Histone variant H2B.Z acetylation is necessary for maintenance of Toxoplasma gondii biological fitness

Through regulation of DNA packaging, histone proteins are fundamental to a wide array of biological processes. A variety of post-translational modifications (PTMs), including acetylation, constitute a proposed histone code that is interpreted by “reader” proteins to modulate chromatin structure. Canonical histones can be replaced with variant versions that add an additional layer of regulatory complexity. The protozoan parasite Toxoplasma gondii is unique among eukaryotes in possessing a novel variant of H2B designated H2B.Z. The combination of PTMs and the use of histone variants is important for gene regulation in T. gondii, offering new targets for drug development. In this work, T. gondii parasites were generated in which the 5 N-terminal acetylatable lysines in H2B.Z were mutated to either alanine (c-Myc-A) or arginine (c-Myc-R). c-Myc-A mutant only displayed a mild effect in its ability to kill mice. c-Myc-R mutant presented an impaired ability to grow and an increase in differentiation to latent bradyzoites. This mutant line was also more sensitive to DNA damage, displayed no virulence in mice, and provided protective immunity against future infection. While nucleosome composition was unaltered, key genes were abnormally expressed during in vitro bradyzoite differentiation. Our results show that the N-terminal positive charge patch of H2B.Z is important for these procceses. Pull down assays with acetylated N-terminal H2B.Z peptide and unacetylated one retrieved common and differential interactors. Acetylated peptide pulled down proteins associated with chromosome maintenance/segregation and cell cycle, opening the question of a possible link between H2B.Z acetylation status and mitosis.


46
Histones proteins function to form nucleosomes that regulate packaging of DNA, thereby affecting 47 Tetrahymena termophila, this number is considerably higher (between 5 and 16). Moreover, the presence 93 of a T. gondii double variant nucleosome that altogether carries 15 acetylatable lysines that could be 94 regulated is highly intriguing. 95 In the present work we studied the role of the five T. gondii H2B.Z N-terminal tail lysines, using a 96 mutagenesis strategy; we also pursued a gene knockout strategy for H2B.Z. In an RH strain background, we 97 generated T. gondii that possess mutated versions of H2B.Z: c-Myc-R, in which the five N-terminal 98 acetylatable lysines were replaced by arginines, and c-Myc-A, in which they were replaced by alanines. 99 These lines were analyzed for their ability to grow in vitro, differentiate to bradyzoites in vitro and produce 100 virulence in mice. In addition, expression of key genes was studied, as well as nucleosome composition and 101 DNA damage sensitivity. Our results suggest that regulation of the N-terminal positive charge patch by 102 lysine acetylation, rather than the histone code, is essential for the aforementioned biological processes. 103 We have also found some differential interactors for an acetylated N-terminal H2B.Z peptide, compared to Attempts to obtain a mutant cell line by deletion of the h2b.z gene were unsuccessful (data not 119 shown). The failure to obtain an H2B.Z. knockout is consistent with its CRISPR fitness score of -4.05, strongly 120 suggesting it is essential [27] (ToxoDB, TGGT1_209910). 121 Given the high number of modifiable lysines in the H2B.Z variant unique to apicomplexan parasites, 122 we analyzed their role by using an over-expression strategy of myc-tagged wild-type or mutant forms. We 123 generated parasites over-expressing wild-type H2B.Z (c-Myc-WT-OE) or a mutated form in which all the 124 acetylatable lysines in the N-terminal tail were changed to alanine, which mimics how acetylation ablates the 125 positive charge of the lysine residues. We also over-expressed a mutated version in which the lysines were 126 replaced with arginines, which would generate a constitutive positive charge patch but no longer support 127 acetylation (Fig. 1B). Neither mutant form would be acetylated in this region, therefore would be incapable 128 mutations, which were named c-Myc-A and c-Myc-R ( Fig. 2A-C, Fig. S1C-D). Each form of c-Myc H2B.Z in these 138 parasites localized properly to the nucleus (Fig. 2B). In both CRISPR manipulated clones, a single band 139 corresponding to c-Myc-tagged mutated H2B.Z can be observed by western blot with anti-H2B.Z and anti-c-140 Myc antibodies (Fig. 2C). Quantification of the band corresponding to c-Myc H2B.Z relativized to Sag1 as a 141 charge control, was 1.43 ± 0.77 and 1.65 ± 0.82 for c-Myc-R and c-Myc-A, respectively, indicating comparable 142 levels of expression between both mutants. 143 144

In vitro growth analysis in c-Myc-R and c-Myc-A T. gondii lines 145
We used a competitive growth assay to determine differences in parasite growth between parental but cannot be acetylated, these results indicate that the constitutive positive charge patch on the H2B.Z N-152 terminal tail, but not the lack of acetylation, impairs tachyzoite growth in vitro. Therefore, wild-type T. gondii 153 must regulate the charge patch on H2B.Z N-terminal tail by PTMs such as acetylation for normal growth. 154 155

In vitro differentiation analysis in c-Myc-R and c-Myc-A T. gondii lines 156
We next studied the effect of the mutant H2B.Z variants on in vitro tachyzoite to bradyzoite 157 differentiation using alkaline stress to trigger stage conversion and Dolichos biflorus lectin (DBL) to monitor 158 tissue cyst wall formation. Strikingly, we found that c-Myc-R, but not c-Myc-A, showed an increase in  positive vacuoles compared to the wild-type control (Fig. 3B), suggesting that the impossibility to regulate 160 the presence of a positive charge patch in the H2B.Z N-terminal tail promotes an in vitro differentiation. 161 We also checked whether key stage-specific genes were altered in parasites expressing mutant 162 H2B.Z. We obtained RNA from freshly lysed tachyzoites, early bradyzoites (48 hours post-differentiation), a cystogenic strain with the treatment, and different from RH expression profile (Fig. 3C). Also, the expression 170 of BFD1 showed an increase in c-Myc-R, compared to RH (Fig. 3C). Notably, the expression profile of BFD1 in 171 RH type I strain has not been published to date. These results are consistent with the heightened bradyzoite 172 differentiation rate seen in c-Myc-R parasites. 173 174

Sensitivity to DNA damaging agents in c-Myc-A and c-Myc-R lines 175
In other species, H2A with MMS for c-Myc-R tachyzoites (Fig. 4A). By contrast, topotecan did not produce a significant defect in 184 growth, although c-Myc-R seems to be more sensitive (Fig. 4A). In addition, treatment with MMS produced 185 a disorganization of vacuoles, rounded shapes and a loss of genetic material in both mutants as well as 186 parental (Fig. 4B). T. gondii normally presents a synchronic replication in the parasitophorous vacuole (PV). 187 As a result, PVs usually show 2, 4, 8, 16 and so on, tachyzoites (Tz) per PV. Treatment with MMS and 188 Topotecan also caused a loss of synchronization, with an altered number of Tz per PV, which was also counted 189 as "anomalous vacuole". Anomalous vacuoles and loss of genetic material were also observed with topotecan 190 treatment (Fig. 4B). However, quantification showed that anomalous PV are significantly more abundant in 191 c-Myc-R than parental and c-Myc-A, with both drugs tested (Fig. 4C). Taken together, the constitutive positive 192 charge patch generated in the c-Myc-R parasites alter the DNA damage response after MMS treatment,193 suggesting that acetylation status of H2B.Z N-terminal tail is likely to be modulated for proper DNA repair. 194 195

In vivo analysis in c-Myc-R and c-Myc-A T. gondii lines 196
Proper gene regulation is required for tachyzoite propagation in different tissue environments and 197 differentiation in vivo. Progression of tachyzoite infection of these mutants was analyzed in C57BL/6 mice by 198 inoculating 100 tachyzoites per T. gondii line. As expected, parental parasites displayed high virulence, killing 199 mice within 7 days (Fig. 5A). c-Myc-A tachyzoites were also lethal to mice, but with a delay in the time of 200 death (Fig. 5A). According to Mantel-Cox and Gehan-Breslow-Wilcoxon tests, the survival curves are 201 significantly different (p>0.0001), suggesting that acetylation of at least some of the 5 N-terminal tail lysines 202 is relevant for parasite progression in vivo. 203 In contrast, c-Myc-R and Me49 infected mice, as well as non-infected (PBS group) survived until 204 sacrifice on day 30-35 (Fig. 5A). c-Myc-WT-OE was used a control that the tagged version is not responsible 205 per se of any affect observed. T. gondii line expressing both endogenous H2B.Z and c-Myc-H2B.Z-R (c-Myc-R-206 OE) previous to CRISPR procedure, were as lethal as the parental line (data not shown), suggesting that the 207 randomly integrated ptub-H2B.Z-R gene is not responsible for the loss of virulence and that co-expression of 208 H2B.Z wt and H2B.Z-R mutant histone did not generate a dominant negative T. gondii line. Infection with 209 1,000 and 10,000 c-Myc-R tachyzoites were also nonlethal to mice (Fig S2A). 210 To confirm acute infection took place, hematoxylin and IFA staining of the peritoneal fluids at day 4 211 post-infection were performed (Fig. 5B). Intraperitoneal tachyzoites could be detected only with parental 212 and c-Myc-A T. gondii lines infections, but not with c-Myc-R parasites (Fig. 5B). Surviving animals (c-Myc-R 213 and Me49 T. gondii lines) were analyzed to detect antibodies against T. gondii. An increase in total IgG in 214 mice infected with c-Myc-R tachyzoites was observed, comparable to Me49, which indicated that an initial 215 infection had taken place (Fig. 5C, Fig. S2B). The analysis of IgG subtypes showed a Th1-like humoral response 216 in both cases, associated to IgG2a and higher for IgG2b (normal for C57/BL6 mice), typical for an intracellular 217 parasite infection (Fig. 5C, Fig. S2B). Following sacrifice, brains of surviving mice were processed for cyst 218 detection by direct observation with optical microscopy and DBL staining. Tissue cysts were observed in 219 brains of mice infected with Me49 strain, but not in those infected with c-Myc-R parasites (data not shown). 220 To determine if infection with c-Myc-R parasites elicits an adequate immune response, mice 221 previously infected with 1,000 c-Myc-R tachyzoites were reinfected with 100 tachyzoites of the highly virulent 222 RH parental line. c-Myc-R infected mice survived until sacrifice, while all mice in the control group died at day 223 38 (day 8 after re-infection) (Fig. 5D). These results suggest that c-Myc-R tachyzoites must have been capable 224 of invading cells and producing a stimulation of the cellular immune response adequate to control a challenge 225 infection before clearing. 226 227

H2B.Z acetylation status is involved in regulation of ROP proteins expression 228
The ability of the c-Myc-R line to establish a protective immune response suggest these parasites can 229 invade the host cells but are efficiently controlled by the immune system. Among the T. gondii virulence 230 proteins that exert protection from PV establishment are the polymorphic rhoptry proteins Rop5 and Rop18, 231 which disrupt the IFN-inducible IGR pathway [47][48][49][50][51]. Here, we used antibodies against T. gondii Rop5 232 (TGGT1_411430) and Rop18 (TGGT1_205250) recombinant proteins for western blot analysis. Rop5 was 233 present in parental, c-Myc-A, and c-Myc-R parasites, whereas Rop18 only in the parental line (Fig. 6A). 234 However, quantification of the band intensities showed a significative decrease in Rop5 abundance in c-Myc-235 R parasites and a lack of Rop18 protein in both c-Myc-A and c-Myc-R ( Fig. 6B and Fig. S3A). To test whether 236 the absence of Rop18 was a consequence of the insertion of the H2B.Z construction in that locus, we 237 performed the same experiment using the over-expressing tachyzoites obtained prior to CRISPR 238 manipulation. As shown in Fig.S3 (B, C) both Rop5 and Rop18 are detectable in every clone similar to parental. 239 Co-localization analysis showed a correct localization of Rop5 in all T. gondii lines whereas Rop18 could only 240 be detected in the parental line (Fig. 6C). Of note, we also detected a decrease in the fluorescence intensity 241 of Rop5 in c-Myc-R parasites (Fig. 6D). RT-PCR analysis indicated that both genes were expressed in every T. The positive charge patch on H2B.Z could be associated with a more compact chromatin structure. 268 Since the mark of H2B.Z, either wild type or mutants, and DAPI are coincident in the diameter of the nucleus, 269 to analyze chromatin compaction, we measured the size of the nuclei of these lines compared to the parental 270 in IFAs stained with -H2B.Z antibody. As shown in Fig. 7E, nuclei sizes are significantly smaller that the 271 parental in c-Myc-R parasites in accordance with the hypothesis. However, this significant difference is also 272 seen in c-Myc-A tachyzoites, indicating that the inability of this histone to be post-translationally modified is 273 enough to lead to this state. The sizes of c-Myc-WT-OE parasite nuclei were similar to parental nuclei, 274 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint suggesting that the c-Myc tag is not responsible for the effect observed (Fig. 7E). Taken  line showed a mild defect in virulence in vivo and defects in chromatin compaction. These defects could be 281 associated with the interaction with acetylation "reader" proteins. We therefore examined if there are 282 differential proteins interacting with H2B.Z N-terminal region depending on its acetylation state. We 283 designed two peptides, one containing two acetylated lysines (K14 and K18) and the other unacetylated; 284 both peptides contained a biotin tag towards the C-terminal end (Fig. 8). Peptide design was performed 285 according to Wysocka 2006 [52]. In this work, it was recommended to perform the synthesis of peptides 286 about 20 amino acids long, with biotin conjugated through a linker on the C-terminus for N-terminal histone 287 peptides, and with the modification positioned close to the center of the peptide. In addition, two 288 consecutive modifications were not recommended, explaining our choice of K14 and K18. 289 We used extracellular tachyzoites (RH) to perform the pull-down assays. After resolving on SDS-290 PAGE, samples were analyzed by mass spectrometry. We detected a total of 48 interactors, 18 exclusive of 291 non-acetylated oligo, 17 exclusive of acetylated oligo and 14 present in both (Table S1). While many 292 interactors have nuclear localization, other localizations were detected, and many typical contaminants (e.g. 293 ribosomal subunits). Among the nuclear proteins, we can select a few that can be interacting with the H2B.Z 294 N-terminal tail independent of acetylation status; among them, GCN5-A, histone H4 and a PHD protein (Fig.  295 8). Interestingly, only three nuclear proteins were differentially pulled down with strong evidence (2 296 independent pull downs), one of them TGME49_262620, a RRM protein (unacetylated peptide), H3.3 and a 297 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint putative CDK protein (both with the acetylated peptide). Considering the nuclear genes that only appeared 298 in 1 pull down, the acetylated peptide retrieved only genes that code for proteins associated with the 299 maintenance and segregation of chromosomes during the cell cycle, as peptidase c50 (TGME49_262825), 300 RecF/RecN/SMC N terminal domain-containing protein (TGME49_231170) and meiotic recombination 301 protein DMC1 family (TGME49_216400) ( Table S1). Among the retrieved peptides in both acetylated and 302 non-acetylated pull-downs, we also detected an apicoplast localized acetyl-CoA carboxylase ACC1 303 (TGME49_221320) ( Fig. 8 and Table S1). It is interesting to observe that an apicoplast protein displayed a 304 significant number of peptides, indicative of a consistent interaction which could occur if H2B.Z presents in 305 some situation an extracellular location as has been observed in other systems [53][54][55][56][57]. Acetylation and methylation are associated to opposite impacts in gene expression, the first favoring 316 expression, while the second is often a repressive mark. As an example, acetylation of lysine 31 on histone 317 H4 (H4K31) was associated to the promoter of a nearby active gene, while in the core body of these genes 318 H4K31me1 was detected [64]. Until now, the role of PTMs in T. gondii H2BZ has not been studied. However, 319 T. gondii H2B.Z/H2A.Z double variant nucleosomes were found in the promoter of active genes and in the 320 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. However, in this work we showed that nuclei size was smaller in both clones compared to parental and to c-361 Myc-WT-OE tachyzoites, indicating that the charges in the N-terminal tail are not responsible for chromatin 362 compaction. Chromatin compaction has also been described to be regulated by linker histone H1, that 363 stabilizes the nucleosome structure. A candidate H1 has been recently identified in T. gondii, and interaction 364 of H1 with H2B.Z was observed [69]. The acetylation status of H2B.Z may be important for this interaction, 365 leading to a more compact chromatin in both alanine and arginine mutants because of linker histone H1. 366 Further experiments are needed to test this hypothesis. 367 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint It could be expected that the phenotypic alterations are given by defects in the formation of the 368 nucleosome with an impact on the expression of the genes. In our analysis, both situations are not altered. 369 Although a massive analysis of gene expression is necessary to detect specific changes, part of the study 370 shows that the expression of Rop5 and Rop18 does not occur at the transcriptional level, but rather post- Therefore, it may be likely that acetylated H2A.Z and H2B.Z would associate to this variant histone in active 403 promoters. However, this association was not found in our genome wide analysis on tachyzoite stage [17]. 404 In addition, CDKs have also been linked to mitosis [76, 77] and H3.3 is related to chromosome 405 segregation, nuclear structure, and the maintenance of genome integrity [78]. In this direction, it is 406 interesting that our pull-down assay has also retrieved, only for the acetylated peptide, other genes, all of 407 them associated to chromosome segregation (Table S1) were included (underlined). Both oligos were annealed and cloned into the pU6 CRISPR Universal plasmid 477 (kindly provided by Lourido´s Lab, Whitehead Institute), by using the Bsa I restriction site. The plasmids were 478 confirmed by PCR, using the gRNA as primers, were sequenced (Macrogen) and prepared for transfection in 479 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

Phenotypical assays 511
For growth/competition assays, coverslips seeded with Htert cells were infected with a mix of 50% parental 512 (RHΔhxgprt) and 50% of each clone. 3 coverslips per clone were infected with 0,05 to 0,1 tachyzoite per cell, 513 incubated 10 minutes on ice, and 2 hours at 37°C in incubator for invasion. Slides were washed twice with 514 PBS to remove tachyzoites that did not enter the cells, and fresh DMEM supplemented with 1% SFB was 515 added. Slides were fixed at different times, up to 96 hours and IFA was performed with -c-Myc and -Sag1 516 antibodies to distinguish between parental and c-Myc positive vacuoles. Number of c-Myc positive and total 517 vacuoles in at least 100 vacuoles, randomly choosing different fields of each slide were counted. In the case 518 of competition assays with genotoxic drugs treatment, the experiment was modified as follows: 3 slides per 519 clone mixture were fixed after 24 h, to stablish the initial percentage of c-Myc positive vacuoles. At this time 520 point, media was changed in the rest of the slides for DMEM supplemented with DMSO or drugs at the 521 concentrations indicated. These slides were fixed after 96 h, and IFA was performed and counted as explained 522 before. 523 For in vitro differentiation assays, in a similar way using Htert confluent slides, media was changed by DMEM 524 HEPES pH 8.1, after invasion with 1 parasite every 10 cells, accompanied with deprivation of CO2. Media was 525 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made and -Sag1 antibody to stain the tachyzoites. In the case of collection of parasites for RNA extraction, 528 differentiation assay was performed in a similar way, but in t25 dishes. After 48 or 96 h parasites were forced 529 out of the cells by 23G, 25G and 27G syringe passages. 530 For in vivo survival, virulence and differentiation assays, C57BL/6 female mice were used. For the use of 531 animals, C.I.C.U.A.E-UNSAM 10/22 was approved. Mice were maintained in optimal conditions in the 532 biotherium, with controlled temperature and free access to sterilized water and food. Infection was carried 533 out intra-peritoneal in 10 mice per group with 100, 1000 or 10000 tachyzoites of parental lines or clones, 534 depending on the assay. PBS was injected as negative control in five mice per experiment. Deceases were 535 registered along 30-35 days in each experiment, and signs of illness were monitored daily. Two mice from 536 each group (except PBS) were sacrificed at day 5 (when RH infected mice have symptoms of illness), and 537 intraperitoneal fluids were collected in order to detect acute Toxoplasma infection. Blood samples were 538 taken at days 0, 14, 21 and 35 and sera was frozen for ELISA assays. After sacrifice, surviving mice brains were 539 processed for cyst observance by optical microscopy. Also, a sample was stained using DLB and checked by 540 fluorescence microscopy. 541 In every phenotypical assay, three independent experiments were performed. One representative 542 experiment is shown. 543

RT-PCR and RT-qPCR 544
Tachyzoites or parasites exposed to 48 or 96 h differentiation stress as explained before were conserved in 545 TriZol (Invitrogen) solution at -80°C until use. All experiments were performed in three independent 546 replicates. RNA extraction was performed according to the manufacturer's instructions and cDNA was 547 obtained by means of MMLV reverse transcriptase (Promega) using oligo dT with the protocol provided with 548 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The primers were first assayed for efficiency using a pooled cDNA in 1 to 0.001 dilutions, and the threshold 562 was defined for each set of primers. Melt curves were also obtained for each set. For each set of experiments, 563 SybrGreen master solution (Roche) was used and qPCR was run in StepOne Real time equipment (Applied 564 Biosystems). Actin and tubulin were used as housekeeping genes and data was normalized to those genes 565 by Infostat software. For the experiments in figure 3, all sets of data were relativized to tachyzoite 566 amplification. For experiment shown in figure 7, data from the different clones was relativized to the 567 parental. 568

Pull-down assays 607
Pull-down experiments were performed as detailed in Wysocka [52] after nuclear extraction with NE-PER 608 commercial kit following manufacturer´s instructions (Thermo Scientific #78833) and run in SDS-PAGE for a 609 short time in order to avoid separation. Protein gels were fixed by soaking for 3 hr in 30% methanol, 2% 610 phosphoric acid. Then the gel was washed with deionized water 3 times, 5 minutes each. After removing the 611 deionized water, the staining solution (0.5 g/L Coomasie blue brilliant R250, 18% methanol, 17% (NH4)2SO4, 612 2% phosphoric acid) was added until covering the gel. It was stained for 1 h in gentle shaking. Then the gel 613 was rinsed in deionized water 3 times for 5 minutes each. The background staining was removed with 30% 614 methanol. The gel was stored in deionized water until use. Each lane of the gel was cut into individual slices. 615 Each band was then cut into 1 mm 3 cube and further treated with three washes of 50 mM NH4HCO3 in 50% 616 CH3CN with 10 min incubations. Each group of gel cubes was then dehydrated in CH3CN for 10 min and dried 617 in a Speed Vac. Protein samples were reduced by dithiothreitol (DTT) and alkylated by iodoacetamide [92]. A 618 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint solution of 10 ng/µL trypsin in 50 mM NH4HCO3 was used to re-swell the gel pieces completely at 4 o C for 30 619 min, followed by a 37 o C digestion overnight. A small amount of 10% formic acid was then added to stop the 620 digestion. The sample was then centrifuged at 2,800 x g, and the supernatant was collected for LC-MS/MS. 621

LC-MS/MS Analysis 622
A fused silica microcapillary LC column (15-cm long x 75-µm inside diameter) packed with Halo C18 reversed-623 phase resin (2.7 µm particle size, 90 nm pore size, MichromBioresources.) was used with EASY-nLC 1200 624 system (Thermo Fisher). The nanospray ESI was fitted onto the Thermo Q-Exactive plus mass spectrometer 625 (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ;

693
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint intensities, relative to Sag1 band intensity in each lane. Image J software was employed to quantify relative 745 intensities and graphed with GraphPad Prism 8. Average plus SD of three independent assays is represented. 746 *: p<0.05. C. Immunofluorescence assay. Htert confluent slides were infected with tachyzoites of parental 747 (RHΔhxgprt), c-Myc-A and c-Myc-R and fixed after 24 h for immunofluorescence analysis using anti Rop5 and 748 anti Rop18 antibodies (green). AcTubulin in red, was used to stain parasites and DAPI for the nuclei. D. Rop5 749 intensity quantification. Image J software was used to quantify the antibodies intensities in three 750 independent experiments, by triplicate, at least 10 vacuoles per slide. The graph shows the average intensity 751 in relative units; statistical analysis was performed by GraphPad Prism 8 software. *: p<0.05. 752 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint primers indicated in the graph, using actin as housekeeping control. Data was normalized to actin, and 771 relative quantities to RH tachyzoites in each sample is plotted for each of the genes studied. GraphPad Prism 772 was used to statistically analyze data, by two-way Anova. ***: p< 0.0001; ns: not significant. E. Tachyzoite 773 nuclei size. Tachyzoites of parental (RHΔhxgprt), c-Myc-A, c-Myc-R or c-Myc-WT-OE were allowed to invade 774 hTert confluent slides and replicate for 20 h, and IFA was performed with -H2B.Z antibody to detect the 775 nuclei. Image J software was used to measure the average nuclei size of at least 100 tachyzoites per slide, in 776 three independent experiments, and this was plotted and analyzed with Graphpad Prism 8, by two-way 777 ANOVA. ****: p< 0.0001. 778 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted February 24, 2023. ; https://doi.org/10.1101/2023.02.14.528480 doi: bioRxiv preprint