Analysis of intron-containing genes and spliceosomal proteins in apicomplexans
Alveolates are reported to have high number of introns; however, the genes with intron numbers have not been analyzed in Apicomplexa. Using VEuPathDB (https://veupathdb.org/veupathdb/app/)47, we determined the percentage of the genes with introns in the six apicomplexans (T. gondii, Neospora caninum, Theileria annulata, Babesia microti, Plasmodium falciparum, and Cryptosporidium parvum) and compared to the relatively low intron content of S. cerevisiae. This genome-wide comparison of introns revealed moderate to high intron densities (1–5 introns/gene) in apicomplexans, except C. parvum (Fig. 1A, Supplementary Tables 2 and 3). Compared to other apicomplexans, T. gondii and N. caninum have a wide range of exon distribution, with ~ 25% of genes being intronless and ~ 5% containing > 16 exons/gene. T. annulata and B. microti show similar exon distribution with ~ 29% of genes without intron and ~ 0.7% of genes containing > 16 exons/gene. While P. falciparum contains a relatively similar percentage of genes with no introns (~ 46%) and 2–5 introns (~ 44%), the exon distribution of C. parvum is comparable to S. cerevisiae (Fig. 1A, Supplementary Tables 2 and 3).
The spliceosome is a large RNP machinery composed of five snRNPs (U1, U2, U4, U5, and U6) and numerous proteins, including NTC or Prp19/Cdc5 complex proteins. In apicomplexans, studies suggested that splicing occurs in four steps: assembly (complex A - interactions between snRNPs and pre-mRNA), activation (complex B - release of U1 and U4), splicing (complex B* - NTC or Prp19/Cdc5 complex binding and complex C), and disassembly, as in model organisms; however, several spliceosomal proteins, including snRNPs and non-snRNPs, have not been identified. To identify these proteins, we performed BLASTP homology searches48 of six apicomplexan genomes using amino acid sequences of S. cerevisiae and H. sapiens as queries. While the search analysis revealed that most snRNPs and Prp19/Cdc5 complex proteins are present in these organisms, many have not been annotated (Fig. 1C). This analysis revealed that nearly all primary splicing factors are present in apicomplexan, similar to model organisms.
T. gondii Cdc5 is a conserved splicing factor of large spliceosomal complex
The identified T. gondii Cdc5 (TgME49_275480), a 888-aa protein contains conserved nucleic acid binding Myb-domain and SANT domain in the N-terminus (Fig. 2A). To gain insight into the expression, and localization of TgCdc5, full-length TgCdc5-His protein of ~ 100 kDa was purified (Fig. 2B) and used to generate specific anti-TgCdc5 antibodies. TgCdc5 is robustly expressed (Fig. 2C) in both the asexual stages and localized in the nucleus of the tachyzoite and the perinuclear to cytoplasm in the bradyzoite stage (Fig. 2D). Cdc5 is highly conserved amongst eukaryotes as a part of NTC or Prp19/Cdc5 complex that is required to activate the spliceosome. To identify TgCdc5 interacting proteins in Toxoplasma, we immunoprecipitated TgCdc5 protein from freshly lysed tachyzoites and identified co-purifying proteins by mass spectrometry (Fig. 2E). With the cut-off of two unique peptides, 146 proteins (Supplementary Table 4) were identified after the removal of contaminants and common proteins present in the pre-immune sera immunoprecipitated sample. As most of the proteins were unannotated, we performed homology searches using BLASTP48 and HHpred49 to identify and name these proteins following the human/yeast nomenclature (Fig. 2E). Based on these searches, we identified 52 putative spliceosomal proteins, including eight core proteins (Prp19, Cdc5, SPF27, PRL1, CRN, SKIP, PPIL1b, and SYF1) belonging to the Prp19/Cdc5 complex, and 44 were other spliceosomal proteins (Fig. 2E). Based on the coverage and peptide score, TgPrp19 (TgME49_320210), a conserved splicing factor containing Ubox and WD40 repeat (Fig. 2F), was the major protein co-purified with TgCdc5 (Fig. 2E). Before confirming their interaction, we first bacterially expressed full-length TgPrp19-His6 protein (~ 56 kDa) (Fig. 2G) and generated anti-TgPrp19 polyclonal antibodies. Similar to TgCdc5, TgPrp19 is also highly expressed in both the asexual stages (Fig. 2H) and localized predominantly in the nucleus of the tachyzoite and bradyzoite stages (Fig. 2I), suggesting an interaction of these proteins. Further, the reciprocal pull-down experiment (Fig. 2J,K) and co-localization (Fig. 2L) studies confirmed the strong interaction between TgCdc5 and TgPrp19.
Cdc5 family members associate with the spliceosome throughout the entire splicing reaction50. Given that TgCdc5 contains nucleic acid binding domain51 and interacts with other core members of the spliceosome, indicating TgCdc5 may act as a scaffold linking splicing components and RNAs. To determine the interaction between TgCdc5 and snRNAs, MST assay was performed using fluorescently labeled U1, U2, U4, U5, and U6 snRNAs. As measured, TgCdc5 showed strong binding affinity towards U2 (KD=440 nM) and U6 (KD=481 nM) compared to U5 (KD=1 µM), U1 (KD=1.5 µM), and U4 (KD=3.7 µM) snRNAs (Fig. 5M). These results confirm the specificity of TgCdc5 with U2 and U6 snRNAs.
Next, we performed functional complementation in yeast to confirm that TgCdc5 and TgPrp19 are central to splicing due to their functional conservation across organisms despite phylogenetic distance. We separately cloned the TgCdc5, and TgPrp19 genes into a yeast 2µ TRP1 pYES3 plasmid, and found that 2µ TgCdc5 and TgPrp19 supported the growth of Δcef1, and Δprp19 cells, respectively (Fig. 2N,O). These results demonstrate that Toxoplasma encodes the biologically active Cdc5 protein, the core spliceosomal factor.
TgCdc5 is an essential pre-mRNA splicing factor
To comprehend the role of TgCdc5 in splicing-associated processes, we endogenously tagged TgCdc5 at the C-terminus with mini auxin-inducible degron fused with three copies of HA (TgCdc5-mAID-3HA) in RH strain parasites expressing TIR1, which allows for rapid degradation of the TgCdc5-mAID-HA protein (Fig. 3A) with the addition of IAA. The resulting TgCdc5-mAID-HA strain was confirmed by diagnostic PCR (Fig. 3B) and western blotting (Fig. 3C). Western blot (Fig. 3D) and IF (Fig. 3E) staining using an anti-HA antibody revealed that TgCdc5-mAID-HA protein was completely depleted in < 1 h after adding IAA in the culture medium.
To determine how TgCdc5 affects gene splicing, we generated a reporter (Fig. 3F) in which an exon I-intron I-exon II (e-i-e) containing region (mini-gene) of TgPrp19 was fused with the ORF of Renilla luciferase (Rluc) under the TgTub promoter (e-i-e-Rluc). In the case of normal splicing, TgPrp19 intron is spliced out, leading to functional Rluc protein, whereas if intron is not spliced out, several stop codons appear in the frame, resulting in no functional Rluc protein. To characterize the splicing reporter, we cotransfected TgCdc5-mAID-HA parasites with RLuc plasmid and plasmid containing intronless firefly luciferase (Fluc) reporter (as control) followed by infection to HFFs (Fig. 3F). Further, parasites were treated with IAA and subsequently harvested to measure luciferase activity and mRNA levels of RLuc and Fluc. The relative ratio of Rluc/Fluc activity was significantly reduced (Fig. 3G) in the TgCdc5 depleted parasites consistent with reduced mRNA level of Rluc (Fig. 3H). The greatest change in activity and mRNA level was observed at 8 h of TgCdc5 depletion; hence, 8 h IAA treatment time was used for all the further experiments. To test whether TgCdc5 was required for efficient pre-mRNA splicing of the downregulated e-i-e-Rluc gene, qRT-PCR analyses were performed to measure the relative levels of spliced and unspliced RNA (Prp19 mini-gene) using exon-exon and intron-exon junction-specific primers. We observed that the splicing efficiency of e-i-e-Rluc was considerably reduced by TgCdc5 depletion (Fig. 3I). To test whether reduction in the luciferase activity and splicing efficiency was not due to parasite death, trypan blue staining was performed. While the percentage of trypan blue-positive parasites increased over time, the total dead parasite was < 15% (Fig. 3J), suggesting a specific effect of TgCdc5 on pre-mRNA splicing.
Next, we analyzed the effect of TgCdc5 depletion on parasite-specific processes. The Cdc5-depleted parasites showed the complete arrest of parasite replication (Fig. 3K) and severe morphological defects (Fig. 3L). A parasite growth assay revealed that TgCdc5-depleted parasites produced no visible plaques (Fig. 3M). To determine whether parasites can recuperate from a transient loss of TgCdc5, we performed a plaque assay using six different IAA treatment conditions (Fig. 3N). Parasites grown for 24 h were treated with IAA or a vehicle for 1/2/4/8/12/24 h, replaced with standard parasite medium, and incubated for 5 days. While plaque numbers were comparable for 1 h IAA or vehicle-treated parasites (Fig. 3N), no plaques were observed when treated for 2/4/8/12/24 h IAA (Fig. 3N). Furthermore, TgCdc5 depletion significantly reduced the parasite's invasion efficiency (Fig. 3O); however, the parasites' ability to egress upon inducing egress using calcium ionophore showed a marginal difference (Fig. 3P). Together, these data show that TgCdc5 is essential for Toxoplasma proliferation.
TgCdc5 loss perturbs global gene expression through dysregulation of RNA splicing
To assess the global impact of the severe phenotypic defect obtained after TgCdc5 loss, we performed RNA-seq on TgCdc5 (+ IAA) and vehicle-treated parasites. Differential gene expression (DGE) of the obtained 8111 transcripts using DESeq showed drastic changes in mRNA abundance (log2FC 2, FDR < 0.05) (Fig. 4A, Supplementary Table 5). Of 1125 DEGs identified, 394 were upregulated, and 731 were downregulated in TgCdc5 depleted parasites (IAA-treated) compared with vehicle-treated parasites (Fig. 4A). The k-means clustering of the differentially expressed genes (showed four clusters (A, B, C, and D) with similar expression profiles (n = 2) in IAA-treated and vehicle-treated parasites (Fig. 4B). The genes that showed significantly different gene expressions (up or down) relevant to the study are shown in the volcano plot (Fig. 4C). The host cell invasion genes RON2, RON3, RON4, and RON5 were found to be downregulated (Fig. 4C), which was consistent with compromised invasion for TgCdc5-depleted parasites. Other downregulated genes were PAN domain-containing genes involved in protein ubiquitination/proteolysis’, PFK domain domain-containing genes responsible for a decreased rate of protein synthesis, and Golgi enzyme CD39 (Fig. 4C). Also, genes related to rhoptries and their trafficking; ClpB, necessary for suppressing and reversing protein aggregation; SNF1, a histone kinase required for transcriptional activation and repression of gene expression; DNA replication-related proteins; AP2 transcription factor AP2IX5, essential for cell cycle; SPM2 functions in sub perinuclear microtubules; and ULK kinase that inhibit autophagy were downregulated (Fig. 4C). Surprisingly, we found a few bradyzoites inducing Apetala − 2 (AP2) factors (AP2IV-3 AP2X-9 BRP1, AP2Ibl, and AP2IV-2) and HDAC5 were upregulated, and transcription repressor factor AP2IV-4 for bradyzoite stage differentiation was downregulated (Fig. 4C). Furthermore, we observed intron number bias for DEGs as the downregulated genes had more introns than upregulated or unchanged genes, which had relatively fewer introns (Fig. 4D). Genes with more intron count were more affected, conceivably due to reduced splicing efficiency.
Impaired RNA splicing may directly reduce mature mRNA; therefore, based on RNA-seq data, we selected 9 downregulated candidate genes (Tub1, Rbp1, Rps3, MCM4, Nhe2, Sec62, TDCP, Rab7, and AMA1) and performed qRT-PCR (Fig. 4E). These downregulated genes were essential for parasite processes, suggesting that their decreased expression may directly contribute to the observed phenotypes in TgCdc5 parasites. qRT-PCR results validated the RNA-seq findings and the expression of intronless gene, Srs40E remained unchanged by TgCdc5 knockdown (Fig. 4E). Next, we evaluated whether TgCdc5 was essential for efficient pre-mRNA splicing of the downregulated genes. To test that, we determined the splicing efficiency of the same 9 downregulated intron-containing genes by qRT-PCR as performed in a reporter assay. qRT-PCR analyses were performed to measure the ratio of spliced/unspliced transcripts using the exon-exon and intron-exon junction primers. TgCdc5 depletion leads to a ~ 1.5-to-2-fold decrease in the splicing efficiency of the Tub1, Rbp1, Rps3, MCM4, Nhe2, Sec62, TDCP, Rab7, and AMA1 downregulated genes tested (Fig. 4F).
To understand the processes in which the differentially expressed genes might be involved, we conducted gene ontology (GO) enrichment analyses (Supplementary Table 5) (Fig. 4G). The GO analysis of downregulated genes revealed that depletion of TgCdc5 caused the deregulation of genes involved in the chromosomal organization, cell cycle, cytoskeleton and microtubule, DNA metabolic process, DNA damage response, and organelle organization (Fig. 4H). In contrast, GO analysis of upregulated genes showed enriched processes related to the regulation of transcription, metabolic and biosynthetic processes, and RNA biosynthetic process (Fig. 4H). Together, processes and functions related to cell cycle and parasite replication were compromised, while the processes that help maintain cellular homeostasis were upregulated by TgCdc5 depletion.
TgCdc5 modulates alternative splicing of genes
In many cancerous cells, mutations in the core spliceosomal factors result in aberrant splicing, leading to pervasive intron retention (IR) and aberrant selection of splice sites (ss), two of a few types of alternative splicing. To test whether depletion of TgCdc5 may result in the perturbation of alternative splicing (AS), we performed differential splicing analyses using ASpli program. ASpli analysis revealed significant changes in AS events (n = 20987) (Fig. 5A, Supplementary Table 6) corresponding to 3604 genes (Fig. 5B) for TgCdc5-depleted parasites compared to wild-type parasites. The AS events were detected in 54.8% (n = 3604) of exon-containing transcripts (n = 6581) in TgCdc5-depleted parasites, indicating its extensive role in regulating splicing. Next, we examined different AS types such as intron retention (IR), alternative 5’ splice site (Alt5’SS), alternative 3’ splice site (Alt3’SS), exon skipping (ES), and other unclassified events in the data sets. AS type analysis revealed IR event appeared at the highest frequency (82.18% events; 64.86% genes), followed by exon skipping (0.71% events; 2.65% genes), Alt5’SS/Alt3’SS (0.02% events; 0.09%), and other unclassified events (17.09% events; 32.4% genes). Figures 4C-F depict schematics showing AS event type and gene plot of the affected representative gene. Next, we determined the contribution of AS events for DEGs (n = 1125). The AS events were detected in 29.66% of DEGs (Fig. 5G), whereas 70.34% of DEGs (Fig. 5G) did not have AS events (AS independent).
TgCdc5 depletion leads to the cell cycle arrest and DNA damage in the parasites
GO analysis revealed that loss of TgCdc5 generates major deregulation of genes involved in cell cycle and DNA damage response. Accordingly, the effect of TgCdc5 depletion on the parasite cell cycle was evaluated using Centrin1 (centrosome - G1 stage marker)52 and MORN1 (centrocone - mitosis marker)53. Centrosome duplication marked the S phase (Fig. 6A,B), whereas centrocone duplication indicated the M phase (Fig. 6A,B). Cell cycle progression was tested by depleting the TgCdc5 in either G1 stage or late S phase and followed the cell cycle until its completion (~ 8 h). After 30 min of post-infection (p.i.), the expression of TgCdc5 was depleted by IAA, and parasites were subjected to IFA after 4 h and 8 h. The knockdown of TgCdc5 caused immediate cell cycle arrest in G1 (the actual stage at the time of infection), as most (~ 80%) of TgCdc5-depleted parasites (+ IAA) contained a single centrosome and did not progress to S phase (Fig. 6C,D). In a similar experiment, TgCdc5 was depleted post 4 h p.i. (in the late S phase marked by centrosome duplication and single centrocone stained by MORN1) and IFA was performed after 4 h and 8 h. The deletion of TgCdc5 induced rapid cell cycle arrest in S phase, as most (~ 80%) of TgCdc5-depleted parasites (+ IAA) contained single centrocone (Fig. 6E,F). Together, these results suggest that TgCdc5 is essential for all cell cycle stages, not only for mitosis, as reported for human Cdc5.
DNA fragmentation, a hallmark of DNA damage was tested using a TUNEL assay. Direct labeling of DNA breaks confirmed a significant increase (~ 80) in TgCdc5-depleted parasites (+ IAA) compared to vehicle-treated parasites (~ 10%) (Fig. 6G,H). It is important to mention that not all parasites within the parasitophorous vacuoles examined were TUNEL-positive in either treatments. DNA fragmentation is associated with apoptosis in human cells and is considered an important marker for apoptosis-like cell death in protozoa. Using PI and Annexin staining, we measured the apoptosis in the parasites by flow cytometry. A significant increase of apoptotic parasites (~ 32%) was observed after 12 h of TgCdc5 depletion (+ IAA) compared to vehicle treatment (~ 12%), suggesting apoptosis-like cell death in Toxoplasma (Fig. 6I).
Depletion of TgCdc5 generates protein aggregates that triggers bradyzoite induction program
In metazoans, NMD, an mRNA quality control process, removes erroneous transcripts18. However, recently, in P. falciparum, disruption of core NMD proteins showed no degradation of nonsense transcripts20, suggesting either such transcripts reside in the cytoplasm or may be translated to non-functional proteins. Accordingly, to test whether the large number of erroneous transcripts generated in the TgCdc5-depleted parasites can affect the translation of mRNAs, we examined the global translation by the incorporation of puromycin into nascent translated proteins. Interestingly, no reduction in the global translation (Fig. 7A) was observed even after 8 h of TgCdc5 depletion, indicating sustained translation of either normal mRNAs or erroneous transcripts. The translation of such erroneous transcripts may lead to non-functional proteins with misfolded structures, which form protein aggregates. Using Proteostat, a stain to detect protein aggregates, we demonstrated that loss of TgCdc5 generates protein aggregates (Fig. 7B,C) in a significant number of parasites (~ 22%), suggesting that translation of erroneous transcript generates non-functional misfolded protein aggresomes.
Cellular and environmental stresses are known to develop latent bradyzoites from rapidly growing tachyzoites in Toxoplasma4. Protein aggregates, a kind of cellular stress, and the upregulation of bradyzoite induction genes raised the possibility of conversion of tachyzoites to bradyzoites by TgCdc5 loss. Accordingly, we addressed whether TgCdc5 loss results in stage transition. As expected, most of the parasites in the vacuoles were dead (+ IAA), confirmed by irregular morphology of the parasites and loss of parasites in the vacuoles, and only a few (~ 20%) vacuoles showed standard morphology of the parasites with evidence of bradyzoite formation as determined by BAG1 staining upon IAA-induced TgCdc5 depletion (Fig. 7D). Importantly, those ~ 20% of vacuoles were not fully DBA-positive displayed by partial staining along their periphery (staining increased from day 2 to 5, however, decreased on day ≥ 6), suggesting these vacuoles containing bradyzoites were transitioning to cyst or will remain immature cysts (Fig. 7D, E). In normal conditions, TgCdc5-mAID-HA parasites did not form bradyzoites but could be converted to bradyzoites by stress-induced conversion (in alkaline pH and CO2 depletion) (Fig. 7F). Regardless, stress-induced bradyzoite formation was inefficient in the RH TgCdc5 parasites even after 6 days, which is characteristic of type I RH parental strain.
After 6 days of IAA-induced TgCdc5 depletion, these partially DBA-positive vacuoles showed loosely packed and misshaped bradyzoites (Fig. 7D), suggesting that loss of TgCdc5 may eventually be lethal. To examine whether these bradyzoites formed due to the loss of TgCdc5 were viable, we observed their ability to reconvert to tachyzoites. These bradyzoites could not recover (no plaque formation) even after 15 days from 6 days after loss of TgCdc5 (Fig. 7G). These results suggest that TgCdc5 loss generates protein aggregates due to erroneous splicing, which imparts stress and signals to stage conversion; however, a lack of functional proteins to support the bradyzoite's growth eventually leads to the death of the parasite.
Furthermore, we performed a quantitative proteome analysis of TgCdc5-deficient parasites at 8 h IAA treatment similar to RNA-seq analysis. As predicted for splicing deregulation, the lack of splicing factor TgCdc5 significantly changed global protein expression (DEPs − 636) (Fig. 7H). A comparative analysis of vehicle and IAA-treated parasites showed that more proteins (Fig. 7H) had reduced (n = 424) than elevated expression (n = 212). Downregulated genes contain more introns than upregulated genes (Fig. 7I), consistent with RNA-seq data. Of 636 differentially expressed proteins (Supplementary Table 7), 75 (~ 12%) genes (48 downregulated and 27 upregulated) were also differently expressed, as confirmed by RNA-seq data. To explore more about those 75 genes and their biological roles, we performed a GO analysis (Fig. 7J). TgCdc5-mediated RNA splicing deregulation affects Toxoplasma in multiple ways. Consequently, our analysis detected dominant changes in the cell division-associated factors accompanied by decreased expression of the proteins required to maintain the cytoskeleton and chromatin structure of daughter parasites during division (Fig. 7J). As expected for lowered invasion, rhoptry proteins and kinases showed a significant reduction in the expression, which lowers parasite survival in the host (Fig. 7J). As predicted for protein aggregates, chaperones, and ubiquitin-proteasome system proteins displayed decreased expression, favoring the aggregation of misfolded proteins. We detected reduced production of the factors regulating vesicular transport (between the endoplasmic reticulum and Golgi compartments), redox balance, and translation fidelity. Importantly, IAA-induced depletion of TgCdc5 initiates stage transition to bradyzoite, demonstrated by increased expression of multiple bradyzoite-specific SRS, cyst wall, GRA, and ApiAP2 factors.
Knockdown of TgCdc5 protects mice from lethal toxoplasmosis as well as induces protective immunity
TgCdc5 protein is essential for parasite fitness in cell culture, and to test its essentiality in establishing an infection in the host, we performed mouse infection studies. The experiment had three groups (10 mice/group): two groups with infection and the remaining one without infection control. Mice from two groups (infection groups) were injected intraperitoneally (i.p.) with 50 tachyzoites of RH TgCdc5-mAID-HA, followed by oral treatment with 200 mg/kg/day IAA or vehicle from day 2 to 15 post-infection. (Fig. 8A) and mice from the third no-infection control group were supplemented with IAA. On day 6 pi, the 2 mice from each group were sacrificed, and peritoneal exudate cells (PECs) were collected for IF microscopy. Compared to parasites from vehicle-treated mice, parasites from IAA-treated mice had depleted TgCdc5 levels but normal control protein levels, TgIMC1 (Fig. 8B), confirming effective in vivo depletion of TgCdc5 protein < 6 days. By day 11 post-infection, all mice receiving the vehicle control treatment died from lethal toxoplasmosis (Fig. 8C), whereas IAA treatment rescued all mice from fatal infection (Fig. 8C). The vehicle treatment group showed severe morbidity and complete mortality compared to the IAA treatment group (Fig. 8D and 8C). By day 2 pi, the vehicle treatment group showed weight loss, and a sign of illness, however, no weight loss was observed in IAA treatment group. Depleting TgCdc5 expression effectively blocked T. gondii replication since stopping IAA treatment following day 15 did not result in morbidity (Fig. 8D), mortality (Fig. 8C). We did not observe T. gondii DNA (not shown) and bradyzoite cysts (not shown) in the brain and heart tissues of mice sacrificed on day 30. These results show the essential role of TgCdc5 for parasite survival and replication in the mouse host. The absence of tissue cysts in the brain samples may be attributed to fewer injected parasites (n = 50), potentially leading to effective elimination by the host's immune cells.
To address this, we performed a similar experiment involving two groups of 8 male and female mice injected with 5x103 TgCdc5-mAID-HA tachyzoites (Fig. 8E). After 15 days of IAA treatment, we found no mortality in mice and also could not detect bradyzoite cysts (not shown) and T. gondii DNA (tested by qRT-PCR for 529 repeats) (not shown) in the brain and heart samples of 2 male and female sacrificed mice. In a parallel experiment, two groups of three male and three female mice were injected with tachyzoites, resulting in 100% mortality by day 7. On day 21, we collected serum samples from these mice and performed IgG IFA for both tachyzoite and bradyzoite stages. The sera from all mice showed immunoreactivity against tachyzoite antigens (Fig. 8E) but not against bradyzoite antigens (not shown), indicating that anti-T. gondii antibodies were generated in response to the high dose of parasites. From day 22–25, male and female mice mating was carried out, and mice were further segregated into 4 groups (3 mice each male/female). On day 27, 3 mice, each male/female (1 group each), were injected with 5x103 TgCdc5-mAID-HA tachyzoites, and mice were observed for an additional 33 days. No mortality was observed in either (parasite injected or the control) groups of male mice. In the case of female mice, all females in the control group had a normal pregnancy with an average of 12 pups delivered on ~ 21 days of post-mating; however, only 1 of 3 female mice injected with tachyzoites had a normal pregnancy with 2 pups delivered, and the remaining 2 females did not deliver any pups (Fig. 8E). Until day 60, we found no mortality in mice and could not detect bradyzoite cysts and T. gondii DNA in the sacrificed mouse; however, the sera of all mice were immunoreactive for tachyzoite (Fig. 8E).