Toxoplasma Development of Its Replicative Niche: In Its Host Cell and 1 Beyond 2

3 Ira J. Blader1 and Anita A. Koshy2 4 5 1Departments of Microbiology and Immunology and Ophthalmology, University at 6 Buffalo, Buffalo, NY 7 8 2Departments of Neurology and Immunobiology, BIO5 Institute, University of 9 Arizona, Tucson, AZ 10 11 Corresponding Authors: 12 1Department of Microbiology and Immunology 13 University at Buffalo 14 BRB 347 15 3435 Main Street 16 Buffalo, NY 14127 17 Phone (716) 829-5809; 18 e-mail: iblader@buffalo.edu 19 20 21 22 2Departments of Neurology 23 University of Arizona 24 Keating Bioresearch Building, Rm 229 25

EC Accepts, published online ahead of print on 20 June 2014Eukaryotic Cell doi:10.1128/EC.00081-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

1657 E. Helen St 26 Tucson, AZ 85721-0240 27 Phone (520) 626-1696 28 akoshy@email.arizona.edu 29 30

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

ABSTRACT 31 Intracellular pathogens can only replicate efficiently after they manipulate and 32 modify their host cells to create an environment conducive for its replication. 33 While diverse cellular pathways are targeted by different pathogens, metabolism, 34 membrane and cytoskeletal architecture, and cell death are the three primary 35 cellular processes that are modified by infections. Toxoplasma gondii is an 36 obligate intracellular protozoan that infects ~30% of the world’s population and 37 causes severe and life-threatening disease in developing fetuses, immune-38 comprised patients, and in certain otherwise healthy individuals who are primarily 39 in South America. The high prevalence of Toxoplasma in humans is in large part 40 a result of its ability to modulate these three host cell processes. Here, we will 41 highlight recent work defining the mechanisms by which Toxoplasma interacts 42 with the processes. In addition, we will hypothesize why some processes are 43 modified not only in the infected host cell but also in neighboring uninfected cells. 44 45

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

INTRODUCTION 46 Toxoplasma gondii is a protozoan, obligate intracellular parasite that is 47

considered one of the world’s most successful pathogens (1). Multiple factors 48 contribute to this success including a complex life cycle in which the parasite can 49 be transmitted by both vertical and horizontal transmission, efficient propagation 50 within both its primary (felines) and intermediate hosts, extensive mechanisms to 51 evade and disarm host immunity, an ability to form chronic life-long infections in 52 intermediate hosts, and a wide host tropism in which the parasite can infect most 53 nucleated cells of warm-blooded animals (2). Central to most of these factors is 54 that Toxoplasma has developed the means to replicate efficiently within the 55 hostile intracellular environment of its host cell. In this review, we will highlight 56 recent data that has shed light on how parasite growth is achieved by the 57 parasite interacting with its host cell to manipulate host signaling cascades, 58 transcription, cell survival pathways, and membrane transport. In addition, we 59 will discuss how parasites interact with neighboring host cells and will propose 60 how this may contribute to establishing a permissive microenvironment to 61 improve its overall success. In particular, we will focus on those processes that 62 are essential for the growth of all parasite strains and we refer readers to recent 63 reviews that highlight how polymorphic parasite molecules contribute to 64 Toxoplasma virulence (3-5). 65 66 NUTRIENT ACQUISTION 67

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

As an obligate intracellular parasite that resides within a non-fusogenic 68 vacuole, Toxoplasma must satisfy its nutritional needs by scavenging essential 69 nutrients from its host cell. These nutrients include carbon sources (glucose and 70 glutamine) to fuel its energy demands, specific amino acids, lipids, and other 71 nutrients. Below and in Figure 1, we will discuss each of these and highlight 72 pathways and processes that are unique to the parasite that could serve as novel 73 drug targets. 74 75 Glucose and Glutamine Power the Parasite: 76 Toxoplasma expresses a full complement of glycolytic and TCA enzymes 77 and both metabolic pathways are active in tachyzoites (6). Toxoplasma 78 glycolytic genes function in both glycolysis and in other parasite processes such 79 as parasite motility (7-9). These data led several groups to conclude that 80 glucose was the primary carbon source that was scavenged by Toxoplasma from 81 its host cell. In turn, this conclusion led to questions such as how was the 82 parasite scavenging glucose, what impact did siphoning this nutrient have on the 83 host cell’s physiology, and what was the function of the parasite’s TCA cycle in 84 growth? 85 Toxoplasma expresses a hexose transporter (TgGT1) on its plasma 86 membrane that shows the highest affinity for glucose. Deletion of the TgGT1 87 gene results in a significant defect in glucose uptake and a defect in parasite 88 motility and replication (10). The requirement for glucose in parasite motility is 89 linked to the observation that during motility glycolytic enzymes relocalize to the 90

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

Inner Membrane Complex (a membranous structure that lies directly adjacent to 91 the plasma membrane and serves as an anchor for the acto-myosin machinery to 92 propel the parasite into the host cell) suggesting that glucose provides the energy 93 needed for invasion (8, 9). Surprisingly, loss of TgGT1 had no impact on 94 virulence (10) suggesting that Toxoplasma uses other carbon sources to 95 generate ATP. 96

Identification of this other carbon source came from the observation that 97 motility of the TgGT1 knockout parasites could be restored by the addition of 98 glutamine to the media (10). Together these data suggested that parasites could 99 generate ATP either through glycolysis or glutaminolysis. This hypothesis was 100 confirmed by isotope labeling and metabolite profiling that showed that 101 Toxoplasma uses both host-derived glucose and glutamine to generate ATP via 102 cytosolic glycolysis and mitochondrial oxidative phosphorylation. It is unknown 103 how acetyl-CoA is generated for the TCA cycle since the parasite lacks a 104 mitochondrial pyruvate dehydrogenase complex. Rather this complex is 105 localized within the apicoplast where it generates acetyl-CoA that is used by the 106 fatty acid II biosynthetic pathway (11, 12). See figure 1 for a current model of 107 glucose and glutamine uptake and use by the parasite. 108

Whether the parasite’s TCA cycle is essential for growth is unclear. Fleige 109 and coworkers reported that reduced expression of the TCA enzyme succinyl-110 CoA synthase did not impact parasite growth whereas chemical inhibition of 111 aconitase did (11, 13). These seemingly contradictory data can be resolved by 112 the discovery that Toxoplasma synthesizes γ-aminobutyric acid (GABA) from 113

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

glutamine and this compound can be shunted into the TCA cycle as succinate via 114 succinic-semialdehyde dehydrogenase and thus bypasses succinyl-CoA 115 synthase (11). However, neither the GABA shunt, nor by extension the TCA 116 pathway, appear to be essential since deletion of the gene encoding glutamate 117 decarboxylase (the enzyme that converts glutamate to GABA) has a relatively 118 minor impact on parasite growth and virulence (11). 119 120 LIPIDS: We All Need Fat 121 Cholesterol 122 Sterols are essential components of eukaryotic membranes and 123 cholesterol is the major sterol in mammalian cells. While parasite membranes 124 contain cholesterol, Toxoplasma lacks cholesterol biosynthetic enzymes and 125 must scavenge it from its host (14). Serum-derived cholesterol is the primary 126 source for cholesterol since parasite growth is not reduced in cell lines unable to 127 synthesize cholesterol (14). Parasites scavenge cholesterol from LDL particles 128 and do so by redirecting LDL-receptor trafficking to the PV (14, 15). An 129 unexpected player in this process is the host multidrug resistance efflux pump, P-130 glycoprotein, that appears to be required for parasite uptake of cholesterol at 131 some point after cholesterol delivery to the PV (16). But, how cholesterol 132 crosses the PVM and the parasite plasma membrane remains to be resolved. 133 134 Isoprenoids 135

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

The mevalonate and deoxy-D-xylulose-5-phosphate (DOXP) pathways 136 produce isopentyl pyrophosphate, which is the precursor for the biosynthesis of 137 longer isoprenoids. Only the DOXP pathway is expressed by the parasite and 138 deletion of several genes encoding DOXP enzymes is lethal (17). Toxoplasma 139 expresses a bifunctional farnesyl/geranyl diphosphate synthase (TgFPPS) (18) 140 whose activities are required for isoprenoids to be incorporated into cholesterol, 141 dolichols, or isopentyls. Deletion of the TgFPPS gene results in parasites with 142 growth defects in specific types of host cells (e.g the knockout can grow in 143 human foreskin fibroblasts but cannot in macrophages) (17, 18). TgFPPS 144 knockouts also cannot survive as extracellular parasites for extended periods of 145 time because of a mitochondrial defect that is likely due to a loss of ubiquinone, 146 which is an isoprenylated cofactor of the mitochondrial respiratory chain (18). 147

Why is the DOXP pathway essential while TgFPPS is not? DOXP 148 isoprenoid biosynthesis occurs within the apicoplast and presumably its products 149 are transported from the apicoplast to the mitochondria where TgFPPS is 150 localized. TgFPPS, on the other hand, is not essential because the parasite can 151 salvage longer isoprenoids (e.g farnsyl diphosphate and geranylgeranyl 152 diphosphate from the host cell). This requirement for host isoprenoids renders 153 the parasites highly susceptible to statins, which inhibit HMG-CoA reductase that 154 is specifically expressed by the host but not parasite. Importantly, statin 155 treatment increases murine resistance to Toxoplasma indicating that 156 simultaneous inhibition of host and parasite isoprenoid biosynthesis may be a 157 novel approach to treating Toxoplasma infections. It is also possible that statins 158

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

impact parasite growth in vivo by reducing cholesterol levels in the host although 159 in vitro data suggest that statins primarily act by inhibiting host isoprenoid 160 synthesis (19). The finding that TgFPPS knockout growth is severely restricted 161 in macrophages suggests that isoprenoid scavenging may be restricted in these 162 cells. The fact that the TgFPPS knockout grows in human foreskin fibroblasts 163 suggests that either there are differences in the basal rates of isoprenoid 164 biosynthesis between fibroblasts and macrophages or that restricting isoprenoid 165 availability is a novel innate immune mechanism in macrophages. 166

167 SPHINGOLIPIDS 168

Sphingolipids are a diverse group of lipids that have important functions in 169 cell structure, membrane trafficking, and cell signaling. Toxoplasma, which 170 contains at least 20 different sphingolipid species (primarily sphingomyelin and 171 ceramide), expresses sphingolipid biosynthetic enzymes and thus can synthesize 172 these lipids. Many parasite sphingolipids contain saturated and unsaturated long 173 chain fatty acid moieties (C:20-C24) making them structurally distinct from host 174 sphingolipids whose fatty acids are primarily C:16 and C:18. 175

Addition of fluorescently labeled ceramide to uninfected host cells 176 normally stains the Golgi apparatus as well as punctate cytoplasmic structures. 177 Infected host cells are similarly stained early after labeled ceramide addition but 178 with time the ceramide accumulates in intracellular parasites (20, 21). These 179 data suggested that not only does Toxoplasma synthesize its own sphingolipids 180 but it scavenges host-derived ones too; subsequent radiolabelling assays 181

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

confirmed this model (22). Since infection induces a redistribution of host Golgi 182 to the PV, it was possible that parasites scavenged host sphingolipds though this 183 rerouting of host Golgi membrane trafficking. Rabs are a family of >40 small 184 molecular weight GTPases whose primary functions are to regulate intracellular 185 membrane trafficking. Consistent with Toxoplasma inducing a redistribution of 186 host Golgi, infection altered the localization of 3 Rab GTPases (Rab14, Rab30, 187 and Rab43) established as regulators of Golgi assembly and dynamics. 188 Significantly, expression of dominant negative Rab14 and Rab43, but not Rab30, 189 significantly reduced host-derived sphingolipid accumulation in the parasite (20). 190

191 AMINO ACIDS 192 Tryptophan 193

Tryptophan is an essential amino acid that Toxoplasma scavenges from 194 the host. The first evidence for this requirement came from studies showing that 195 IFNγ, the key cytokine for limiting parasite replication, upregulates indoleamine-196 dioxygenase (IDO), a gene that encodes the first and rate-limiting enzyme in 197 tryptophan catabolism. In addition, parasite growth cannot be controlled in IFNγ-198 treated cells lacking IDO and the repressive effect of IDO on parasite growth can 199 be reversed by the addition of excess tryptophan to the growth medium (23). 200 These data not only highlighted one manner by which IFNγ controls Toxoplasma 201 growth in human cells but also suggested that Toxoplasma is a tryptophan 202 auxotroph that scavenges the amino acid from its host cytosol. Definitive proof 203 that Toxoplasma is a tryptophan auxotroph came from the ability to grow 204

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

parasites in tryptophan-limited medium when they express the E. coli trpB gene, 205 which encodes tryptophan synthase (24). Moreover, in silico metabolic pathway 206 reconstruction indicates that the parasite lacks tryptophan biosynthetic enzymes 207 (http://www.genome.jp/kegg-208 bin/show_pathway?org_name=tgo&mapno=00400&mapscale=&show_descriptio209 n=hide). 210

More recent work, however, has suggested that IDO is not a universally 211 utilized anti-Toxoplasma control mechanism in human cells. Niedelman et al 212 reported that while IDO could restrict parasite growth in IFNγ-treated HeLa cells 213 IDO was not involved in killing Toxoplasma in the human fibroblasts that they 214 used (25). Since the work discussed above on the function of IDO during 215 Toxoplasma infections by Pfefferkorn and colleagues also used human 216 fibroblasts (23), a likely explanation for the discrepancy between these studies is 217 that genetic and/or epigenetic differences between the different fibroblasts dictate 218 the anti-parasitic mechanism used by IFNγ to kill Toxoplasma. 219 220 Arginine 221

Arginine plays a unique role in Toxoplasma growth and virulence. First, it 222 is an essential amino acid that the parasite scavenges from its host and a 223 decrease in its availability triggers bradyzoite development (26). Besides being 224 an essential amino acid, arginine must also be metabolized by the host cell to 225 generate polyamines (e.g spermine, spermadine, etc…) that are then transported 226 into the parasite (27, 28). Like tryptophan and other amino acids that the 227

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

parasite scavenges from its host, arginine and polyamine transporters remain 228 to be identified. 229

Arginase 1 is a host cell enzyme that catabolizes arginine to ornithine and 230 urea. Its expression in macrophages is the hallmark of alternatively activated 231 macrophages whereas its lack of expression and the macrophage’s exposure to 232 IFNγ produces classically activated macrophages (29). Arginase 1 expression is 233 upregulated by Toxoplasma types I and III strains due to their expression of the 234 polymorphic serine/threonine kinase ROP16 that is secreted into the host cell 235 where it phosphorylates and activates the STAT3 and STAT 6 transcription 236 factors (29-35). In contrast, type II parasites trigger the development of 237 classically activated macrophages by limiting STAT3/6 activity presumably by 238 expressing a less efficient ROP16 allele as well as by activating other proteins 239 such as the Suppressor for Cytokine Signaling 3 (SOCS3) proteins that act to 240 limit STAT3 activity (36) and also by expressing GRA15 which activates NF-κB, 241 which controls the expression of genes that help skew the macrophages towards 242 becoming classically activated (34). Because arginine availability is rate limiting 243 for parasite growth, up regulation of Arginase 1 would be predicted to limit 244 parasite growth. Indeed, arginine limitation reduces growth of wild-type type 1 245 parasites but not ROP16 knockouts (35). But Arginase 1 expression would also 246 act to reduce polyamine levels in the host cell and how parasites handle 247 decreased availability of these nutrients, which also must be scavenged, remains 248 to be determined (27). 249 250

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

THE HOST PLASMA MEMBRANE AS A KEY TARGET FOR TOXOPLASMA 251 TO REGULATE ITS HOST CELL 252

Being the initial site of contact, the host plasma membrane represents a 253 key interface between Toxoplasma and its host cell both during and after parasite 254 invasion. Yet, little is known about how they interact. Before parasites begin to 255 invade a host cell, unknown signals induce rhoptry and microneme secretion. 256 Amongst the secreted rhoptry proteins, the RONs are a complex of proteins that 257 are injected into the host cell and localize to the host cell surface (37). The 258 RONs then bind AMA1, which is a micronemal protein that is secreted onto the 259 parasite surface. Once AMA1 and the RON complex engage, the parasite can 260 then propel itself into the host cell. Thus, the parasite places its own receptor on 261 the surface of its host cell, which likely explains Toxoplasma’s diverse host cell 262 tropism. But this does not mean that the host cell plasma membrane plays a 263 passive role during invasion. Other microneme proteins (e.g MIC2) are adhesins 264 that mediate parasite-host cell attachment by binding to as yet unidentified host 265 cell surface factors (38). 266

A primary function of the plasma membrane is to activate cellular 267 responses to extracellular cues by triggering intracellular signaling pathways. 268 Interactions between micronemal and parasite surface antigens with the host 269 plasma membrane suggested that the parasite might regulate host cell signaling 270 by engaging host plasma membrane receptors. This hypothesis was supported 271 by the findings that addition of parasite secreted factors (mainly composed of 272 micronemal-derived factors) to uninfected host cells led to changes in gene 273

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

expression (39, 40). The fact that many of these host genes encoded 274 chemokines, cytokines, and other immune-response associated proteins 275 suggested a role for TLR and/or other pathogen detection receptors in this 276 response. More recent studies have, however, provided more direct evidence for 277 how Toxoplasma uses host cell plasma membrane receptors to help in 278 establishing its replicative niche. 279

Concomitant with Toxoplasma penetrating into its host cell, it forms its PV, 280 while avoiding the endolysosomal pathway since that route would result in 281 autophagy-mediated degradation of the PV and parasite (41-43). Parasite 282 avoidance of the phagolysosome is inhibited by treatment of cells with tyrosine 283 kinase inhibitors and the Epidermal Growth Factor (EGF) receptor, which is a 284 receptor tyrosine kinase, was identified as at least one target of these inhibitors 285 (44). Significantly, parasite growth was attenuated in cells transfected with 286 siRNAs targeting the EGF receptor and the PV in these cells appeared as if they 287 were undergoing autophagic destruction. AKT kinase activation of PI-3 appears 288 to be the critical downstream target of the EGF receptor signaling. These data 289 support a model where EGF/AKT signaling either prevents the invading parasite 290 from entering the endolysosomal pathway or prevents lysosomal recruitment to 291 the PV. While the parasite ligand(s) that activate the EGF receptor are unknown 292 the receptor is not activated by parasites with knockout mutations in the 293 micronemal proteins MIC1 and MIC3 (44). 294

Besides allowing the PV to properly develop, host plasma membrane 295 signaling-dependent reprogramming of host gene expression and intracellular 296

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

signaling is likely also to be important for Toxoplasma to establish its replicative 297 niche. As an example, initial DNA microarray studies revealed that Toxoplasma 298 infection causes dramatic alterations to the host cell transcriptome (45, 46). 299 While modulation of many of these genes are most likely important for either 300 promoting host resistance or allowing the parasite to evades host defenses, 301 others likely act to modify the host cell’s intracellular environment to make it 302 hospitable for parasite growth. Host genes predicted to promote parasite growth 303 would include those that prevent host cell death and those that function in 304 biosynthetic pathways that provide the parasite with a necessary nutrient. 305

One clade of upregulated host genes that fulfilled the criteria to possibly 306 being important for parasite replication included those that encoded VEGF, 307 glucose transporter, and glycolytic transcripts (45). These genes are targets for 308 the Hypoxia-inducible factor-1 (HIF-1) transcription, which is a heterodimer 309 composed of α and β subunits that regulates cellular responses to decreased 310 oxygen availability (47, 48). Toxoplasma activates HIF-1 via host- or parasite-311 derived soluble secreted factor that signals through a family of plasma 312 membrane-localized, serine/threonine kinase receptors named the activin-like 313 kinase receptors (ALK4,5,7) (49). Following infection, ALK4,5,7 signaling 314 triggers HIF-1 by increasing the stability of the HIF-1 α subunit, which is a protein 315 with an inherently short half-life. The mechanism by which HIF-1 α is degraded 316 is a well-studied pathway where immediately after the protein is synthesized two 317 proline residues are hydroxylated by a prolyl hydroxylase (PHD) (50). Prolyl 318 hydroxylated HIF-1 α is then recognized by and ubiquitylated by the Von Hippel-319

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

Lindau (VHL) ubiquitin ligase, which targets HIF-1α to the proteasome. 320 Toxoplasma infection stabilizes HIF-1 α by blocking its prolyl hydroxylation and 321 this correlates to decreased abundance of PHD2, which is the PHD most critical 322 for controlling HIF-1 α protein levels (49). The importance for HIF-1 in parasite 323 growth was demonstrated by the finding that parasite growth is reduced in HIF-1324 α deficient cells (48). Interestingly, Toxoplasma dependence on HIF-1α is 325 increasingly important at physiological O2 levels but it is unknown how HIF-1 326 promotes parasite growth under this condition. 327 328 KEEPING THE HOST CELL ALIVE: APOPTOSIS AND THE INFLAMMASOME 329

Toxoplasma must ensure that its host cell remains alive long enough for 330 the parasite to replicate and then egress and invade the next host cell. Thus, 331 death of infected host cells is a key weapon in the metazoan host’s defense 332 against infection (51). The two best-characterized forms of host cell death during 333 infection are apoptosis and pyroptosis and both are modulated during infection. 334 Apoptosis, which is dependent on the activation of a signaling cascade that leads 335 to caspase-3 activation, is typified by membrane blebbing, nuclear condensation, 336 and DNA fragmentation. The membrane blebbing results in the formation of 337 apoptotic bodies that are taken up by phagocytic cells. In contrast, pyroptosis is 338 dependent on the inflammasome activating caspase-1, which leads to the 339 release of IL-1β and IL-18 and membrane permeabilization following by cell 340 swelling and osmotic lysis (51, 52). 341 342

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

Apoptosis 343 Apoptosis is a non-inflammatory form of programmed cell death that been 344 implicated as an innate defense mechanism to eliminate intracellular pathogens 345 (51). There are two major apoptotic pathways. The first, the intrinsic pathway, is 346 triggered by cytotoxic stress and DNA damage (e.g UV irradiation, 347 chemotherapy) and involves increased expression and mitochondrial localization 348 of pro-apoptotic Bcl-2 family members. This leads to release of cytochrome C 349 that promotes assembly of the apoptosome, which is a multiprotein complex that 350 activates caspase-9. The second is the extrinsic pathway that is triggered by 351 death receptor activation (e.g FasL and TRAIL) and involves activation of 352 caspase-8 (53-55). Activated caspase-8 or 9 can then cleave caspase 3, which 353 then allows it to trigger apoptosis by cleaving cellular targets. 354 Toxoplasma infection renders the host cells resistant to stimuli that 355 activate either the intrinsic or extrinsic pathways (56-58). Consistent with these 356 findings Toxoplasma-infected cells show decreased levels of cleaved caspase-3, 357 as well as caspases-8 (extrinsic) and 9 (intrinsic pathway) (57, 59). A variety of 358 potential upstream changes have also been noted including expression 359 increases in inhibitor of apoptosis proteins (IAPs) (60) and in anti-apoptotic 360 members of the Bcl-2 family (45), activation of PI 3-kinase signaling (61), an NF-361 κB-dependent degradation of pro-apoptotic Bcl-2 family members (BAX, BAD, 362 and BID) (59, 62), and upregulation and expression of STAT6-dependent specific 363 serine protease inhibitors, including SERPIN B3 and B4, which are known to 364 protect against TNF-α induced apoptosis (63). 365

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

Although Toxoplasma clearly impacts host cell apoptosis, a key question 366 that remains to be addressed is whether this effect on the host cell’s physiology 367 provides an advantage for parasite growth and/or immune evasion. Or, are 368 these effects on host cell apoptosis an off target effect of the parasite modulating 369 other host cell pathways? Directly testing these possibilities awaits identification 370 of the parasite factor(s) that impact host cell apoptosis. 371 372 The Inflammasome 373

The inflammasome is a multiprotein complex first described in 2002 that 374 assembles in the cytoplasm after a sensor protein within the complex detects a 375 microbial or environmental factor danger signal (52, 64, 65). Inflammasome 376 activation requires two signals: signal 1, which is often initiated by TLR binding of 377 PAMPs (pathogen-associated molecular patterns) or other receptors sensing 378 DAMPs (danger-associated molecular patterns). This signal leads to an 379 upregulation of pro-IL-1β through activation of the transcription factor NF-κB. 380 Then signal 2 triggers the activation of an intracellular sensor that leads to 381 inflammasome assembly and caspase-1 activation, which then leads to the 382 processing and secretion of IL-1β and IL-18 (See Figure 2 for a schematic of 383 inflammasome activation.) Several inflammasome sensors have been defined 384 but the ones pertinent to this review belong to the nod-like receptor family 385 (NLRs), and are called NLRP1 (NALP1) and NLRP3 (NALP3). Unlike other 386 intracellular sensors that respond only to foreign molecules (e.g RIG-I binding to 387 dsRNA), inflammasome sensors respond to both PAMPs and DAMPs (52, 66). 388

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

In some cells, such as macrophages, inflammasome activation can trigger 389 pyroptosis, a rapid, inflammatory cell death that is caspase-1-dependent (52, 66). 390 In humans, polymorphisms in the nlrp1 gene have been linked to 391 susceptibility in congenital toxoplasmosis (67). Moreover, parasite growth was 392 enhanced while host cell viability and IL-1β and IL-18 expression were reduced in 393 monocytes engineered to express decreased NLRP1 protein (67, 68). Further 394 supporting the link between the inflammasome and human toxoplasmosis was 395 data showing that IL-1β expression in human monocytes was dependent on 396 caspase-1 and ASC, which is an adaptor protein that mediates caspase-1 397 binding to either the NLRP1/Caspase 5 or NLRP3/CARDB inflammasomes (64, 398 68). IL-1β was significantly upregulated by type II strain parasites (i.e. Pru 399 strain) and this polymorphic effect was dependent on GRA15, which mostly likely 400 is upregulating expression of IL-1β by virtue of its ability to activate NF-κB (68). 401 Infection of human monocytes with Types I, II, or III strain Toxoplasma 402 tachyzoites did not result in rapid cell death as the assays in the above studies 403 were carried out at 24 and 36 hours (67, 68) suggesting that in these conditions, 404 inflammasome activation was not triggering pyroptosis. Thus, in human 405 monocytes, activation of the inflammasome restricts parasite growth either 406 through IL-1β and IL-18-dependent mechanisms or through another unknown 407 mechanism. This manner of controlling Toxoplasma is distinct from that seen in 408 macrophages from Toxoplasma-resistant rats, in which NLRP1 inflammasome 409 activation led to rapid cell death (within 3 – 10 hours) after infection with 410 Toxoplasma, consistent with pyroptosis (73,74). 411

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

In rats, it had previously been noted that diverse strains differed in their 412 susceptibility to Toxoplasma, and this difference was linked to a specific 1.7-cM 413 genetic locus named the toxo1 locus (69). The rat nlrp1 gene lies within this 414 locus and polymorphisms in this gene determine how macrophages from 415 Toxoplasma-susceptible and resistant rats differentially respond to Toxoplasma. 416 In susceptible rats, macrophages infected by Toxoplasma do not undergo 417 pyroptosis nor secrete IL-1β while in resistant rats the macrophages do secrete 418 IL-1β and undergo pyroptosis (70, 71). This difference in triggering the 419 inflammasome then leads to a difference in parasite expansion within 420 macrophages (70). Most Toxoplasma strain types are similarly detected by the 421 resistant rat inflammasome(70) regardless of whether or not the macrophages 422 were first primed with LPS (73). In mice, both NLRP1 and NLRP3 contribute to 423 inflammasome activation by Toxoplasma as evidenced by induction of IL-1β 424 maturation and secretion from macrophages (71, 72). But unlike rats and akin to 425 humans, inflammasome activation did not trigger pyroptosis (71). Interestingly, in 426 mice, few strain-specific differences in inflammasome activation were noted if the 427 macrophages were primed with substances such as LPS or Pam3CSK4 (71, 72). 428 Finally, in mice, Toxoplasma does not activate NLRP1 by cleaving its N-terminus 429 as Bacillus anthracis lethal factor does (70, 71, 73), the only previously known 430 mechanism for activation of NLRP1 (78). Thus, even though Toxoplasma 431 activates the inflammasome through NLRP1 (and NLRP3 in mice), this activation 432 is occurring through novel mechanisms. 433

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

Collectively these studies clearly implicate inflammasome activation as 434 playing an important role in innate defenses against Toxoplasma, but their 435 contradictions also raise questions. Why does priming murine macrophages 436 eliminate the strain-specific initiation of the NLRP1 inflammasome seen in 437 unprimed murine macrophages? In mouse macrophages, certain strains can 438 clearly give both signal 1 and signal 2 (e.g type II Pru and GRA15-dependent NF-439 κB activation), but other strains may only trigger signal 2 (71), which means they 440 do not activate the inflammasome in vitro unless the cell has been exogenously 441 primed to activate signal 1 (i.e. LPS or Pam3CSK4). This may also explain 442 differences reported in human monocytic cells, in which neither study primed the 443 cells but one found that type I parasites triggered the inflammasome by 36 hpi 444 (67) and the other study found strain-specific differences in inflammasome 445 activation at 24 hpi (68). Thus, without priming, type I strains (RH) may only 446 activate signal 1 and 2 of the inflammasome by 36 hpi whereas other strain types 447 (type II) can do so more rapidly. Currently, it is unknown if pre-stimulation of the 448 human macrophages/monocytes with LPS or Pam3CSK4 would eliminate these 449 strain differences.(71, 74-77) 450 While these are very exciting developments in the Toxoplasma-451 inflammasome story, there is much that remains to be understood. Macrophages 452 have been the major focus of the inflammasome work, but it is possible (and 453 likely) that the inflammasome is important in Toxoplasma resistance in other cell 454 types. In addition, definitive proof that macrophage- (or other cells) 455 inflammasome activation drives Toxoplasma susceptibility or resistance remains 456

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

to be developed possibly through bone marrow chimeric rats and mice. In 457 addition, understanding how Toxoplasma is sensed by NLRP1 will offer insights 458 into why polymorphisms in this gene impacts infection outcomes in humans 459 (congenital disease) and rodents. Clearly, Toxoplasma’s effect on both 460 apoptosis and pyroptosis/inflammasome activation underscore the importance of 461 host cell viability to parasite growth and survival. 462 463 TOXOPLASMA REGULATES HOST CELL NUCLEAR FUNCTIONS 464 As described in the preceding sections, modulating host cell gene 465 expression is one important way for the parasite to develop its replicative niche. 466 This is achieved by the activation of host transcription factors such as STAT3/6, 467 NF-κB, and HIF-1. In contrast to factors that activate NF-κB (GRA15) and HIF-1 468 (identity currently unknown), STAT3/6 is activated by ROP16, which is a 469 polymorphic protein that translocates to the nucleus after it is injected into a host 470 cell (32). This was reminiscent of another rhoptry-localized protein phosphatase 471 2C (PP2c) homolog that also translocates to the host cell nucleus following 472 invasion (78), although the function for this protein is unknown. Spurred by these 473 findings, Hakimi and colleagues used a in silico approach to identify parasite 474 proteins that, like ROP16 and the rhoptry PP2c homolog, contained both a signal 475 sequence (to facilitate export into the host cell) and a nuclear localization signal. 476 This screen led to the identification of GRA16 and GRA24 that are released from 477 the dense granules, which are constitutively secreting, apicomplexan specific 478 organelles. GRA16 binds to a complex composed of a host deubiquitinase 479

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

(HAUSP) and a host PP2A phosphatase (79). Together this complex acts to 480 maintain levels of the host p53 protein, which may impact host cell growth and 481 cycle progression and/or pro-inflammatory responses (79, 80). Importantly 482 deletion of GRA16 severely attenuates virulence of type II strain parasites. Thus, 483 GRA16 is the first non-polymorphic factor that is secreted into host cells to be 484 identified that impacts parasite virulence. 485

GRA24 was the second protein identified by this screen and it binds to 486 and promotes and maintains activation of the host p38 MAP kinase. GRA24-487 dependet activation of p38 MAP kinase results in the activation of several 488 transcription factors including the Early Growth Response proteins (81). GRA24 489 deletion leads to a significant reduction in the expression of chemokines 490 including those critically required for recruitment of inflammatory monocytes (81). 491 Since inflammatory monocytes are required for resistance to Toxoplasma 492 infection, (82, 83), it was surprising that loss of GRA24 had no discernable 493 impact on virulence and further experiments are required to establish the basis 494 for this (81). 495 Studies on immune evasion have largely revealed that many (but not all) 496 of the known Toxoplasma virulence factors act by disengaging the immune 497 response from properly detecting and responding to the infection. IFNγ is the 498 key cytokine in mediating host cell defense and does so by upregulating the 499 expression of IFNγ effectors that kill Toxoplasma by a number of distinct 500 mechanisms. These include limiting nutrient scavenging, degraded the 501 parasitophorous vacuole, and increasing infected cell antigen presentation so 502

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

that they can be recognized by Toxoplasma specific T-cells. Recent work in both 503 human and murine cells demonstrated that virulent Toxoplasma strains can 504 prevent IFNγ from triggering the degradation of their PV by inhibiting IFNγ 505 stimulated GTPases (the IRGs and GBPs) from becoming activated and 506 associating with the PVM (25, 84-89). But all known strains escape IFNγ-507 dependent killing if they infect the host cell before it becomes activated by IFNγ. 508 This evasion is due to the parasite dysregulating IFNγ-induced gene expression 509 including blocking upregulation of the IRGs, GBPs, nitric oxide synthase, IDO, 510 and MHC Class I and II (90, 91). STAT1 is the major transcription factor 511 downstream of the IFNγ receptor but its activation (as assessed by its 512 phosphorylation) is not affected in parasite-infected cells (90, 92). Rather 513 Toxoplasma inhibits STAT1 by altering histone acetylation and other chromatin 514 modifications at STAT1-activated promoters (93). It was also reported that 515 Toxoplasma can also prevent dissociation of STAT1 from DNA, would limits it 516 recycling between STAT1 responsive genes (94). 517 518 TOXOPLASMA MODULATION OF UNINFECTED CELLS: IS THE PARASITE 519 SETTING UP A MICROENVIRONMENT? 520 While the primary focus of this review has been on the interaction between 521 Toxoplasma and its infected host cell, in vivo the parasite grows in a diverse and 522 dynamic setting. Analogous to the tumor microenvironment, parasite survival in 523 such an environment would likely require that the parasite manipulate not only 524 the cell in which it is currently residing but also neighboring resident tissue cells 525

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

as well as immune-derived cells recruited to the site of infection. Perhaps the 526 best-described example of this interaction is in the gut where Toxoplasma 527 triggers the expression dendritic cell-and monocyte-recruiting chemokines. While 528 these cells are important for host resistance, the parasite also infects and uses 529 them to disseminate in the host via a Trojan Horse-like mechanism (95-97). In 530 addition, early microarray studies demonstrated that a subset of the host genes 531 modulated by infection were regulated as a result of parasite- and/or host-derived 532 factors secreted released into the culture medium following infection (45). 533

As noted in the previous section, several host cell plasma membrane 534 receptors are activated by Toxoplasma. ALK4,5,7 signaling is required to 535 activate HIF-1 and HIF-1 is activated in both infected and neighboring uninfected 536 cells, suggesting that the parasite either secretes an ALK4,5,7-inducing ligand or 537 induces the host cell to release one. The finding that HIF-1 can be induced even 538 when direct contact between the parasite and host cell is prevented supports this 539 prediction (48). Similarly, a secreted host- or parasite-derived low molecular 540 weight factor modifies cell cycle progression of both infected host cells and 541 neighboring uninfected host cells by having them enter S-phase (98). Why 542 Toxoplasma induces this by-stander effect on S-phase is unclear but could be 543 related to the long standing observation that parasites prefer to invade cells that 544 are in S-phase (99, 100). 545

EGF receptor activation is dependent on the expression of at least 2 546 micronemal proteins MIC1 and MIC3, and addition of recombinant MIC1 and 547 MIC3 proteins to cells infected with mic1/3 double knockout parasites restores 548

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

EGF receptor-dependent inhibition of CD40L-induced autophagy (44). Both of 549 these proteins are shed from the parasite’s plasma membrane during invasion 550 (101) and it is therefore possible that the released ectodomains can interact with 551 and activate EGF receptor signaling in non-infected cells and thus initiate an anti-552 autophagic response in a host cell prior to its infection. . 553

Besides host plasma membrane receptors, intracellular host proteins are 554 also targeted during infection and this is due in part to the secretion of rhoptry 555 effector proteins (102). The previously established model predicted that rhoptry 556 proteins only functioned in the infected host cell since in theory they were 557 injected concomitant with invasion (103). This paradigm has recently been 558 challenged by a system in which Toxoplasma parasites were engineered to 559 secrete Cre recombinase (Cre) into host cells. Using these Toxplasma-Cre 560 parasites to infect reporter cells that only express a Green Fluorescent Protein 561 (GFP) after Cre excises a stop codon, GFP could be detected in both infected 562 and uninfected cells (104). In addition, pSTAT6 nuclear translocation, which is 563 dependent on ROP16, can be observed in vitro and in vivo in a percentage of 564 uninfected cells, consistent with the idea that multiple rhoptry proteins are 565 entering these uninfected cells (105). Importantly, rhoptry secretion into 566 uninfected cells appears to be a widespread phenomenon and can be observed 567 in diverse types of immune and non-immune cells (105). 568

The host-pathogen interaction is a continuous battle and we propose that 569 Toxoplasma modulation of its microenvironment provides it two important 570 advantages to winning this battle. First, activation of host cell processes prior to 571

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

parasite invasion provides additional time and opportunity for the parasite to 572 establish its replicative niche. This would include altering host cell metabolism in 573 a way that would help the parasite gain access to necessary nutrients and to 574 activate mechanisms to evade intrinsic immune defenses such as autophagy, 575 pyroptosis, and apoptosis. Second, this would allow the parasite to disarm IFNγ 576 and other immune effector killing mechanisms before the parasite enters the host 577 cell. Finally, the ability to interact with and regulate immune cells may provide 578 another mechanism for the parasite to evade the immune response. For 579 example, HIF-1 activation can dampen T-cell Receptor signaling in effector T-580 cells (106, 107) and also promote regulatory T-cell development (108, 109). Not 581 only would negatively regulating T-cells aid in immune evasion but it also could 582 limit the collateral immune-mediated tissue damage. Testing whether the 583 parasite truly modulates its microenvironment is therefore an important issue that 584 needs to be addressed. 585

586 Conclusion 587

As an obligate intracellular microbe, Toxoplasma must contend with a 588 variety of pressures in order to successfully survive in the intracellular 589 environment. In this review, we primarily focused on those processes that 590 predominantly act in a parasite-strain independent manner - nutrient acquisition, 591 keeping the host cell alive, and the microenvironment. Each likely represents 592 critical areas in which Toxoplasma has co-evolved with its host cells in order to 593 ensure that both survive. Studies that continue to define the host cell processes 594

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

targeted by infection will continue to provide insights into the fundamental biology 595 of these cellular processes. We also believe that host cell pathways that are rate 596 limiting for Toxoplasma growth, the parasite factors that activate them, and the 597 parasite processes dependent on these host cell pathways are potentially 598 important and untapped drug targets. 599

600 Acknowledgements 601

IJB is funded in part by NIH grants R01-AI069986, R21-AI107257, and 602 R21-AI087485 (IJB). AAK is funded in part by NIH grant K08-NS065116. 603 604

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

LITERATURE CITED 605 606 607 1. Dubey JP, Su C. 2009. Population biology of Toxoplasma gondii: what's out 608 and where did they come from. Memorias do Instituto Oswaldo Cruz 609 104:190-195. 610 2. Kim K, Weiss LM. 2004. Toxoplasma gondii: the model apicomplexan. 611 International journal for parasitology 34:423-432. 612 3. Dubremetz JF, Lebrun M. 2012. Virulence factors of Toxoplasma gondii. 613 Microbes and infection / Institut Pasteur 14:1403-1410. 614 4. Hunter CA, Sibley LD. 2012. Modulation of innate immunity by Toxoplasma 615 gondii virulence effectors. Nature reviews. Microbiology 10:766-778. 616 5. Sibley LD, Qiu W, Fentress S, Taylor SJ, Khan A, Hui R. 2009. Forward 617 genetics in Toxoplasma gondii reveals a family of rhoptry kinases that 618 mediates pathogenesis. Eukaryotic cell 8:1085-1093. 619 6. Denton H, Roberts CW, Alexander J, Thong KW, Coombs GH. 1996. 620 Enzymes of energy metabolism in the bradyzoites and tachyzoites of 621 Toxoplasma gondii. FEMS Microbiol Lett 137:103-108. 622 7. Al-Anouti F, Tomavo S, Parmley S, Ananvoranich S. 2004. The expression 623 of lactate dehydrogenase is important for the cell cycle of Toxoplasma gondii. 624 The Journal of biological chemistry 279:52300-52311. 625 8. Pomel S, Luk FC, Beckers CJ. 2008. Host cell egress and invasion induce 626 marked relocations of glycolytic enzymes in Toxoplasma gondii tachyzoites. 627 PLoS pathogens 4:e1000188. 628 9. Starnes GL, Coincon M, Sygusch J, Sibley LD. 2009. Aldolase is essential 629 for energy production and bridging adhesin-actin cytoskeletal interactions 630 during parasite invasion of host cells. Cell host & microbe 5:353-364. 631 10. Blume M, Rodriguez-Contreras D, Landfear S, Fleige T, Soldati-Favre D, 632 Lucius R, Gupta N. 2009. Host-derived glucose and its transporter in the 633 obligate intracellular pathogen Toxoplasma gondii are dispensable by 634 glutaminolysis. Proceedings of the National Academy of Sciences of the 635 United States of America 106:12998-13003. 636 11. MacRae JI, Sheiner L, Nahid A, Tonkin C, Striepen B, McConville MJ. 637 2012. Mitochondrial metabolism of glucose and glutamine is required for 638 intracellular growth of Toxoplasma gondii. Cell host & microbe 12:682-692. 639 12. Mazumdar J, H. Wilson E, Masek K, A. Hunter C, Striepen B. 2006. 640 Apicoplast fatty acid synthesis is essential for organelle biogenesis and 641 parasite survival in Toxoplasma gondii. Proceedings of the National 642 Academy of Sciences 103:13192-13197. 643 13. Fleige T, Pfaff N, Gross U, Bohne W. 2008. Localisation of gluconeogenesis 644 and tricarboxylic acid (TCA)-cycle enzymes and first functional analysis of 645 the TCA cycle in Toxoplasma gondii. International journal for parasitology 646 38:1121-1132. 647

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

14. Coppens I, Sinai AP, Joiner KA. 2000. Toxoplasma gondii exploits host low-648 density lipoprotein receptor- mediated endocytosis for cholesterol 649 acquisition. The Journal of cell biology 149:167-180. 650 15. Coppens I, Dunn JD, Romano JD, Pypaert M, Zhang H, Boothroyd JC, 651 Joiner KA. 2006. Toxoplasma gondii sequesters lysosomes from 652 mammalian hosts in the vacuolar space. Cell 125:261-274. 653 16. Bottova I, Hehl AB, Stefanic S, Fabrias G, Casas J, Schraner E, Pieters J, 654 Sonda S. 2009. Host cell P-glycoprotein is essential for cholesterol uptake 655 and replication of Toxoplasma gondii. The Journal of biological chemistry 656 284:17438-17448. 657 17. Nair SC, Brooks CF, Goodman CD, Sturm A, McFadden GI, Sundriyal S, 658 Anglin JL, Song Y, Moreno SN, Striepen B. 2011. Apicoplast isoprenoid 659 precursor synthesis and the molecular basis of fosmidomycin resistance in 660 Toxoplasma gondii. The Journal of experimental medicine 208:1547-1559. 661 18. Brumlik MJ, Pandeswara S, Ludwig SM, Jeansonne DP, Lacey MR, Murthy 662 K, Daniel BJ, Wang RF, Thibodeaux SR, Church KM, Hurez V, Kious MJ, 663 Zhang B, Alagbala A, Xia X, Curiel TJ. 2013. TgMAPK1 is a Toxoplasma 664 gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and 665 interferon-gamma-mediated nitric oxide production. Experimental 666 parasitology 134:389-399. 667 19. Li ZH, Ramakrishnan S, Striepen B, Moreno SN. 2013. Toxoplasma gondii 668 relies on both host and parasite isoprenoids and can be rendered sensitive to 669 atorvastatin. PLoS pathogens 9:e1003665. 670 20. Romano JD, Sonda S, Bergbower E, Smith ME, Coppens I. 2013. 671 Toxoplasma gondii salvages sphingolipids from the host Golgi through the 672 rerouting of selected Rab vesicles to the parasitophorous vacuole. Molecular 673 Biology of the Cell 24:1974-1995. 674 21. de Melo EJ, de Souza W. 1996. Pathway of C6-NBD-Ceramide on the host 675 cell infected with Toxoplasma gondii. Cell Struct Funct 21:47-52. 676 22. Bisanz C, Bastien O, Grando D, Jouhet J, Marechal E, Cesbron-Delauw MF. 677 2006. Toxoplasma gondii acyl-lipid metabolism: de novo synthesis from 678 apicoplast-generated fatty acids versus scavenging of host cell precursors. 679 The Biochemical journal 394:197-205. 680 23. Pfefferkorn ER. 1984. Interferon gamma blocks the growth of Toxoplasma 681 gondii in human fibroblasts by inducing the host cells to degrade tryptophan. 682 Proceedings of the National Academy of Sciences of the United States of 683 America 81:908-912. 684 24. Sibley LD, Messina M, Niesman IR. 1994. Stable DNA transformation in the 685 obligate intracellular parasite Toxoplasma gondii by complementation of 686 tryptophan auxotrophy. Proceedings of the National Academy of Sciences of 687 the United States of America 91:5508-5512. 688 25. Niedelman W, Gold DA, Rosowski EE, Sprokholt JK, Lim D, Arenas AF, 689 Melo MB, Spooner E, Yaffe MB, Saeij JPJ. 2012. The Rhoptry Proteins 690 ROP18 and ROP5 Mediate Toxoplasma gondii Evasion of the Murine, But Not 691 the Human, Interferon-Gamma Response. PLoS pathogens 8. 692

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

26. Fox BA, Gigley JP, Bzik DJ. 2004. Toxoplasma gondii lacks the enzymes 693 required for de novo arginine biosynthesis and arginine starvation triggers 694 cyst formation. International journal for parasitology 34:323-331. 695 27. Cook T, Roos D, Morada M, Zhu G, Keithly JS, Feagin JE, Wu G, Yarlett N. 696 2007. Divergent polyamine metabolism in the Apicomplexa. Microbiology 697 153:1123-1130. 698 28. Pfaff AW, Villard O, Klein JP, Mousli M, Candolfi E. 2005. Regulation of 699 Toxoplasma gondii multiplication in BeWo trophoblast cells: cross-regulation 700 of nitric oxide production and polyamine biosynthesis. International journal 701 for parasitology 35:1569-1576. 702 29. Classen A, Lloberas J, Celada A. 2009. Macrophage activation: classical 703 versus alternative. Methods in molecular biology 531:29-43. 704 30. Ong YC, Reese ML, Boothroyd JC. 2010. Toxoplasma rhoptry protein 16 705 (ROP16) subverts host function by direct tyrosine phosphorylation of STAT6. 706 The Journal of biological chemistry 285:28731-28740. 707 31. Yamamoto M, Standley DM, Takashima S, Saiga H, Okuyama M, Kayama 708 H, Kubo E, Ito H, Takaura M, Matsuda T, Soldati-Favre D, Takeda K. 709 2009. A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 710 determines the direct and strain-specific activation of Stat3. The Journal of 711 experimental medicine 206:2747-2760. 712 32. Saeij JP, Coller S, Boyle JP, Jerome ME, White MW, Boothroyd JC. 2007. 713 Toxoplasma co-opts host gene expression by injection of a polymorphic 714 kinase homologue. Nature 445:324-327. 715 33. Saeij JP, Boyle JP, Coller S, Taylor S, Sibley LD, Brooke-Powell ET, Ajioka 716 JW, Boothroyd JC. 2006. Polymorphic secreted kinases are key virulence 717 factors in toxoplasmosis. Science (New York, N.Y 314:1780-1783. 718 34. Jensen KD, Wang Y, Wojno ED, Shastri AJ, Hu K, Cornel L, Boedec E, Ong 719 YC, Chien YH, Hunter CA, Boothroyd JC, Saeij JP. 2011. Toxoplasma 720 polymorphic effectors determine macrophage polarization and intestinal 721 inflammation. Cell host & microbe 9:472-483. 722 35. Butcher BA, Fox BA, Rommereim LM, Kim SG, Maurer KJ, Yarovinsky F, 723 Herbert DBR, Bzik DJ, Denkers EY. 2011. Toxoplasma gondii Rhoptry 724 Kinase ROP16 Activates STAT3 and STAT6 Resulting in Cytokine Inhibition 725 and Arginase-1-Dependent Growth Control. PLoS pathogens 7:e1002236. 726 36. Whitmarsh RJ, Gray CM, Gregg B, Christian DA, May MJ, Murray PJ, 727 Hunter CA. 2011. A critical role for SOCS3 in innate resistance to 728 Toxoplasma gondii. Cell host & microbe 10:224-236. 729 37. Alexander DL, Mital J, Ward GE, Bradley P, Boothroyd JC. 2005. 730 Identification of the Moving Junction Complex of Toxoplasma gondii: A 731 Collaboration between Distinct Secretory Organelles. PLoS pathogens 1:e17. 732 38. Huynh MH, Rabenau KE, Harper JM, Beatty WL, Sibley LD, Carruthers 733 VB. 2003. Rapid invasion of host cells by Toxoplasma requires secretion of 734 the MIC2-M2AP adhesive protein complex. The EMBO journal 22:2082-735 2090. 736

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

39. Channon JY, Suh EI, Seguin RM, Kasper LH. 1999. Attachment ligands of 737 viable Toxoplasma gondii induce soluble immunosuppressive factors in 738 human monocytes. Infection & Immunity 67:2547-2551. 739 40. Stutz A, Kessler H, Kaschel ME, Meissner M, Dalpke AH. 2012. Cell 740 invasion and strain dependent induction of suppressor of cytokine signaling-741 1 by Toxoplasma gondii. Immunobiology 217:28-36. 742 41. Jones TC, Hirsch JG. 1972. The interaction between Toxoplasma gondii and 743 mammalian cells. II. The absence of lysosomal fusion with phagocytic 744 vacuoles containing living parasites. The Journal of experimental medicine 745 136:1173-1194. 746 42. Sibley LD, Weidner E, Krahenbuhl JL. 1985. Phagosome acidification 747 blocked by intracellular Toxoplasma gondii. Nature 315:416-419. 748 43. Ling YM, Shaw MH, Ayala C, Coppens I, Taylor GA, Ferguson DJ, Yap GS. 749 2006. Vacuolar and plasma membrane stripping and autophagic elimination 750 of Toxoplasma gondii in primed effector macrophages. The Journal of 751 experimental medicine 203:2063-2071. 752 44. Muniz-Feliciano L, Van Grol J, Portillo JA, Liew L, Liu B, Carlin CR, 753 Carruthers VB, Matthews S, Subauste CS. 2013. Toxoplasma gondii-754 induced activation of EGFR prevents autophagy protein-mediated killing of 755 the parasite. PLoS pathogens 9:e1003809. 756 45. Blader IJ, Manger ID, Boothroyd JC. 2001. Microarray Analysis Reveals 757 Previously Unknown Changes in Toxoplasma gondii-infected Human Cells. J. 758 Biol Chem 276:24223-24231. 759 46. Gail M, Gross U, Bohne W. 2001. Transcriptional profile of Toxoplasma 760 gondii-infected human fibroblasts as revealed by gene-array hybridization. 761 Mol Genet Genomics 265:905-912. 762 47. Semenza GL. 2012. Hypoxia-inducible factors in physiology and medicine. 763 Cell 148:399-408. 764 48. Spear W, Chan D, Coppens I, Johnson RS, Giaccia A, Blader IJ. 2006. The 765 host cell transcription factor hypoxia-inducible factor 1 is required for 766 Toxoplasma gondii growth and survival at physiological oxygen levels. 767 Cellular microbiology 8:339-352. 768 49. Wiley M, Sweeney KR, Chan DA, Brown KM, McMurtrey C, Howard EW, 769 Giaccia AJ, Blader IJ. 2010. Toxoplasma gondii activates hypoxia inducible 770 factor by stabilizing the HIF-1 alpha subunit via type I activin like receptor 771 kinase receptor signaling. The Journal of biological chemistry 285:26976-772 26986. 773 50. Kaelin WG, Jr., Ratcliffe PJ. 2008. Oxygen sensing by metazoans: the 774 central role of the HIF hydroxylase pathway. Molecular cell 30:393-402. 775 51. Sridharan H, Upton JW. 2014. Programmed necrosis in microbial 776 pathogenesis. Trends in microbiology. 777 52. Lamkanfi M. 2011. Emerging inflammasome effector mechanisms. Nature 778 reviews. Immunology 11:213-220. 779 53. Estaquier J, Vallette F, Vayssiere JL, Mignotte B. 2012. The mitochondrial 780 pathways of apoptosis. Advances in experimental medicine and biology 781 942:157-183. 782

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

54. Wilson NS, Dixit V, Ashkenazi A. 2009. Death receptor signal transducers: 783 nodes of coordination in immune signaling networks. Nature immunology 784 10:348-355. 785 55. Boatright KM, Salvesen GS. 2003. Mechanisms of caspase activation. 786 Current opinion in cell biology 15:725-731. 787 56. Carmen JC, Hardi L, Sinai AP. 2006. Toxoplasma gondii inhibits ultraviolet 788 light-induced apoptosis through multiple interactions with the 789 mitochondrion-dependent programmed cell death pathway. Cellular 790 microbiology 8:301-315. 791 57. Goebel S, Gross U, Luder CG. 2001. Inhibition of host cell apoptosis by 792 Toxoplasma gondii is accompanied by reduced activation of the caspase 793 cascade and alterations of poly(ADP-ribose) polymerase expression. Journal 794 of cell science 114:3495-3505. 795 58. Nash PB, Purner MB, Leon RP, Clarke P, Duke RC, Curiel TJ. 1998. 796 Toxoplasma gondii-infected cells are resistant to multiple inducers of 797 apoptosis. Journal of Immunology 160:1824-1830. 798 59. Payne TM, Molestina RE, Sinai AP. 2003. Inhibition of caspase activation 799 and a requirement for NF-{kappa}B function in the Toxoplasma gondii-800 mediated blockade of host apoptosis. Journal of cell science 116:4345-4358. 801 60. Molestina RE, Payne TM, Coppens I, Sinai AP. 2003. Activation of NF-802 {kappa}B by Toxoplasma gondii correlates with increased expression of 803 antiapoptotic genes and localization of phosphorylated I{kappa}B to the 804 parasitophorous vacuole membrane. Journal of cell science 116:4359-4371. 805 61. Kim L, Denkers EY. 2006. Toxoplasma gondii triggers Gi-dependent PI 3-806 kinase signaling required for inhibition of host cell apoptosis. Journal of cell 807 science 119:2119-2126. 808 62. Carmen JC, Sinai AP. 2011. The Differential Effect of Toxoplasma Gondii 809 Infection on the Stability of BCL2-Family Members Involves Multiple 810 Activities. Frontiers in microbiology 2:1. 811 63. Ahn HJ, Kim JY, Ryu KJ, Nam HW. 2009. STAT6 activation by Toxoplasma 812 gondii infection induces the expression of Th2 C-C chemokine ligands and B 813 clade serine protease inhibitors in macrophage. Parasitology research 814 105:1445-1453. 815 64. Martinon F, Burns K, Tschopp J. 2002. The inflammasome: a molecular 816 platform triggering activation of inflammatory caspases and processing of 817 proIL-beta. Molecular cell 10:417-426. 818 65. Aachoui Y, Sagulenko V, Miao EA, Stacey KJ. 2013. Inflammasome-819 mediated pyroptotic and apoptotic cell death, and defense against infection. 820 Current opinion in microbiology 16:319-326. 821 66. Gross O, Thomas CJ, Guarda G, Tschopp J. 2011. The inflammasome: an 822 integrated view. Immunological reviews 243:136-151. 823 67. Witola WH, Mui E, Hargrave A, Liu S, Hypolite M, Montpetit A, Cavailles 824 P, Bisanz C, Cesbron-Delauw MF, Fournie GJ, McLeod R. 2011. NALP1 825 influences susceptibility to human congenital toxoplasmosis, 826 proinflammatory cytokine response, and fate of Toxoplasma gondii-infected 827 monocytic cells. Infection and immunity 79:756-766. 828

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

68. Gov L, Karimzadeh A, Ueno N, Lodoen MB. 2013. Human innate immunity 829 to Toxoplasma gondii is mediated by host caspase-1 and ASC and parasite 830 GRA15. mBio 4. 831 69. Cavailles P, Sergent V, Bisanz C, Papapietro O, Colacios C, Mas M, Subra 832 JF, Lagrange D, Calise M, Appolinaire S, Faraut T, Druet P, Saoudi A, 833 Bessieres MH, Pipy B, Cesbron-Delauw MF, Fournie GJ. 2006. The rat 834 Toxo1 locus directs toxoplasmosis outcome and controls parasite 835 proliferation and spreading by macrophage-dependent mechanisms. 836 Proceedings of the National Academy of Sciences of the United States of 837 America 103:744-749. 838 70. Cirelli KM, Gorfu G, Hassan MA, Printz M, Crown D, Leppla SH, Grigg ME, 839 Saeij JP, Moayeri M. 2014. Inflammasome Sensor NLRP1 Controls Rat 840 Macrophage Susceptibility to Toxoplasma gondii. PLoS pathogens 841 10:e1003927. 842 71. Ewald SE, Chavarria-Smith J, Boothroyd JC. 2014. NLRP1 is an 843 inflammasome sensor for Toxoplasma gondii. Infection and immunity 844 82:460-468. 845 72. Gorfu G, Cirelli KM, Melo MB, Mayer-Barber K, Crown D, Koller BH, 846 Masters S, Sher A, Leppla SH, Moayeri M, Saeij JP, Grigg ME. 2014. Dual 847 Role for Inflammasome Sensors NLRP1 and NLRP3 in Murine Resistance to 848 Toxoplasma gondii. mBio 5. 849 73. Chavarria-Smith J, Vance RE. 2013. Direct proteolytic cleavage of NLRP1B 850 is necessary and sufficient for inflammasome activation by anthrax lethal 851 factor. PLoS pathogens 9:e1003452. 852 74. Babelova A, Moreth K, Tsalastra-Greul W, Zeng-Brouwers J, Eickelberg 853 O, Young MF, Bruckner P, Pfeilschifter J, Schaefer RM, Grone HJ, 854 Schaefer L. 2009. Biglycan, a danger signal that activates the NLRP3 855 inflammasome via toll-like and P2X receptors. The Journal of biological 856 chemistry 284:24035-24048. 857 75. Cruz CM, Rinna A, Forman HJ, Ventura AL, Persechini PM, Ojcius DM. 858 2007. ATP activates a reactive oxygen species-dependent oxidative stress 859 response and secretion of proinflammatory cytokines in macrophages. The 860 Journal of biological chemistry 282:2871-2879. 861 76. Lees MP, Fuller SJ, McLeod R, Boulter NR, Miller CM, Zakrzewski AM, Mui 862 EJ, Witola WH, Coyne JJ, Hargrave AC, Jamieson SE, Blackwell JM, Wiley 863 JS, Smith NC. 2010. P2X7 receptor-mediated killing of an intracellular 864 parasite, Toxoplasma gondii, by human and murine macrophages. Journal of 865 Immunology 184:7040-7046. 866 77. Correa G, Marques da Silva C, de Abreu Moreira-Souza AC, Vommaro RC, 867 Coutinho-Silva R. 2010. Activation of the P2X(7) receptor triggers the 868 elimination of Toxoplasma gondii tachyzoites from infected macrophages. 869 Microbes and infection / Institut Pasteur 12:497-504. 870 78. Gilbert LA, Ravindran S, Turetzky JM, Boothroyd JC, Bradley PJ. 2007. 871 Toxoplasma gondii targets a protein phosphatase 2C to the nuclei of infected 872 host cells. Eukaryotic cell 6:73-83. 873

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

79. Bougdour A, Durandau E, Brenier-Pinchart MP, Ortet P, Barakat M, 874 Kieffer S, Curt-Varesano A, Curt-Bertini RL, Bastien O, Coute Y, Pelloux 875 H, Hakimi MA. 2013. Host cell subversion by Toxoplasma GRA16, an 876 exported dense granule protein that targets the host cell nucleus and alters 877 gene expression. Cell host & microbe 13:489-500. 878 80. Bougdour A, Tardieux I, Hakimi MA. 2014. Toxoplasma exports dense 879 granule proteins beyond the vacuole to the host cell nucleus and rewires the 880 host genome expression. Cellular microbiology 16:334-343. 881 81. Braun L, Brenier-Pinchart MP, Yogavel M, Curt-Varesano A, Curt-Bertini 882 RL, Hussain T, Kieffer-Jaquinod S, Coute Y, Pelloux H, Tardieux I, 883 Sharma A, Belrhali H, Bougdour A, Hakimi MA. 2013. A Toxoplasma 884 dense granule protein, GRA24, modulates the early immune response to 885 infection by promoting a direct and sustained host p38 MAPK activation. The 886 Journal of experimental medicine 210:2071-2086. 887 82. Dunay IR, Damatta RA, Fux B, Presti R, Greco S, Colonna M, Sibley LD. 888 2008. Gr1(+) inflammatory monocytes are required for mucosal resistance 889 to the pathogen Toxoplasma gondii. Immunity 29:306-317. 890 83. Robben PM, LaRegina M, Kuziel WA, Sibley LD. 2005. Recruitment of Gr-891 1+ monocytes is essential for control of acute toxoplasmosis. The Journal of 892 experimental medicine 201:1761-1769. 893 84. Virreira Winter S, Niedelman W, Jensen KD, Rosowski EE, Julien L, 894 Spooner E, Caradonna K, Burleigh BA, Saeij JP, Ploegh HL, Frickel EM. 895 2011. Determinants of GBP recruitment to Toxoplasma gondii vacuoles and 896 the parasitic factors that control it. PloS one 6:e24434. 897 85. Selleck EM, Fentress SJ, Beatty WL, Degrandi D, Pfeffer K, Virgin HWt, 898 Macmicking JD, Sibley LD. 2013. Guanylate-binding protein 1 (Gbp1) 899 contributes to cell-autonomous immunity against Toxoplasma gondii. PLoS 900 pathogens 9:e1003320. 901 86. Fleckenstein MC, Reese ML, Konen-Waisman S, Boothroyd JC, Howard 902 JC, Steinfeldt T. 2012. A Toxoplasma gondii pseudokinase inhibits host IRG 903 resistance proteins. PLoS biology 10:e1001358. 904 87. Fentress SJ, Behnke MS, Dunay IR, Mashayekhi M, Rommereim LM, Fox 905 BA, Bzik DJ, Taylor GA, Turk BE, Lichti CF, Townsend RR, Qiu W, Hui R, 906 Beatty WL, Sibley LD. 2010. Phosphorylation of immunity-related GTPases 907 by a Toxoplasma gondii-secreted kinase promotes macrophage survival and 908 virulence. Cell host & microbe 8:484-495. 909 88. Behnke MS, Khan A, Wootton JC, Dubey JP, Tang K, Sibley LD. 2011. 910 Virulence differences in Toxoplasma mediated by amplification of a family of 911 polymorphic pseudokinases. Proceedings of the National Academy of 912 Sciences of the United States of America 108:9631-9636. 913 89. Etheridge RD, Alaganan A, Tang K, Lou HJ, Turk BE, Sibley LD. 2014. The 914 Toxoplasma Pseudokinase ROP5 Forms Complexes with ROP18 and ROP17 915 Kinases that Synergize to Control Acute Virulence in Mice. Cell host & 916 microbe 15:537-550. 917 90. Kim SK, Fouts AE, Boothroyd JC. 2007. Toxoplasma gondii dysregulates 918 IFN-gamma-inducible gene expression in human fibroblasts: insights from a 919

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

genome-wide transcriptional profiling. Journal of Immunology 178:5154-920 5165. 921 91. Luder CG, Walter W, Beuerle B, Maeurer MJ, Gross U. 2001. Toxoplasma 922 gondii down-regulates MHC class II gene expression and antigen 923 presentation by murine macrophages via interference with nuclear 924 translocation of STAT1alpha. European journal of immunology 31:1475-925 1484. 926 92. Lang C, Algner M, Beinert N, Gross U, Luder CG. 2006. Diverse 927 mechanisms employed by Toxoplasma gondii to inhibit IFN-gamma-induced 928 major histocompatibility complex class II gene expression. Microbes and 929 infection / Institut Pasteur 8:1994-2005. 930 93. Lang C, Hildebrandt A, Brand F, Opitz L, Dihazi H, Luder CG. 2012. 931 Impaired chromatin remodelling at STAT1-regulated promoters leads to 932 global unresponsiveness of Toxoplasma gondii-Infected macrophages to IFN-933 gamma. PLoS pathogens 8:e1002483. 934 94. Rosowski EE, Nguyen QP, Camejo A, Spooner E, Saeij JP. 2014. 935 Toxoplasma gondii Inhibits gamma interferon (IFN-gamma)- and IFN-beta-936 induced host cell STAT1 transcriptional activity by increasing the association 937 of STAT1 with DNA. Infection and immunity 82:706-719. 938 95. Courret N, Darche S, Sonigo P, Milon G, Buzoni-Gatel D, Tardieux I. 2006. 939 CD11c- and CD11b-expressing mouse leukocytes transport single 940 Toxoplasma gondii tachyzoites to the brain. Blood 107:309-316. 941 96. Hitziger N, Dellacasa I, Albiger B, Barragan A. 2005. Dissemination of 942 Toxoplasma gondii to immunoprivileged organs and role of Toll/interleukin-943 1 receptor signalling for host resistance assessed by in vivo bioluminescence 944 imaging. Cellular microbiology 7:837-848. 945 97. Bierly AL, Shufesky WJ, Sukhumavasi W, Morelli AE, Denkers EY. 2008. 946 Dendritic cells expressing plasmacytoid marker PDCA-1 are Trojan horses 947 during Toxoplasma gondii infection. Journal of Immunology 181:8485-8491. 948 98. Lavine MD, Arrizabalaga G. 2009. Induction of mitotic S-phase of host and 949 neighboring cells by Toxoplasma gondii enhances parasite invasion. 950 Molecular and biochemical parasitology 164:95-99. 951 99. Dvorak JA, Crane MS. 1981. Vertebrate cell cycle modulates infection by 952 protozoan parasites. Science (New York, N.Y 214:1034-1036. 953 100. Grimwood J, Mineo JR, Kasper LH. 1996. Attachment of Toxoplasma 954 gondii to host cells is host cell cycle dependent. Infection and immunity 955 64:4099-4104. 956 101. Carruthers VB, Giddings OK, Sibley LD. 1999. Secretion of micronemal 957 proteins is associated with toxoplasma invasion of host cells. Cellular 958 microbiology:225-235. 959 102. Boothroyd JC, Dubremetz JF. 2008. Kiss and spit: the dual roles of 960 Toxoplasma rhoptries. Nature reviews. Microbiology 6:79-88. 961 103. Carruthers V, Boothroyd JC. 2007. Pulling together: an integrated model of 962 Toxoplasma cell invasion. Current opinion in microbiology 10:83-89. 963

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

104. Koshy AA, Fouts AE, Lodoen MB, Alkan O, Blau HM, Boothroyd JC. 2010. 964 Toxoplasma secreting Cre recombinase for analysis of host-parasite 965 interactions. Nature methods 7:307-309. 966 105. Koshy AA, Dietrich HK, Christian DA, Melehani JH, Shastri AJ, Hunter CA, 967 Boothroyd JC. 2012. Toxoplasma co-opts host cells it does not invade. PLoS 968 pathogens 8:e1002825. 969 106. Neumann AK, Yang J, Biju MP, Joseph SK, Johnson RS, Haase VH, 970 Freedman BD, Turka LA. 2005. Hypoxia inducible factor 1 alpha regulates 971 T cell receptor signal transduction. Proceedings of the National Academy of 972 Sciences of the United States of America 102:17071-17076. 973 107. Lukashev D, Klebanov B, Kojima H, Grinberg A, Ohta A, Berenfeld L, 974 Wenger RH, Ohta A, Sitkovsky M. 2006. Cutting Edge: Hypoxia-Inducible 975 Factor 1{alpha} and Its Activation-Inducible Short Isoform I.1 Negatively 976 Regulate Functions of CD4+ and CD8+ T Lymphocytes. Journal of 977 Immunology 177:4962-4965. 978 108. Ben-Shoshan J, Maysel-Auslender S, Mor A, Keren G, George J. 2008. 979 Hypoxia controls CD4+CD25+ regulatory T-cell homeostasis via hypoxia-980 inducible factor-1alpha. European journal of immunology 38:2412-2418. 981 109. Clambey ET, McNamee EN, Westrich JA, Glover LE, Campbell EL, Jedlicka 982 P, de Zoeten EF, Cambier JC, Stenmark KR, Colgan SP, Eltzschig HK. 983 2012. Hypoxia-inducible factor-1 alpha-dependent induction of FoxP3 drives 984 regulatory T-cell abundance and function during inflammatory hypoxia of the 985 mucosa. Proceedings of the National Academy of Sciences of the United 986 States of America 109:E2784-2793. 987 988 989 on A

pril 11, 2021 by guesthttp://ec.asm

.org/D

ownloaded from

FIGURE LEGENDS 990 991 Figure 1. Glucose and Glutamine Utilization by Toxoplasma gondii. 992 Glucose (Glc) is scavenged from the host cell by the GT1 glucose transporter 993 and metabolized by either the apicoplast or cytosolic pathways. Glutamine (Gln) 994 is scavenged by an unknown transporter where it is converted to glutamate that 995 can be utilized by the mitochondrial Krebs Cycle as either α-ketoglutarate (α-KG) 996 or succinate (via the GABA shunt). 997 998 Figure 2. Schematic of inflammasome activation by Toxoplasma gondii. 999 Increased production of secreted IL-1β via the inflammasome requires two 1000 steps. Signal 1 causes activation of NF-κB leading to increased expression of 1001 pro-IL-1β (*pro-IL-18 is constitutively expressed). In Toxoplasma studies the 1002 triggering of Signal 1 is achieved either through natural parasite sensors/factors 1003 (e.g type II GRA15 in human monocytes) or priming of the cell by exogenous 1004 factors (e.g LPS & TLR-4). Signal 2 triggers the activation of an intracellular 1005 sensor that leads to assembly of the inflammasome, which causes activation of 1006 caspase-1, which then cleaves pro-IL-1β and pro-IL-18, and allows the secretion 1007 of processed IL-1β and IL-18. NLRP1 is an inflammasome sensor for human, 1008 rat, and mouse immune cells. In mice, NLRP3 is also activated by Toxoplasma. 1009 How Toxoplasma triggers either sensor is currently unknown. NLRP3 and 1010 CARDB are italicized to indicate that they interact with each other. 1011 1012

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

Figure 3. Modulation of Host Gene Expression by Non-Polymorphic 1013 Parasite Factors. Toxoplasma can modulate gene expression by either 1014 modulating host transcriptional regulators (EGRs, p53, or HIF-1) or indirectly by 1015 affecting chromatin remodeling. 1016 1017

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from

on April 11, 2021 by guest

http://ec.asm.org/

Dow

nloaded from