An ortholog of P. falciparum chloroquine resistance transporter (PfCRT) plays a key role in maintaining the integrity of the endolysosomal system in Toxoplasma gondii to facilitate host invasion

Toxoplasma gondii is an apicomplexan parasite with the ability to use foodborne, zoonotic, and congenital routes of transmission that causes severe disease in immunocompromised patients. The parasites harbor a lysosome-like digestive vacuole, termed the “Vacuolar Compartment/Plant-Like Vacuole” (VAC/PLV), which plays an important role in maintaining the lytic cycle and virulence of T. gondii. The VAC supplies proteolytic enzymes that are required to mature the parasite’s invasion effectors and that digest autophagosomes and endocytosed host proteins. Previous work identified a T. gondii ortholog of the Plasmodium falciparum chloroquine resistance transporter (PfCRT) that localized to the VAC. Here, we show that TgCRT is a membrane transporter that is functionally similar to PfCRT. We also genetically ablate TgCRT and reveal that TgCRT protein plays a key role in maintaining the integrity of the parasite’s endolysosomal system by controlling morphology of the VAC. When TgCRT is absent, the VAC dramatically increases in size by ~15-fold and co-localizes with its adjacent endosome-like compartment. Presumably to reduce aberrant swelling, transcription and translation of endolysosomal proteases are decreased in ΔTgCRT parasites. Expression of one endolysosomal subtilisin protease is quite significantly reduced, which impedes trimming of micronemal proteins, and significantly decreases parasite invasion. Chemical and genetic inhibition of proteolysis within the VAC reverses these effects, reducing VAC size and partially restoring the endolysosomal system, micronemal protein trimming, and invasion. Taken together, these findings reveal for the first time a physiological role of TgCRT in controlling VAC volume and the integrity of the endolysosomal system in T. gondii. Author Summary Toxoplasma gondii is an obligate intracellular protozoan parasite that belongs to the phylum Apicomplexa and that infects virtually all warm-blooded organisms. Approximately one-third of the human population is infected with Toxoplasma. The parasites invade host cells via processed invasion effectors in order to disseminate infection. A lysosome-like digestive vacuole (VAC) is involved in refining these invasion effectors to reach their final forms. A T. gondii ortholog of the malarial chloroquine resistance transporter protein (TgCRT) was found to be localized to the VAC membrane. Although the mutated version of the malarial chloroquine resistance transporter (PfCRT) has been shown to confer resistance to chloroquine treatment, its physiologic function remains poorly understood. Comparison between the related PfCRT and TgCRT proteins facilitates definition of the physiologic role of CRT proteins. In this study, we report that TgCRT plays a key role in regulating the integrity and proteolytic activity of the VAC and adjacent organelles, the secretion of invasion effectors, and parasite invasion and virulence. To relieve osmotic stress caused by VAC swelling when TgCRT is deleted, parasites repress proteolytic activities within this organelle to decrease solute accumulation, which then has secondary effects on parasite invasion. Our findings highlight a common function for PfCRT and TgCRT proteins in regulating apicomplexan parasite vacuolar size and function.

abnormal secretion of micronemal proteins alters their intracellular trafficking patterns, we stained pulse 247 invaded and replicated parasites with TgMIC2 and TgM2AP antibodies. Both microneme proteins trafficked 248 to the apical end of the parasites and showed normal staining patterns (Fig 3C). 249 250 Prior to secretion, some membrane-anchored micronemal proteins are released via proteolytic 251 cleavage by intramembrane rhomboid proteases such as TgROM4. The deletion of TgROM4 leads to 252 retention of some micronemal proteins on the parasite's plasma membrane, such as TgMIC2 and TgAMA1 253 (Toxoplasma apical membrane antigen 1) [6][7][8]28]. To test whether the aberrant endolysosomal system 254 alters the retention of micronemal proteins on the surface of parasites, we stained the purified, non-255 permeabilized extracellular parasites with anti-TgMIC2 antibody. Immunofluorescence microscopy did not 256 reveal excess TgMIC2 on the plasma membrane of ∆crt parasites (Fig S1), suggesting that the reduced 257 secretion of micronemal proteins is not due to inefficient intramembrane cleavage of micronemal proteins 258 on the parasite's plasma membrane. 259

260
The endosome-like compartment is involved not only in the trafficking of micronemal proteins, but also 261 rhoptry contents [29]. We stained newly invaded and replicated parasites with anti-TgROP7 antibodies to 262 examine the trafficking of rhoptry proteins and the morphology of the rhoptry. The TgROP7 staining 263 revealed typical rhoptry patterns located at the apical end of the parasites (Fig 3D), excluding the possibility 264 of aberrant trafficking of rhoptry contents and possible defects in biogenesis. Taken together, our data 265 suggest that invasion defects for ∆crt parasites are caused by incomplete trimming and consequent 266 inefficient secretion of micronemal proteins, but not by altered intracellular maturation, trafficking, or 267 intramembrane cleavage of micronemal proteins, nor by altered rhoptry morphology. 268 269 5. TgSUB1 transcript and protein levels are decreased for ∆crt parasites. 270 The inefficient proteolytic processing of TgMIC2 and TgM2AP in RH∆ku80∆crt ESAs led us to 271 investigate the abundance of Toxoplasma subtilisin 1 (TgSUB1) in the ∆crt parasites. A previous 272 publication reported that parasites lacking TgSUB1 showed defective trimming patterns for secreted 12 micronemal proteins, such as TgMIC2 and TgM2AP [3], which seemed, to us, to be similar to the secretion 274 patterns observed for the ∆crt mutant. Therefore, we quantified secreted TgSUB1 in both constitutive and 275 induced ESAs by probing them with an anti-SUB1 antibody, previously found to specifically react against 276 TgSUB1 and PfSUB1 [30]. Immunoblotting analysis revealed that there was no detectable TgSUB1 in the 277 ESAs of ∆crt parasites (Fig 4A). Non-permeabilized extracellular parasites were also stained with anti-278 SUB1 to evaluate the amount of surface-anchored TgSUB1. Similarly, there was no detectable TgSUB1 279 staining on the plasma membrane of ∆crt parasites (Fig 4B). These data suggest that TgSUB1 is not 280 efficiently delivered to the surface of parasites in the ∆crt mutant. 281 282 TgSUB1 is a micronemal protein that also traffics through the parasite's endolysosomal system [3,11]. 283 The aberrant endolysosomal system in ∆crt parasites potentially alters intracellular trafficking and/or 284 maturation of TgSUB1 that then reduces expression. To test these possibilities, first, we stained pulse 285 invaded and replicated parasites with anti-SUB1 to examine TgSUB1 intracellular trafficking patterns. 286 TgMIC5 localization was used as a reference for typical expected microneme staining. Surprisingly, we 287 observed much less TgSUB1 staining in ∆crt parasites compared to the WT strain (Fig 4C). Next, we 288 quantified abundance of TgSUB1 in parasite cell lysates and found that TgSUB1 was decreased by 289 approximately 90% in ∆crt parasites compared to WT parasites (Fig 4D). To further understand how 290 TgSUB1 expression is suppressed in the ∆crt mutant, we performed qPCR to measure TgSUB1 mRNA 291 for WT, ∆crt, and ∆crtCRT parasites. TgSUB1 transcript was reduced ~10-fold upon deletion of TgCRT 292 ( Fig 4E). Collectively, our findings suggest that arrested co-localization of the VAC and ELC dramatically 293 decreases the abundance of TgSUB1 protein, which then alters the proteolytic processing of normally 294 secreted micronemal protein invasion effectors, thereby reducing invasion efficiency. 295 296 6. VAC alterations reduce endolysosomal protease proteins and transcripts. 297 The swollen VAC and its aberrant co-localization with the ELC in the ∆crt parasites could conceivably 298 lead to altered gene transcription to assist in the adaptation of these parasites. We conducted 299 transcriptome sequencing to detect global alterations in gene transcription for ∆crt parasites relative to WT. 300

316
To determine the subcellular locations of these down-regulated proteases, we tagged endogenous 317 TgAMN and TgSCP with 3xHA and 3xmyc epitope tags at their C-termini in WT parasites, respectively 318 ( Fig S2). After drug-selection, we probed cell lysates from these tagged strains with anti-HA and anti-myc 319 antibodies, respectively, to test expression. Immunoblotting revealed that the observed molecular mass of 320 both proteins was similar to the predicted size based on primary sequences (Fig 5D). Next, the tagged 321 strains were co-stained with antibodies recognizing the epitope tags along with anti-TgCPL and anti-VP1 322 antibodies to determine subcellular location. Immunofluorescence microscopy revealed both TgSCP and 323 TgAMN to be in the VAC/ELC (Fig 5D) TgASP1 subcellular location was also determined to be within the 324 VAC (data not shown; Dou, Z. et al., in preparation). Collectively, these data suggest that the swollen VAC 325 in ∆crt parasites causes reduced transcription and translation of several endolysosomal proteases. 326 327 7. Suppression of proteolysis within the swollen ∆crt VAC partially restores VAC size, organellar 328 separation, and invasion. 329 We suspected that inhibition of proteolysis might reduce the size of the swelled VAC. We tested this 330 hypothesis by chemically and genetically suppressing VAC proteolysis. First, we treated WT, ∆crt, and 331 ∆crtCRT parasites with 1 µM LHVS, an irreversible inhibitor of TgCPL protease [23]. As mentioned TgCPL 332 is a major endopeptidase involved in the maturation of micronemal proteins and digestion of host proteins 333 [4,10]. Infected host cells were incubated with LHVS for 48 hrs to allow full inhibition of TgCPL. Treated 334 parasites were liberated from host cells and used to infect new host cells for 30 minutes, followed by TgCPL 335 staining to quantify the size of the VAC. As expected, LHVS-treated ∆crt parasites displayed smaller VACs 336 than DMSO-treated ∆crt parasites (Fig 6A). TgCPB is another known VAC-localizing protease, displaying 337 both endo-and exo-peptidase activities [13,23]. Due to its carboxypeptidase activity, it is expected that 338 TgCPB generates more small solutes relative to TgCPL. We used CRISPR-Cas9 editing to generate a 339 ∆crt∆cpb double knockout (Fig 6B). The replacement of TgCPB with a pyrimethamine resistance cassette 340 was confirmed by PCR and immunoblotting (Fig 6B). The resulting ∆crt∆cpb mutant showed a smaller 341 concave subcellular structure compared to the ∆crt mutant ( Fig S3). The size of the VAC in WT, ∆crt, and 342 ∆crt∆cpb was quantified based on the TgCPL staining as described above and the ∆crt∆cpb parasite VAC 343 (0.85 ± 0.15 µm) was reduced by ~30% compared to ∆crt parasites (1.15 ± 0.10 µm) (Fig 6C). The 344 moderate decrease in the size of the VAC in the ∆crt∆cpb strain also reduced the number of parasites 345 showing partial overlap between the VAC and ELC. Approximately 44% of ∆crt∆cpb parasites showed 346 partial overlap between TgCPL and proTgM2AP staining compared to 62% in the ∆crt strain (Fig 6D), with 347 both significantly higher than the 19% and 25% seen for WT and ∆crtCRT strains, respectively. TgSUB1 348 showed comparable expression in both the WT and ∆crt∆cpb strains (Fig 6E). Similarly, TgSUB1 was 349 observed in both constitutive and induced ESA fractions in the ∆crt∆cpb parasites (Fig 6F). TgM2AP and 350 TgMIC2 were cleaved by TgSUB1 in ∆crt∆cpb, and their secretion patterns were similar to those seen in 351 the WT strain (Fig 6F). These partially restored phenotypes in the ∆crt∆cpb mutant improved the invasion 352 efficiency by ~ 60% compared to the ∆crt strain, although invasion was still significantly lower than that of 353 WT parasites (Fig 6G). Collectively, these data show a close association between the size of the VAC, 354 15 altered morphology of the parasite's endolysosomal system, protein abundance of TgSUB1, and parasite 355 invasion. 356 357

TgCRT is a functional transporter. 358
Finally, we attempted to express TgCRT in S. cerevisiae yeast following previously described 359 strategies for PfCRT [31,32]. Native TgCRT cDNA did not express well in S. cerevisiae yeast (Fig S5), 360 however, following a previously published strategy for difficult to express PfCRT mutants [20] we created 361 a fusion gene that replaced the 300 most N-terminal residues of the TgCRT sequence with 111 most N-362 terminal residues from S. cerevisiae plasma membrane ATPase (PMA), which harbors a yeast plasma 363 membrane localization sequence (Fig S4B and S4C). Via alignment with PfCRT (Fig S4A), removing the 364 300 N-terminal TgCRT residues that are non-homologous to PfCRT preserves all putative 365 transmembraneous domains and inter helical loop regions. The fusion protein was well expressed in S. 366 cerevisiae (Fig S5). Following an approach previously described for PfCRT and PfCRT mutants [20,33] 367 we assayed PMA-TgCRT expressing yeast for chloroquine (CQ) transport (Fig. 7A). Via alignment with 368 PfCRT (Fig S4A), TgCRT T369 corresponds to the well-studied K76 residue within PfCRT; previously, 369 mutation of PfCRT K76 to T has been shown to increase the efficiency of CQ transport by PfCRT [20,34,35]. 370 We individually expressed both WT TgCRT and a T369K variant in the yeast to measure their transport 371 efficiencies. Both the wild type protein and a TgCRT T369K mutant were found to transport CQ slower 372 than PfCRT under similar conditions and to require higher external [CQ] (80 mM versus 16 mM for PfCRT) 373 to achieve similar levels of transport (Fig 7A). These initial data have shown that mutation of the 374 corresponding TgCRT threonine to lysine affects CQ transport similarly. 375

376
We also exchanged threonine for lysine in the WT TgCRT complementation construct and transfected 377 ∆crt to examine the extent to which TgCRT T369K affects VAC size in Toxoplasma parasites. Interestingly, 378 in contrast to full recovery of VAC size in the WT TgCRT complementation strain, TgCRT T369K only partially 379 restored the swollen VAC (Fig 7B). These findings, along with the TgCRT transport data, strongly suggest 380 that the swollen VAC is caused by luminal osmolyte excess, similar to findings for PfCRT as described in 381 "Discussion". 382

383
In summary, our findings strongly suggest a role for TgCRT in small solute transport that regulates 384 VAC volume, similar to the role proposed for PfCRT [16,17]. However, at least for T. gondii, osmotic 385 pressure within this organelle regulated by the TgCRT protein governs proper segregation of other 386 organelles within the endolysosomal system that, in turn, facilitates microneme secretion and parasite 387 invasion. The data also indicate that the invasion deficiency exhibited by the ∆crt mutant is likely due to 388 multiple factors, since the recovery of TgSUB1 expression and micronemal trimming in ∆crt∆cpb did not 389 completely reverse invasion defects. To our best knowledge, this is the first observation of regulation of Here, we created a TgCRT knockout that completely removes TgCRT from the VAC membrane. The 405 resulting ∆crt strain shows a dramatic increase in VAC size, and the organelle aberrantly co-localizes with 406 the adjacent endosome-like compartment (Fig 8). Although a previous study reported that parasites deliver 407 minor amounts of TgCPL to the ELC which then contributes to maturation of some micronemal proteins 408 We also measured the retention and secretion of micronemal proteins on the parasite's surface and in 414 the medium executed by TgROM4 and TgSUB1, respectively. We found that the micronemal proteins were 415 improperly trimmed on the surface of ∆crt parasites. Patterns of secreted micronemal proteins observed 416 for the ∆crt mutant were similar to those for ∆sub1 parasites, which led us to examine the expression of 417 TgSUB1 in ∆crt parasites and ESAs. As expected, levels of TgSUB1 were decreased on the surface of 418 ∆crt parasites and in the medium during secretion. Interestingly, the steady state abundance of TgSUB1 419 was also significantly decreased in the ∆crt mutant. Surprisingly, we found that the reduction of TgSUB1 420 was due to a decrease in the transcription level of TgSUB1 in the ∆crt strain, suggesting that the parasites 421 utilize a feedback transcriptional mechanism to regulate TgSUB1. in an increase in osmotic pressure within the hybrid VAC/ELC organelle (Fig 8). During this scenario, the 432 parasites may utilize a feedback mechanism to repress additional expression of TgSUB1 in order to avoid 433 further VAC swelling. Moreover, we also discovered that the ∆crt parasites had reduced protein and/or 434 transcript levels of several other proteases, including two known VAC proteases, TgCPL and TgCPB. 435 Therefore, the parasites down-regulate a number of endolysosomal-VAC proteases to suppress proteolytic 436 activities in the swollen VAC, presumably to reduce osmotic pressure and thereby control VAC size. 437 Among these proteases, TgSUB1 has been shown to be involved in parasite invasion and virulence defects 438 but not replication and egress [3]. Additionally, TgCPL plays a role in parasite invasion by maturing several 439 micronemal proteins [4]. Therefore, the invasion defects exhibited in the ∆crt mutant could be due to 440 several factors. 441 442 Altered endolysosomal protease transcript levels in ∆crt parasites suggest that parasites repress 443 transcription factors or enhance transcription repressors to respond to increased VAC size. RNA-Seq 444 analysis did not reveal any changes in the AP2-family of transcription factors (data not shown). In 445 mammalian cells, the transcription factor EB (TFEB) is a master regulator that drives gene expression for 446 autophagy and lysosome biogenesis [37]. Search of the Toxoplasma genome did not reveal a TgTFEB 447 ortholog, suggesting that these parasites may adopt an alternative strategy for regulating lysosomal gene 448 expression. Interestingly, our differential gene expression analysis identified that the transcript levels of 449 two zinc finger (CCCH) type motif-containing proteins, TGGT1_246200 and TGGT1_226310, were 450 increased and decreased by 2-fold and 3-fold (Table S1), respectively, in the ∆crt mutant. The CCCH type 451 zinc finger motif-containing protein is known to regulate the stability of mRNA [38]. For example, 452 tristetraprolin inhibits the production of tumor necrosis factor-α in macrophages by destabilizing its mRNA 453 via an interaction with AU-rich elements at the 3'-untranslated region [39]. Further investigation to identify 454 transcription factor(s) and regulator(s) that govern the expression of Toxoplasma lysosomal genes will help 455 elucidate how these parasites regulate the biogenesis and function of the VAC. 456

457
In this study, we have determined that TgCRT-deficient parasites have reduced expression of several 458 endolysosomal proteases. We have also found that suppression of proteolytic activities within the swollen 459 VAC decreases the size of the organelle. These findings, along with data verifying that TgCRT is indeed a 460 transporter with function similar to that of PfCRT, support the idea that TgCRT functions to transport 461 essential VAC osmolytes, similar to proposals for PfCRT [16,17,40]. Likely candidate osmolytes include 462 ions and/or amino acids. We suggest that when TgCRT is absent on the membrane of the VAC, protein 463 degradation products (short peptides, amino acids) likely accumulate within the VAC and increase osmotic 464 pressure, thereby leading to the swollen phenotype. Consistent with this idea, and similar to related . Therefore, it seems likely that ∆crt parasites express less 480 aquaporin to reduce water transport into the VAC/PLV, as an additional tactic to limit VAC swelling. We 481 also found that two putative protein phosphatase 2C (TGGT1_276920 and TGGT1_201520) transcripts 482 are down-regulated in the ∆crt mutant. Both carry signal peptides, indicating endosomal trafficking. 483 TGGT1_276920 and TGGT1_201520 are homologous to PTC3 and PTC1 in S. cerevisiae, respectively. 484 Interestingly, both PTC1 and PTC3 proteins are involved in yeast osmosensory regulation. A mitogen-485 activated protein kinase pathway is activated when yeast cells experience hyperosmotic conditions. PTC1 486 and PTC3 negatively regulate this pathway [43,44]. Furthermore, PTC1 was found to control the function 487 and morphology of the yeast vacuole, which further alters its biogenesis [45]. The dramatic change in 488 20 Toxoplasma VAC volume indicates induced osmotic stress in the ∆crt parasites. The knockout parasites 489 appear to be utilizing a similar mechanism to suppress these protein phosphatases and enhance similar 490 osmoregulatory signaling. We suggest similar studies for P. falciparum and other apicomplexan parasites 491 that express CRT orthologs would be informative. proteoliposomes, have revealed that PfCRT may act as a proton gradient dependent, polyspecific nutrient 499 exporter for small solutes including amino acids, oligopeptides, glutathione, and small drugs [18,19]. These 500 studies also demonstrate that CQR-associated PfCRTs display altered transport efficiency relative to CQ-501 associated PfCRT. Our study has revealed that TgCRT mediates CQ transport similar to PfCRT. The ∆crt 502 strain appears more sensitive to CQ relative to WT parasites (Fig S6), further suggesting that TgCRT is a 503 functional transporter of small solutes across the membrane of the VAC. We suggest that alteration of 504 proteolytic activities in the enlarged VAC of the ∆crt mutant reveals a similar scenario relative to the CQR 505 P. falciparum DV. Given the similarity in components and functionality of the VAC and DV found in 506 Toxoplasma and Plasmodium, this Toxoplasma TgCRT-deficient mutant should prove useful for further 507 studying the native function of CRT orthologs from other apicomplexan parasites. 508 509 In sum, our findings reveal that the Toxoplasma TgCRT protein is indeed a small molecule transporter 510 that plays an essential role in regulating the size and morphology of the VAC. Unexpectedly, this regulation 511 maintains integrity of the parasite's endolysosomal system, which is essential for the trafficking of invasion 512 effectors. Co-localization of the VAC and endosome-like compartment in the TgCRT knockout led to a 513 reduction in transcript and protein levels for several endolysosomal proteases. We found that blocking 514 normal proteolysis within the swollen VAC reduced the size and partially restored the morphology of the 515 organelle. Taken together, these findings suggest that TgCRT mediates the transport of small solutes in 516 order to regulate VAC size and morphology. The data show that the integrity of the parasite endolysosomal 517 system is critical for parasite virulence. We suggest that pharmaceutical modulation of the VAC could serve 518 as a novel strategy for managing toxoplasmosis. To complement ∆crt parasites, we modified the plasmid pTub-TgCRT-mCherry-3xmyc (a gift from the 548 van Dooren lab), which expresses a C-terminally mCherry-3xmyc epitope-tagged TgCRT under the 549 Toxoplasma tubulin promoter. The plasmid was restricted with HpaI and MfeI to remove the tubulin 550 promoter and a segment of TgCRT. The remaining DNA fragment served as the backbone for subsequent 551 Gibson assembly to incorporate a PCR amplified ~1 kb region upstream of the Tgku80 gene, the ~1 kb 552 fragment of the Tgcrt 5'-UTR region, and the removed partial Tgcrt coding sequence to produce the TgCRT 553 complementation plasmid, pCRT-TgCRT-mCherry-3xmyc. The complemented TgCRT is driven by its 554 cognate promoter to maintain physiologic similarity to native TgCRT expression in WT parasites (see text). 555 The 1 kb region located ~6 kb upstream of the Tgku80 gene was used to facilitate a single integration of 556 the TgCRT complementation plasmid into this specific locus by single crossover homologous 557 recombination. The TgCRT complementation construct was digested with SwaI restriction enzyme, gel-558 extracted, purified, and transfected into ∆crt parasites by electroporation. 559 560 To introduce NanoLuc ® luciferase (nLuc) into parasites, we PCR-amplified and assembled the 561 Cas9-GFP and the PCR product were co-transfected into WT parasites. The stop codon of TgAMN was 585 replaced by the 3xHA epitope tag and pyrimethamine resistance cassette. Stable populations were 586 generated after multiple rounds of pyrimethamine selection and TgAMN-3xHA fusion protein was 587 confirmed by immunoblotting analysis. 588 589 TgSCP was endogenously tagged with a 3xmyc epitope tag via single crossover. An approximately 1 590 kb region upstream of the TgSCP stop codon was PCR amplified and fused in frame with a 3xmyc epitope 591 to assemble TgSCP-3xmyc. A pyrimethamine resistance cassette was also included, the resulting plasmid 592 was linearized and transfected into WT parasites. The correct tagging was confirmed by immunoblotting. 593 594

Site-directed mutagenesis 595
Threonine 369 was mutated to lysine in the WT TgCRT complementation construct, via site directed 596 mutagenesis according to the Q5 ® site-directed mutagenesis procedure (NEB). Linear PCR product was 597 phosphorylated, circularized, and transformed into E. coli. Correct clones were identified by direct DNA 598 sequencing. 599 600 Transfection of Toxoplasma parasites 601 T. gondii parasites were allowed to grow in HFF cells for 48 hrs at 37 °C with 5% CO2. Freshly egressed 602 parasites were syringed, filter purified, and harvested in Cytomix buffer (25 mM HEPES, pH 7.6, 120 mM 603 KCl, 10 mM K2HPO4/ KH2PO4, 5 mM MgCl2. 0.15 mM CaCl2, and 2 mM EGTA). Parasites were pelleted 604 at 1,000x g for 10 min, washed once in Cytomix buffer, and resuspended in Cytomix buffer at 2.5 x 10 7 605 parasites per ml. 400 μL of parasite suspension was mixed with 20 μg DNA and 2 mM ATP/5 mM reduced 606 glutathione to a final volume of 500 µL. The mixture was electroporated at 2 kV and 50 ohm resistance 607 using the BTX Gemini X2 (Harvard Apparatus). Transfectants were inoculated into a T25 flask pre-seeded 608 with confluent monolayer of HFF cells and the cells allowed to recover. Drug selection was applied 24 hrs 609 post transfection. 610 611 Immunofluorescence 612 Freshly lysed parasites were used to infect confluent HFF cells pre-seeded in an 8-well chamber slide 613 for 1 hr (pulse invaded parasites) or 18-24 hrs (replicated parasites). The extracellular parasites were 614 attached to chamber slides using 0.1% (w/v) poly-L-lysine. Immunofluorescence was performed as 615 described previously [10,13]. Images were viewed and digitally captured using a Leica ® CCD camera 616 equipped with a DMi8 inverted epifluorescence microscope and processed with Leica ® LAS X software. 617 618

Excretory secretory antigens (ESAs) preparation 619
Freshly egressed parasites were syringed, filter purified, and resuspended at 5 x 10 8 parasites/ml in 620 D1 medium (DMEM medium supplemented with 1% FBS). 100 µL of parasite suspension was transferred 621 to a microfuge tube and incubated at 37 °C for 30 min to prepare constitutive ESAs. To isolate induced 622 ESAs, the parasite suspension was incubated in D1 medium supplemented with 1% ethanol for 2 min at 623 37ºC. ESAs were separated from intact parasites by centrifugation at 1,000 x g for 10 min. ESA fractions 624 were transferred to a new microfuge tube, mixed with SDS-PAGE sample loading buffer, and boiled for 5 625 min for immunoblotting analysis. 626

SDS-PAGE and Immunoblotting 628
Parasite lysates and ESA fractions were prepared in 1x SDS-PAGE sample buffer and boiled for 5 min 629 before resolving on standard SDS-PAGE gels. For immunoblotting, gels were transferred to PVDF 630 membranes by semi-dry protein transfer methods. Blots were blocked with 5% non-fat milk and incubated 631 with primary antibody diluted in 1% non-fat milk. Goat anti-mouse or anti-rabbit IgG antibodies conjugated 632 with horseradish peroxidase were used as secondary antibody. Immunoblots were developed with 633 SuperSignal TM WestPico chemiluminescent substrate (Thermo). The chemiluminescence signals were 634 captured using the Azure ® Imaging System. Bands were quantified by densitometry using LI-COR ® Image 635 Studio software. 636 637

Parasite invasion assay 638
The red-green invasion assay was used to measure the efficiency of parasite invasion. Freshly purified 639 parasites were syringed, filter purified, and resuspended at 5 x 10 7 parasites/ml in invasion medium (DMEM 640 supplemented with 3% FBS). 200 µL of parasite resuspension was inoculated into each well of an 8-well 641 chamber slide pre-seeded with HFF cells, and parasites were allowed to invade host cells for 30, 60, and 642 120 min before fixation with 4% formaldehyde for 20 min. Before membrane permeabilization, slides were 643 stained with mouse anti-TgSAG1 monoclonal antibody (1:1,000) for 1 hr to label attached parasites. After 644 treatment with 0.1% Triton X-100 for 10 min, the parasites were stained with rabbit polyclonal anti-TgMIC5 645 antibody (1:1,000) for 1 hr to stain both invaded and attached parasites. Subsequently, slides were stained 646 with goat anti-mouse IgG conjugated with Alexa 594 (red) (Invitrogen, 1:1,000) and goat anti-rabbit IgG 647 conjugated with Alexa 588 (green) (Invitrogen, 1:1,000) along with DAPI for nuclear staining. Freshly lysed parasites were filter-purified and resuspended in D10 medium at 5 x 10 5 parasites/ml. 100 682 µL of parasite suspension was inoculated into each well of a 96-well plate pre-seeded with HFF cells. The 683 parasites were allowed to replicate for 18-24 hrs, washed, and incubated with 50 µl of Ringer's buffer (10 684 mM HEPES, pH 7.2, 3 mM HaH2PO4, 1 mM MgCl2, 2 mM CaCl2, 3 mM KCl, 115 mM NaCl, 10 mM glucose, 685 and 1% FBS) for 20 min. Subsequently, an equal volume of 1 mM Zaprinast dissolved in Ringer's buffer 686 was added to the wells and incubated for 5 min at 37ºC and 5% CO2. Uninfected wells were treated with 687 50 µl of Ringer's buffer containing 1% Triton X-100 or normal Ringer's buffer, serving as positive and 688 negative controls, respectively. The released lactate dehydrogenase was centrifuged at 1,000 x g for 5 689 min twice to pellet insoluble cell debris. Six-to eight-week-old, outbred CD-1 mice were infected by subcutaneous or intravenous injection with 720 100 WT or mutant parasites diluted in PBS. The infected mice were monitored for symptoms daily for a 721 total of 30 days. Mice that appeared moribund were humanely euthanized via CO2 overdose, in compliance 722 with IACUC's approved protocol. The seroconversion of the surviving mice was tested by enzyme-linked 723 immunosorbent assay (ELISA). The surviving mice were allowed to rest for 10 days, prior to subcutaneous 724 injection with a challenge dose of 1000 WT parasites, and were monitored daily for survival for 30 days. 725 726

Generation of TgCRT expression construct in yeast 727
TgCRT cDNA was PCR amplified from pTub-TgCRT-mCherry-3xmyc plasmid using a forward primer 728 that introduced a 5' KpnI site and S. cerevisiae Kozak sequence, and a reverse primer that omitted the 729 mCherry-3xmyc tag and introduced a 3' XmaJI site. The PCR amplified DNA was digested with KpnI and 730 XmaJI and subcloned into pYES2-6xHis-BAD-V5 (hexa His, biotin acceptor domain, V5 tags) plasmid 731 behind the GAL1 promoter and in front of the His-BAD-V5 epitope tags to generate the plasmid 732 pYES/TgCRT-hbv. To generate the plasmid pYES/PMA-TgCRT-hbv, DNA encoding TgCRT-hbv was PCR 733 amplified using a forward primer that omitted the first 900 bases of TgCRT and introduced a 5' SacI site, 734 and a reverse primer that included a 3' NotI site and His-BAD-V5 tags. The amplified DNA was digested 735 with SacI and NotI and subcloned into a SacI/NotI-digested pYES/PfHB3PMA (from [32]; modified via site-736 directed mutagenesis to introduce a SacI site at the PMA-PfCRT interface). Mutagenesis reactions were 737 performed using reagents obtained from Agilent (Santa Clara, CA).  TgCRT was deleted by double crossover homologous recombination ∆crtCRT RH∆ku80∆hxg∆crt::TgCRT-mCherry-3xmyc Ectopic expression of a C-termially epitopetagged TgCRT in ∆crt for complementation ∆crtCRT T369K RH∆ku80∆hxg∆crt::TgCRT T369K -mCherry-3xmyc Ectopic expression of a C-termially epitopetagged TgCRT mutant in ∆crt for complementation. The original threonine at position 369 within TgCRT was changed to lysine by site-directed mutagenesis.