vent IFN-� from triggering the degradation of their PVs by inhib-iting IFN-�-stimulated GTPases (the immunity-related GTPase[IRG] proteins and guanylate-binding proteins [GBP]) from be-coming activated and associating with the PVM (25, 84–89).However, all known strains escape IFN-�-dependent killing ifthey infect the host cell before it becomes activated by IFN-�. Thisevasion is due to the parasite dysregulating IFN-�-induced gene
FIG 3 Modulation of host gene expression by nonpolymorphic parasite factors. Toxoplasma can modulate gene expression either by modulating host tran-scriptional regulators (EGRs, p53, or HIF-1) or indirectly by affecting chromatin remodeling.
expression, including blocking upregulation of the IRGs, GBPs,nitric oxide synthase, IDO, and major histocompatibility complex(MHC) class I and II (90, 91). STAT1 is the major transcriptionfactor downstream of the IFN-� receptor, but its activation (asassessed by its phosphorylation) is not affected in parasite-in-fected cells (90, 92). Rather, Toxoplasma inhibits STAT1 by alter-ing histone acetylation and by other chromatin modifications atSTAT1-activated promoters (93). It was also reported thatToxoplasma can also prevent dissociation of STAT1 from DNA,which would limit its recycling between STAT1-responsive genes(94).
TOXOPLASMA MODULATION OF UNINFECTED CELLS: IS THEPARASITE SETTING UP A MICROENVIRONMENT?
While the primary focus of this review has been on the inter-action between Toxoplasma and its infected host cell, the parasitegrows in diverse and dynamic settings in vivo. In analogy to thetumor microenvironment, parasite survival in such an environ-ment would likely require that the parasite manipulate not onlythe cell in which it is currently residing but also neighboring res-ident tissue cells as well as immune-derived cells recruited to thesite of infection. Perhaps the best-described example of this inter-action is in the gut, where Toxoplasma triggers the expressiondendritic-cell- and monocyte-recruiting chemokines. While thesecells are important for host resistance, the parasite also infects anduses them to disseminate in the host via a Trojan horse-like mech-anism (95–97). In addition, early microarray studies demon-strated that a subset of the host genes modulated by infection wereregulated as a result of secreted parasite- and/or host-derived fac-tors being released into the culture medium following infection(45).
As noted in the previous section, several host cell plasma mem-brane receptors are activated by Toxoplasma. ALK4,5,7 signaling isrequired to activate HIF-1, and HIF-1 is activated in both infectedand neighboring uninfected cells, suggesting that the parasite ei-ther secretes an ALK4,5,7-inducing ligand or induces the host cellto release one. The finding that HIF-1 can be induced even whendirect contact between the parasite and host cell is prevented sup-ports this prediction (48). Similarly, a secreted host- or parasite-derived low-molecular-weight factor modifies the cell cycle pro-gression of both infected host cells and neighboring uninfectedhost cells by having them enter the S phase (98). Why Toxoplasmainduces this bystander effect in the S phase is unclear but could berelated to the long-standing observation that parasites prefer toinvade cells that are in the S phase (99, 100).
EGF receptor activation is dependent on the expression of atleast 2 micronemal proteins, MIC1 and MIC3, and addition ofrecombinant MIC1 and MIC3 proteins to cells infected with mic1mic3 double-knockout parasites restores EGF receptor-depen-dent inhibition of CD40L-induced autophagy (44). Both of theseproteins are shed from the parasite’s plasma membrane duringinvasion (101), and it is therefore possible that the released ect-odomains can interact with and activate EGF receptor signaling innoninfected cells and thus initiate an antiautophagic response in ahost cell prior to its infection.
Besides host plasma membrane receptors, intracellular hostproteins are also targeted during infection and this is due in part tothe secretion of rhoptry effector proteins (102). The previouslyestablished model predicted that rhoptry proteins functioned onlyin the infected host cell since in theory they were injected concom-
itant with invasion (103). This paradigm has recently been chal-lenged by a system in which Toxoplasma parasites were engineeredto secrete Cre recombinase (Cre) into host cells. Using these Tox-oplasma-Cre parasites to infect reporter cells that express only agreen fluorescent protein (GFP) after Cre excises a stop codon,GFP could be detected in both infected and uninfected cells (104).In addition, pSTAT6 nuclear translocation, which is dependenton ROP16, can be observed in vitro and in vivo in a percentage ofuninfected cells, consistent with the idea that multiple rhoptryproteins are entering these uninfected cells (105). Importantly,rhoptry secretion into uninfected cells appears to be a widespreadphenomenon and can be observed in diverse types of immune andnonimmune cells (105).
The host-pathogen interaction is a continuous battle, and wepropose that Toxoplasma modulation of its microenvironmentprovides it two important advantages for winning this battle. First,activation of host cell processes prior to parasite invasion providesadditional time and an opportunity for the parasite to establish itsreplicative niche. This would include altering host cell metabolismin a way that would help the parasite gain access to necessarynutrients and to activate mechanisms to evade intrinsic immunedefenses such as autophagy, pyroptosis, and apoptosis. Second,this would allow the parasite to disarm IFN-� and other immuneeffector killing mechanisms before the parasite enters the host cell.Finally, the ability to interact with and regulate immune cells mayprovide another mechanism for the parasite to evade the immuneresponse. For example, HIF-1 activation can dampen T-cell recep-tor signaling in effector T cells (106, 107) and can also promoteregulatory T-cell development (108, 109). Not only would nega-tively regulating T cells aid in immune evasion, but it also couldlimit the collateral immune-mediated tissue damage. Whether theparasite truly modulates its microenvironment is therefore an im-portant issue that needs to be addressed.
CONCLUSION
As an obligate intracellular microbe, Toxoplasma must contendwith a variety of pressures in order to survive in the intracellularenvironment. In this review, we primarily focus on those pro-cesses that predominantly act in a parasite strain-independentmanner—nutrient acquisition, keeping the host cell alive, andmanipulation of the microenvironment. Each likely represents acritical area in which Toxoplasma has coevolved with its host cellsin order to ensure that both survive. Studies that define the hostcell processes targeted by infection will continue to provide in-sights into the fundamental biology of these cellular processes. Wealso believe that the host cell pathways that are rate limiting forToxoplasma growth, the parasite factors that activate them, andthe parasite processes dependent on these host cell pathways arepotentially important and untapped drug targets.
ACKNOWLEDGMENTS
I.J.B. is funded in part by NIH grants R01-AI069986, R21-AI107257, andR21-AI087485. A.A.K. is funded in part by NIH grant K08-NS065116.
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