Depletion of splicing factor Cdc5 in Toxoplasma disrupts transcriptome integrity, induces stress-driven abortive bradyzoite formation, and triggers host protective immunity

Depletion of splicing factor Cdc5 in Toxoplasma disrupts transcriptome integrity, induces stress-driven abortive bradyzoite formation, and triggers host protective immunity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Depletion of splicing factor Cdc5 in Toxoplasma disrupts transcriptome integrity, induces stress-driven abortive bradyzoite formation, and triggers host protective immunity Abhijit Deshmukh, Kalyani Aswale This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4811664/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 Apr, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Toxoplasma gondii , a member of the Apicomplexa phylum, has over 75% of genes with predicted introns; however, RNA splicing, a major source of post-transcriptional regulation of gene expression during stage transitions, is not fully understood. Here, we demonstrate the role of pre-mRNA splicing factor Cdc5 in maintaining transcriptome integrity by harmonizing interaction with spliceosomal proteins and snRNAs in Toxoplasma . TgCdc5 is an essential splicing factor, and its depletion generates significant alternative splicing with widespread changes in gene expression demonstrated by RNA-seq and proteomic studies. Loss of TgCdc5 leads to catastrophic effects on the parasites, concomitantly triggering a switch from rapidly replicating tachyzoite to dormant bradyzoite cysts in many parasites, likely due to the formation of misfolded protein aggregates caused by the translation of erroneous transcripts. However, these dormant state parasites could not survive due to lacking functional proteins for bradyzoite development. Remarkably, the knockdown of TgCdc5 in vivo protects mice from lethal infection, and the immune response generated during initial parasite exposure completely protects these mice from future infection and offers partial protection in vertical transmission. Overall, this study unveils a novel role of TgCdc5-mediated pre-mRNA splicing in governing Toxoplasma stage conversion, providing new insights into developmental stage gene regulation. Biological sciences/Microbiology/Parasitology/Parasite biology Biological sciences/Molecular biology/RNA metabolism/RNA splicing Apicomplexan Toxoplasma gondii Cdc5 RNA splicing RNA modifications RNA sequencing Bradyzoite Mouse infection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Toxoplasma gondii is a member of the phylum Apicomplexa, a diverse group of several human (e.g., Plasmodium , Cryptosporidium ) and animal (e.g., Neospora , Theileria , Babesia ) pathogens 1 . T . gondii causes lifelong chronic infections in warm-blooded animals, including humans, and severe disease in fetuses and immunocompromised individuals 2 . The life cycle of T . gondii is complex, involving sexual replication in feline definitive hosts and asexual replication in other intermediate hosts with three infective stages: tachyzoites, bradyzoites, and sporozoites 3 . During infection, parasites disseminate as tachyzoites, causing acute disease, and then differentiate into bradyzoite cysts, leading to a long-lived latent infection 4 . Toxoplasma displays distinct gene expression programs during stage transitions to provide the required diverse protein repertoire, potentially controlled through RNA-based regulatory mechanisms 5–7 . Most parasites in the Apicomplexan genome have genes with a high number of introns 8–10 . Toxoplasma has over 75% of genes with introns compared to 5% in yeast 6 . Apicomplexan introns follow the standard 5′ GU-AG 3′ splice junction with some nucleotide variations at the branch point and are primarily excised by the U2-type spliceosome 6,7,11 . Recent studies suggest that the RNA splicing mechanism is largely conserved in apicomplexans, and the few identified splicing factors acquired divergent features 6,7 . Regardless, the presence of intron-rich genes in these species is intriguing, given the reductive mode of evolution observed in diverse parasite clans 7 . Introns generally regulate the expression of genes at several levels 12 , including mRNA export, stability, and translation efficiency, indicating intron selection in evolution in apicomplexans, particularly Toxoplasma, aids in different developmental stages and thrives in diverse host species. Pre-mRNA splicing is an important step during eukaryotic gene expression. The spliceosome, a multisubunit complex of five small nuclear RNPs (snRNPs) and several non-snRNP proteins, catalyzes RNA splicing by coordinating the removal of introns and joining adjacent exons to create mRNA 13–15 . In higher eukaryotes, most pre-mRNAs undergo alternative splicing (AS), producing different mRNA isoforms, contributing to expanding the proteome and also serving regulatory functions 16,17 . In contrast, recent studies suggest that alternative splicing is less prevalent in apicomplexans 7 , and AS events do not produce multiple alternative proteins but lead to the generation of aberrant or noncoding transcripts 7 . Disrupting conserved regulators of AS has shown lethal effects in apicomplexans 7 , indicating a biological role for some observed alternative splicing. In addition to protein diversity, AS can also generate non-functional products by introducing toxic exons or transcripts with a premature stop codon (PTC) that might undergo nonsense-mediated decay (NMD) 18,19 . A recent study in P . falciparum showed NMD is not essential and reported no change in the nonsense transcripts levels after deleting two core NMD proteins, indicating the regulatory role of the erroneous transcripts in gene expression 20 . The observed discrepancy between transcripts and protein levels in apicomplexan parasites highlights posttranscriptional gene regulation, mainly RNA splicing. The regulation of gene expression through RNA splicing is well-documented in model eukaryotes; however, studies on Toxoplasma splicing are limited, resulting in a poor understanding of this regulatory mechanism. In addition to the snRNPs, several non-snRNP splicing proteins play critical roles in the splicing process. Among those is cell division cycle 5 (Cdc5), a highly conserved splicing factor in animals, plants, and fungi 21 . Cdc5 is an essential splicing factor required for catalytic activation of the spliceosome by forming a heteromeric protein complex known as the Nineteen Complex (NTC) in S . cerevisiae , yeast 22 , and Prp19/Cdc5L complex in humans 23 . The protein composition of human and yeast complexes differs; however, they share the subunits Prp19, Cdc5L/Cef1, Spf27/Snt309, and Prl1/Prp46 (human/yeast) 22,24,25 . In addition to splicing, Cdc5 participates in diverse molecular processes, such as cell cycle 26–28 , DNA repair 29 , and RNA transcription 30 . In yeast and humans, mutation or knockdown of Cdc5 leads to the accumulation of retained introns in partially spliced pre-mRNAs, causing G2/M cell cycle arrest 26 . In plants ( Arabidopsis ), Cdc5 is required for development and immunity to bacterial infection 31,32 ; however, its role in splicing is unclear. Despite Cdc5's pleiotropic function in model organisms, its role in splicing or other related processes in apicomplexans has yet to be studied. Considering the importance of RNA splicing for apicomplexans, a potential new regulatory role of Cdc5 in gene expression or parasite-centric processes cannot be discounted. Here, we report why RNA splicing is crucial for the intron-rich Toxoplasma , show how depleting the core splicing factor Cdc5 affects various parasite processes, and offer protection in the mouse host. TgCdc5 is part of a large spliceosomal complex of snRNPs and non-snRNP with several unidentified proteins in Toxoplasma . We demonstrate that TgCdc5 is an essential pre-mRNA splicing factor, and its depletion results in significant alternative splicing and dysregulated gene expression associated with various parasite functions, including unproductive differentiation into slow-growing bradyzoite cyst in non-cystogenic strain. Finally, we demonstrate that TgCdc5 is essential for T . gondii survival in mice, as depleting TgCdc5 provides complete protection against a lethal dose of tachyzoites, and interestingly, the protective immune response generated against them offers total protection against future infections and partial protection during pregnancy. Materials and Methods Parasite culture T. gondii strains RH, ME49, RH-TIR1-3FLAG were maintained in confluent monolayers of human foreskin fibroblast (HFFs, ATCC) cells in DMEM containing 10% foetal bovine serum, 10 µg/ml gentamicin, 1% penicillin-streptomycin, and 2 mM L-glutamine at 37°C and 5% CO 2 . The stage conversion from tachyzoite to bradyzoite was carried out by incubating the ME49 tachyzoites in bradyzoite induction medium (RPMI pH 8.2) at 37°C for 5 days without CO 2 33 . Cloning, expression, and protein purification A synthetic DNA encoding T . gondii Cdc5 1–2664 and Prp19 1–1545 (superscript number denotes nucleotide coordinates) were purchased from Life Technologies (USA). The synthetic TgCdc5 gene containing C-terminal His 6 was codon-optimized for expression in E . coli and was initially cloned into a pMS vector between NdeI - BamH I sites, which was subsequently subcloned into the pET-21a vector (Novagen, USA). The synthetic TgPrp19 gene containing C-terminal His 6 was initially cloned into the pMA-T vector between Nde I- Xho I sites, which was further subcloned into the pET-28a vector. The recombinant TgCdc5-His (~ 100 kDa) TgMorn1-His (~ 41 kDa) proteins were expressed in E . coli BL21 Rosetta and purified on a Ni 2+ -NTA agarose resin column (Qiagen). TgPrp19 protein was purified from the inclusion bodies. The inclusion bodies were solubilized in 6 M GuHCl and the TgPrp19-His (~ 57 kDa) protein was purified over Ni 2+ -NTA agarose under denaturing condition 34 . The protein was then greatly diluted and refolded in the refolding buffer (50 mM Tris-HCl pH8.5, 9.6 mM NaCl, 0.4 mM KCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM DTT). The purified proteins were dialyzed in 1X PBS and stored at -80°C. Polyclonal antibody raising Mouse and/or rabbit polyclonal antibodies to recombinant TgCdc5, TgPrp19 and TgMorn1 were generated by primary injection with 30 µg (mouse) or 60 µg (rabbit) of purified recombinant protein in Freund's complete adjuvant (Sigma) followed by four boosts of 10 µg (mouse) or 25 µg (rabbit) each in Freund's incomplete adjuvant (Sigma) at 2-week intervals. Serum was collected after day 60 post-immunization. Polyclonal antibodies for Tg- IMC1 35 , SAG1 34 , BAG1 36 , CST1 34 , and Aldolase (ALD) 36 antibodies were used from previous studies. Immunoblotting Filter-purified 10 5 parasites were suspended in SDS-PAGE sample buffer, boiled for 10 min, and run on a single lane of an 8 or 10% polyacrylamide gel. The gel was then transferred to a 0.2 µm PVDF membrane using a Trans-Blot System (BioRad) for 2 h at 150 V. The membrane was blocked in 5% (w/v) non-fat milk in PBS for 60 min and probed with a primary antibody (α-TgCdc5/TgPrp19/TgSAG1/TgBAG1/TgALD-1:1000; αHA-1:5,000) in PBS overnight at 4°C. The membrane was washed 3x with PBS plus Tween-20 detergent (PBST; 0.1% Tween-20) and probed with HRP-conjugated α-rabbit IgG or α-mouse IgG antibodies (Invitrogen). The blot was developed using the SuperSignal West Pico PLUS (ThermoScientific) and visualized on a ChemiDoc Imager. Immunofluorescence (IF) staining T . gondii RH/ME49/TgCdc5-mAID-HA parasites infected HFFs were fixed with 4% paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS for 15 min at room temperature (RT), and blocked with 5% (w/v) BSA in PBS for 60 min at RT. Samples were first incubated with the primary antibody (α- TgCdc5/TgPrp19-1:100 and α-TgCentrin/Morn1/IMC1/BAG1//HA-1:1,000) at RT for 60 min, washed 5x with PBS, and then incubated with fluorescent secondary antibodies (Alexa Fluor 488/594 1:1000) along with 4',6-diamidino-2-phenylindole (DAPI; 300 nM) and Dolichos biflorus agglutinin (DBA) as appropriate at RT for 60 min. Samples were then washed 5x with PBS and coverslips were mounted on a glass slide using Vectashield medium (Vector Laboratories). IF staining was visualised using a Leica confocal microscope with a 100X oil immersion objective. Images were processed using las x software (Leica Microsystems). Immunoprecipitation (IP) The IP was performed using Crosslink IP Kit (ThermoScientific). TgCdc5/TgPrp19 antibodies (5 µg) were crosslinked to protein A/G agarose beads by using disuccinmidyl suberate. Parasite protein lysate was prepared from 2 x 10 8 RH parasites using IP lysis buffer supplemented with complete protease inhibitor cocktail (Sigma). The parasite protein lysate diluted with IP buffer was first pre-cleared by mixing it with protein A/G agarose. Supernatant was added to the TgCdc5/TgPrp19 antibody-linked beads and mixture was incubated for 10 h at 4°C. The beads were collected and washed 3x with IP buffer to remove any unbound proteins. TgCdc5/TgPrp19 associated proteins were eluted and samples were prepared in SDS-PAGE sample buffer. The eluates were separated by 10% SDS-PAGE for Western blot analysis. LC-MS/MS and interactome analyses TgCdc5 IP elute protein concentration was determined using BCA method and further used for LC-MS sample preparation according to the standard protocol. IP elutes was reduced (100 mM DTT for 1 h at 55°C) and alkylated (400 mM Iodoacetamide for 1 h at 25°C in the dark). One µg of proteomics grade trypsin (NEB) was then added per sample and digestion was carried out overnight at 37°C. Digested peptides were desalted and purified using C18 spin column (ThermoScientific). Total 40 µl elute was then dried using a speed vacuum and stored at -80°C. For analysis, the samples were redissolved in 0.1% formic acid and 5 µL of each digest was run by LC-MS/MS using a 3 h gradient on a 75µm x 50cm C18 column (PepMap RSLC C18) feeding into a Q-Exactive HF mass spectrometer (Orbitrap). MS/MS data was analyzed using Proteome Discoverer 2.2 software package (Thermo) against "UniProt-proteome_TgGT1_UP000005641" with 8450 entries and a common lab contaminants database having 244 common contaminants proteins. Sequest HT searches were performed with fragment ion mass tolerance of 1 Da and precursor mass tolerance of 20 ppm. Cysteine carbamidomethylation due to iodoacetamide was set as the default modification, and methionine oxidation was set as a differential/variable modification. Sequest HT-identified proteins were validated by the percolator validation algorithm and grouped according to the significance of peptide evidence. Identified proteins with a minimum of two unique peptides (min #unique peptide = > 2) and False Discovery Rate (FDR) equal to or less than 1% were considered along with percolator validation, which separates the identified PSMs into high-, medium-, and low-confidence identifications. Protein groups with no unique peptides (with similar peptides) that cannot be classified based on unique peptides were grouped by applying the strict parsimony principle parameter of protein grouping in the consensus workflow. After the contaminants were removed, the identified proteins list was moved to Excel and further analyzed using ToxoDB tools 37 . The resulting proteins were normalized by the proteins obtained in the pre-immune sera IP. The proteins having high abundances in common with those of pre-immune sera were omitted/not considered the interactome hits. For the whole proteome analysis for the TgCdc5 mAID-HA vehicle or IAA treated parasites were suspended in IP lysis buffer containing complete protease inhibitor cocktail and incubated on ice for 20 min with intermittent mixing. Subsequently, the protein-containing supernatant was collected by centrifugation and subjected to reduction, alkylation, trypsin digestion, and peptide purification as per the protocol described above. Label-free quantification was performed to analyze the whole cell proteome of TgCdc5-depleted parasites (+ IAA) compared with the vehicle-treated parasites (-IAA) for comparative analysis. Microscale thermophoresis (MST) assay MST experiments were performed with a Monolith NT.115 (Nanotemper). The 50 nM of each 5' cyanine-labeled U1/U2/U4/U5/U6 snRNA (Supplementary Table 1) was titrated against the serial dilution of TgCdc5 protein. The reaction mixtures were incubated at 25°C for 15 min and then loaded into glass capillaries (Nanotemper). The fluorescence intensity for each reaction was measured at 23°C using 15% LED power and 40% MST power. The fluorescence values were analyzed using the Affinity Analysis software version 2.3 (Nano Temper) to determine the binding affinity (dissociation constant: K D ) between TgCdc5 and snRNA variants. Yeast complementation For complementation assays, S . cerevisiae Δcef1 , and Δprp19 chromosomal copy deletion mutant strains (Supplementary Table 1) carrying the wild-type copy in a plasmid with URA marker were utilized 38 . Full-length T . gondii cdc5 , and prp19 genes were cloned into pYES3/CT vector between Bam I- EcoR I and BamH I- Xho I, respectively. The cef1 and prp19 , yeast mutant stains were transformed with respective plasmid carrying respective T . gondii gene or empty plasmid. Single Trp + transformants were patched to agar plates lacking tryptophan (-Trp) and then patched on agar medium containing FOA (-Trp + FOA). FOA-resistant colonies were picked and streaked on -Trp + FOA agar medium. Generation of auxin-inducible TgCdc5-mAID-HA transgenic parasites TgCdc5-mAID-3HA transgenic parasites were generated by CRISPR/Cas9-mediated site-specific gene editing using the T . gondii line RH TIR1-3FLAG 39 . A TgCdc5 CRISPR/Cas9 plasmid with a specific guide RNA targeting the 3’ end of Cdc5 was generated from the pSAG1::Cas9-U6::sgUPRT plasmid (Addgene, #54467). The PCR fragment containing the mAID-3HA tag and the HXGPRT selection was amplified from the plasmid pTUB1:YFP-mAID-3HA with 40 bp of homology with the 3′ end of Cdc5 to facilitate direct insertion of the PCR fragment and double homologous recombination. Fifteen µg each of TgCdc5-mAID-3HA amplicon and pSAG1::Cas9-U6::sgCdc5 were transfected into 10 7 RH TIR1-3FLAG parasites by electroporation using the Gene Pulser Xcell Total System (Biorad, #1652660). Parasites were drug-selected using mycophenolic acid (25 µg/ml) and xanthene (50 µg/ml) for three growth cycles and subsequently cloning out by serial dilution. Endogenous tagging of TgCdc5-mAID-HA was verified using sequencing, diagnostic PCR, immunoblotting, and IF staining. The auxin-inducible degradation of TgCdc5-mAID-HA was tested by culturing the parasites in a medium containing 500 µM indole-3-acetic acid (IAA) (Sigma), followed by immunoblotting and IF staining. Luciferase splicing assay A dual luciferase reporter gene system was employed for the in vivo splicing assay. An intron-containing TgPrp19 minigene (522bp) with exon1 (172bp)-intron1 (225bp)-exon2 (125bp) was introduced between Bgl II and Avr II restriction sites in plasmid pTUB1:YFP-mAID-3HA (Addgene #87259) replacing YFP coding sequence. Next, the renilla luciferase (Rluc) gene was amplified from the pmirGLO plasmid (Promega #E1330) and cloned in frame and tandem with TgPrp19 minigene (exon I-intron I-exon II) between Avr II and Nde I restriction sites. For control, the firefly luciferase (Fluc) gene was amplified from the pmirGLO plasmid and cloned (from the pmirGLO plasmid, Promega) between Bgl II and Avr II restriction sites in plasmid pTUB1:YFP-mAID-3HA replacing YFP coding sequence. Around 10 µg of each reporter plasmid construct were used to co-transfect 10 7 TgCdc5-mAID-HA parasites. Transfected parasites were immediately transferred to a new flask containing a confluent HFF monolayer and incubated for 24 hours before supplementing with vehicle or IAA and then harvested at different time points as indicated. The luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega, # E1910). Plaque assay Freshly egressed 100 TgCdc5-mAID-HA parasites were inoculated on HFF monolayers. After 24 h, the medium was removed, and parasites were grown in the presence 500 µM IAA or an equivalent volume of MeOH vehicle for 5 days prior to fixation with 4% paraformaldehyde in PBS and stained with 1% crystal violet for 20 min. A number of plaques were measured from 50 random fields. The plaque areas were quantified using ImageJ. A similar procedure was followed for the TgCdc5-mAID-HA rescue experiment; however, the IAA-containing medium was replaced with a normal medium at the indicated time points. Intracellular replication assay HFF monolayers on glass coverslips were inoculated with freshly egressed parasites. After 14 h, the medium was removed, and parasites were grown in the presence 500 µM IAA or MeOH vehicle for 18 h fixation with 4% paraformaldehyde in PBS and stained with α-TgIMC1 (1:2,000) and DAPI to detect individual parasites. Results are shown as the mean and the standard deviation of the number of parasites per vacuole determined by counting the parasites from 50 random vacuoles in triplicate from three independent biological replicates. Morphologically defective parasites were counted by following a similar procedure. Invasion assay Freshly egressed TgCdc5-mAID-HA parasites 10 3 were incubated with a medium containing 500 µM IAA or MeOH vehicle at 25 o C for 30 min. Subsequently, these parasites were allowed to infect HFFs grown on glass coverslips in a 6-well plate for 30 min at 37°C before fixation with 4% paraformaldehyde. Standard non-permeabilizing IFA was performed by staining extracellular parasites with rabbit α-TgIMC1 (1:2,000) antibody, followed by permeabilization with PBS/Triton and subsequent staining of invaded parasites with mouse α-TgIMC1 (1:2,000). HFFs were stained with AF-conjugated secondary antibodies (anti-mouse IgG AF 488 and anti-rabbit IgG AF 568) and DAPI. For each experiment, at least 50 parasites were counted, each time distinguishing non-invaded (green) from invaded (red) parasites from three independent biological replicates. Egress assay Freshly egressed TgCdc5-mAID-HA parasites were inoculated on HFF monolayers grown on glass coverslips. After 30 h, the medium was replaced with DMEM containing 500 µM IAA or MeOH vehicle, and cells were further incubated for 8 h. Parasite egress was initiated by adding 3 µM calcium ionophore A23187 to infected HFFs for 2 min. Cells were fixed, permeabilized, and stained with α-TgGRA2 (1:1000) and α-TgIMC1 (1:2000). The number of egressed versus non-egressed vacuoles was calculated by counting 150 vacuoles in triplicate from three independent biological replicates. Vacuoles containing > 2 parasites were considered intact. RNA sequencing and transcriptome analyses TgCdc5-mAID-HA parasites (5 x 10 7 ) were harvested from infected HFFs grown in DMEM containing 500 µM IAA or MeOH vehicle for 8 h. Total RNA was isolated using the RNeasy Plus mini kit (Qiagen) and quantified using a Qubit fluorometer (Thermofisher #Q33238). RIN values of the samples were determined using tape station 4150 and HS RNA screen tape. The cDNA libraries were prepared using a TruSeq Stranded Total RNA kit (Illumina #15032618, Illumina #20020596). Final libraries were quantified using a Qubit 4 Fluorometer (Thermofisher #Q33238), and the samples were subjected to sequencing of paired-end reads on the Illumina NovaSeq 6000 platform. The raw fastq reads of the sample were quality assessed using FastQC, and summarization was performed using MultiQC. The processed reads from the Illumina seq were mapped against the T . gondii reference genome (GCF_000006565.2) using STAR v2.7.9. The rRNA and tRNA features were removed from the GTF file of the T . gondii . The alignment file (BAM) from individual samples was quantified using feature Counts v. 2.0.1 to obtain transcript counts. These transcript counts were used as inputs to DESeq2 for differential expression estimation, keeping a threshold of statistical significance of < 0.05 p-value adjustment using the Benjamini and Hochberg method 40 . The resulting abundance counts were imported into R using the Bioconductor package ‘tximport’. The 'regularized log' transformation in DESeq2 was used for principal component and clustering analyses. An adjusted p-value (FDR) threshold < = 0.05 and log2 fold change of ∓ 2.0 were used for statistical estimation of gene expression. The k-Means clustering was done using iDEP.96, with the count file provided as an input (default parameters: most variable genes to include- 2000; number of clusters- 4; normalize by gene-mean center). For Gene Ontology (GO) annotation 41 , the protein sequences were extracted based on the DESeq2 result (Adjusted p-value < = 0.05 and Log Fold Change ∓ 2) and were subjected to the ToxoDB database 37 to generate GO terms utilizing the integrated GO tool. A threshold of p-value 0.05 and q-value less than 0.25 were set to define statistically significant GO terms, which were further used to generate GO plots in GraphPad prism. Two types of splicing analyses were performed i) Exon usage analysis by aligning the reads against the reference genome “ Toxoplasma _ gondii _TGA4.fna” and indexed using STAR 42 v2.7.9a. The generated bam files were used to perform Differential exon usage analysis using the “DEXSeq” package in R and ii) Splicing event analysis” using ASpli (v.1.5.1), a Bioconductor computational suite using the default commands as previously described 43 . RNA isolation and quantitative RT-PCR (qRT-PCR) Total RNA was isolated using the RNeasy Plus mini kit (Qiagen) followed by reverse transcriptase PCR using SuperScript III First-strand Synthesis System (Invitrogen) to produce cDNA following the manufacturer’s protocol. The qRT-PCR was performed on the 7500 ABI apparatus (Applied Biosystems) using cDNA, gene-specific forward and reverse primers with SYBR green PCR Master Mix (Applied Biosystems). The levels of unspliced and spliced transcripts of indicated genes were determined using primers targeting specific exon-intron and exon-exon junctions, respectively. The transcript level of the intron-less gene SRS40E is expected to be unaltered (based on RNA-seq data) and used as a normalizing control. Duplicate reactions were performed for each sample using the following cycle conditions: 95°C, 15 min followed by 40 cycles of 94°C, 30 s; 55°C, 40 s and 68°C, 50 s. Relative transcript levels were analyzed using the 2 −ΔΔCT method. The obtained transcript values were used to calculate splicing efficiency as the ratio of mRNA (spliced)/pre-mRNA (unspliced) and compared for vehicle and IAA-treated parasites. Apoptosis assay Parasite apoptosis was determined using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences # 556547). Briefly, filter-purified TgCdc5-mAID-HA parasites from the vehicle or IAA-treated at indicated time points were washed two times with cold PBS, centrifuged, and parasite pellets were resuspended in 100 µl 1× Annexin V binding buffer. Subsequently, 5µl of PE-Annexin V was added to the parasite suspension, mixed gently, and incubated in the dark for 30 minutes at room temperature. Later, 400 µl of 1× Annexin V binding buffer was added to each sample tube, and samples were analyzed immediately using a FACSCalibur flow cytometer (BD Biosciences). Flow cytometric data was analyzed using FlowJo software. TUNEL assay DNA fragmentation in the intracellular parasites was detected using the One-step TUNEL In Situ Apoptosis Kit (Elab Science #E-CK-A321). The assay uses the IFA-based terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) method. Briefly, HFF monolayer-infected TgCdc5-mAID-HA parasites were grown for 12 h. Later, the medium was replaced with DMEM containing 500 µM IAA or MeOH vehicle, and cells were further incubated for 8 h. Cells were fixed, permeabilized, equilibrated with TdT buffer at 37°C for 30 min, stained with labeling solution containing TdT enzyme at 37°C for 60 min. For the positive control, cells were treated with DNaseI enzyme prior to the TdT equilibration step. For each experiment, 100 parasites were counted from three independent biological replicates. Puromycin incorporation assay The parasite's nascent RNA synthesis levels were determined using puromycin labeling 44 . Parasites were grown on HFF monolayers in the presence of a vehicle or IAA for the indicated time points. Prior to paraformaldehyde fixation, cells were treated with 10 µg/ml puromycin for 30 min and then subjected to IFA. The complete block of protein synthesis in the control was achieved by treating the infected cells with 100 mg/ml cycloheximide (CHX) for 2 h before adding puromycin and followed by standard IFA. An anti-puromycin antibody was used to stain the newly synthesized proteins in the parasites and HA to visualize the parasite TgCdc5 protein. Protein aggregation assay Parasite protein aggregates were determined using the PROTEOSTAT Aggresome detection kit (Enzo Life Sciences #ENZ-51035-0025) according to the manufacturer’s instructions. Parasites were grown on HFF monolayers in the presence of a vehicle or IAA, which was fixed and permeabilized. Proteostat reagent was used to stain the protein aggregates and the HA to visualize the TgCdc5 protein. Mouse infection The auxin-inducible TgCdc5-mAID-HA protein degradation in mice was performed as described previously 45,46 with some modifications. A 30-day survival experiment was performed using six-week BALB/c male mice with three groups 10 mice/group. Mice from two groups were injected intraperitoneally (i.p.) with 50 tachyzoites of RH TgCdc5-mAID-HA. On the second day of post-infection (pi), 1 of 2 groups was given IAA, and the other group was administered an equivalent volume of MeOH vehicle in the drinking water containing 5% sucrose. Third group was no infection control with IAA. The IAA was administered for 14 days (2 days pi to 15 days pi) in two ways: i) in drinking water (0.5 mg/ml) and ii) by oral gavage (12.5 mg/mL). All mice were weighed and monitored daily. To test the TgCdc5 protein depletion, on day 6 pi, 2 mice each were sacrificed by CO 2 asphyxiation, and the peritoneal exudate cells (PECs) were collected and examined by IF staining using α-HA and α-TgIMC1 antibodies. All survived mice were sacrificed on day 30 and brain samples were processed to examine bradyzoite cyst. The relative weight loss of the mouse was calculated based on the initial body weight on the day of infection. T . gondii challenge experiment - A 60-day survival experiment was performed using six-week BALB/c mice - two IAA groups of 8 male and female mice and two without IAA groups (-IAA) of 3 male and female mice. Mice were injected with 5x10 3 TgCdc5-mAID-HA tachyzoites i.p. and treated with IAA or vehicle from day 2 to 15. On day 16, 2 mice from each group (+ IAA) were sacrificed by CO 2 asphyxiation, and heart and brain tissues were collected. Tissue samples were processed to detect bradyzoite cysts by microscopy. Genomic DNA was isolated by using a tissue DNA extraction kit (QIAmp, Qiagen), and qRT-PCR was performed for the T . gondii 529 repeat region. On day 21, serum from all mice was collected, and IgG-IFA was performed for tachyzoite and bradyzoite stages, as mentioned previously. From day 22–25, male and female mice mating was carried out. Further, male and female mice were segregated into 2 groups each (3 mice male or female/group). On day 27, 3 mice from 1 group each were injected with 5x10 3 TgCdc5-mAID-HA tachyzoites i.p, and mice were observed for another 33 days. Female mice in the control group delivered 12 pups on 21 days post-mating, and only 1 of 3 female mice injected with tachyzoites had a normal pregnancy with 2 pups. On day 60, 1 female and one male mouse were sacrificed by CO 2 asphyxiation. Sera and heart and brain tissues were collected to detect anti- T . gondii antibodies, bradyzoite cyst, and T . gondii DNA. Data analyses All data analyses, including graph preparation and statistics, were performed using GraphPad Prism 9. Results 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. 1 A, 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. 1 A, 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 searches 48 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. 1 C). 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. 2 A). To gain insight into the expression, and localization of TgCdc5, full-length TgCdc5-His protein of ~ 100 kDa was purified (Fig. 2 B) and used to generate specific anti-TgCdc5 antibodies. TgCdc5 is robustly expressed (Fig. 2 C) in both the asexual stages and localized in the nucleus of the tachyzoite and the perinuclear to cytoplasm in the bradyzoite stage (Fig. 2 D). 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. 2 E). 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 BLASTP 48 and HHpred 49 to identify and name these proteins following the human/yeast nomenclature (Fig. 2 E). 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. 2 E). Based on the coverage and peptide score, TgPrp19 (TgME49_320210), a conserved splicing factor containing Ubox and WD40 repeat (Fig. 2 F), was the major protein co-purified with TgCdc5 (Fig. 2 E). Before confirming their interaction, we first bacterially expressed full-length TgPrp19-His 6 protein (~ 56 kDa) (Fig. 2 G) and generated anti-TgPrp19 polyclonal antibodies. Similar to TgCdc5, TgPrp19 is also highly expressed in both the asexual stages (Fig. 2 H) and localized predominantly in the nucleus of the tachyzoite and bradyzoite stages (Fig. 2 I), suggesting an interaction of these proteins. Further, the reciprocal pull-down experiment (Fig. 2 J,K) and co-localization (Fig. 2 L) studies confirmed the strong interaction between TgCdc5 and TgPrp19. Cdc5 family members associate with the spliceosome throughout the entire splicing reaction 50 . Given that TgCdc5 contains nucleic acid binding domain 51 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 (K D =440 nM) and U6 (K D =481 nM) compared to U5 (K D =1 µM), U1 (K D =1.5 µM), and U4 (K D =3.7 µM) snRNAs (Fig. 5 M). 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. 2 N,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. 3 A) with the addition of IAA. The resulting TgCdc5-mAID-HA strain was confirmed by diagnostic PCR (Fig. 3 B) and western blotting (Fig. 3 C). Western blot (Fig. 3 D) and IF (Fig. 3 E) 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. 3 F) 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. 3 F). 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. 3 G) in the TgCdc5 depleted parasites consistent with reduced mRNA level of Rluc (Fig. 3 H). 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. 3 I). 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. 3 J), 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. 3 K) and severe morphological defects (Fig. 3 L). A parasite growth assay revealed that TgCdc5-depleted parasites produced no visible plaques (Fig. 3 M). To determine whether parasites can recuperate from a transient loss of TgCdc5, we performed a plaque assay using six different IAA treatment conditions (Fig. 3 N). 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. 3 N), no plaques were observed when treated for 2/4/8/12/24 h IAA (Fig. 3 N). Furthermore, TgCdc5 depletion significantly reduced the parasite's invasion efficiency (Fig. 3 O); however, the parasites' ability to egress upon inducing egress using calcium ionophore showed a marginal difference (Fig. 3 P). 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. 4 A, 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. 4 A). 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. 4 B). The genes that showed significantly different gene expressions (up or down) relevant to the study are shown in the volcano plot (Fig. 4 C). The host cell invasion genes RON2, RON3, RON4, and RON5 were found to be downregulated (Fig. 4 C), 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. 4 C). 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. 4 C). 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. 4 C). 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. 4 D). 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. 4 E). 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. 4 E). 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. 4 F). To understand the processes in which the differentially expressed genes might be involved, we conducted gene ontology (GO) enrichment analyses (Supplementary Table 5) (Fig. 4 G). 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. 4 H). 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. 4 H). 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. 5 A, Supplementary Table 6) corresponding to 3604 genes (Fig. 5 B) 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 4 C-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. 5 G), whereas 70.34% of DEGs (Fig. 5 G) 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. 6 A,B), whereas centrocone duplication indicated the M phase (Fig. 6 A,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. 6 C,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. 6 E,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. 6 G,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. 6 I). Depletion of TgCdc5 generates protein aggregates that triggers bradyzoite induction program In metazoans, NMD, an mRNA quality control process, removes erroneous transcripts 18 . However, recently, in P . falciparum , disruption of core NMD proteins showed no degradation of nonsense transcripts 20 , 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. 7 A) 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. 7 B,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 Toxoplasma 4 . 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. 7 D). 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. 7 D, 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 CO 2 depletion) (Fig. 7 F). 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. 7 D), 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. 7 G). 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. 7 H). A comparative analysis of vehicle and IAA-treated parasites showed that more proteins (Fig. 7 H) had reduced (n = 424) than elevated expression (n = 212). Downregulated genes contain more introns than upregulated genes (Fig. 7 I), 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. 7 J). 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. 7 J). As expected for lowered invasion, rhoptry proteins and kinases showed a significant reduction in the expression, which lowers parasite survival in the host (Fig. 7 J). 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. 8 A) 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. 8 B), 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. 8 C), whereas IAA treatment rescued all mice from fatal infection (Fig. 8 C). The vehicle treatment group showed severe morbidity and complete mortality compared to the IAA treatment group (Fig. 8 D and 8 C). 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. 8 D), mortality (Fig. 8 C). 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 5x10 3 TgCdc5-mAID-HA tachyzoites (Fig. 8 E). 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. 8 E) 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 5x10 3 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. 8 E). 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. 8 E). Discussion Pre-mRNA splicing is a crucial step in eukaryotic gene expression. This study focuses on the role of the essential pre-mRNA splicing factor Cdc5 in maintaining transcriptional homeostasis of the intron-rich genome of T . gondii . Despite high intron densities, the splicing mechanism in T . gondii appears to be largely conserved. Like model eukaryotes, TgCdc5 is part of a large spliceosomal complex involving both proteins and RNA. TgCdc5 is an essential splicing factor, and its depletion generates erroneous splicing, leading to significant alternative splicing events with widespread defects in gene expression for various parasite processes, including DNA replication, cell cycle, DNA damage, invasion, egress, protein degradation, and bradyzoite differentiation in non-cystogenic RH strain. Notably, TgCdc5 is essential for parasite survival in mice, as depleting TgCdc5 provides full protection against a lethal dose of tachyzoites. Interestingly, the immune response generated during this first exposure offers complete protection against future Toxoplasma infections and partially protects vertical transmission. The genes of Toxoplasma , Plasmodium , and Theileria show remarkable conservation of intron positions, suggesting that shared introns are predominantly ancestral and play an essential role in gene expression 9 . Plasmodium and Theileria ancestors experienced significant intron loss, while Toxoplasma showed no intron loss or gain 9 . Given the high energetic burden of intron-splicing, it's intriguing to consider what advantage apicomplexans gain from having genomes rich in introns. Introns play a crucial role in regulating gene expression by influencing mRNA export, stability, and translation efficiency 12,54–56 . Additionally, introns can affect transcriptional output by regulating the promoter-proximal chromatin profiles 57,58 and RNA Pol II occupancy 59 . Some introns also contain non-coding RNAs, whose processing from introns can speed up or slow down the rate of gene expression 60–62 . Consequently, it is tempting to speculate that the complex gene architecture in apicomplexans, particularly T . gondii , allows for more intricate RNA processing and gene regulation, possibly through alternative splicing or non-coding RNAs. This molecular adaptation of intron selection in evolution may hold the key to Toxoplasma 's success in its complex life cycle and ability to thrive in diverse host species. The immunoaffinity purification of TgCdc5 has enabled the identification of ~ 80 spliceosomal proteins, including eight core proteins. Based on the coverage and peptide score, TgPrp19, TgSpf27, and TgPrl1 were identified as the major core proteins co-purified with TgCdc5. Particularly, the presence of Prp19, Cdc5, Spf27, and Prl1 is consistent across humans, yeast, Trypanosoma parasite, and Toxoplasma complexes, indicating that these four proteins form the conserved core of the complex 63–65 . Given the diverse phylogenetic lineages of Toxoplasma , Trypanosoma , and model organisms from yeast to humans (Alveolata, Excavata, and Opishokonta, respectively), it is evident that the spliceosome's composition and mechanism are highly conserved over evolutionary time. Besides, Toxoplasma Cdc5 has retained a conserved role in the catalytic phase of splicing, as evidenced by its strong interaction with U2, U6, and U5 snRNAs. A loss-of-function mutation in the S . cerevisiae Cef1 (Cdc5 homolog) leads to slower growth, thermosensitivity, and cell cycle arrest in the G2/M phase 21 . Similarly, knockdown of human Cdc5L causes mitotic arrest, chromosome misalignments, and DNA damage, ultimately resulting in mitotic catastrophe 27 . These cell cycle arrests in yeast 66 and humans 27 are associated with defective splicing in cytoskeleton genes crucial for microtubule stability during mitosis. Likewise, TgCdc5 regulates the expression (RNA and protein) and splicing efficiency of cell cycle-specific ApiAP2 factors and a set of genes involved in maintaining the cytoskeleton; however, unlike yeast and humans, its depletion causes stage-independent cell cycle arrest. In addition to parasite replication arrest, depletion of TgCdc5 causes reduced parasite invasion, egress, ER-Golgi vesicular transport, translation fidelity, chaperone-mediated protein folding, ubiquitin-mediated protein degradation, redox balance, and induces severe DNA damage, ultimately leading to death, similar to apoptosis. The impact of TgCdc5 depletion on myriad parasite processes highlights the necessity of pre-mRNA splicing of genes involved in these processes, as every two out of three genes require splicing in Toxoplasma owing to the intron-rich genome. Erroneous splicing often generates transcripts containing PTC, which are usually targeted for degradation by NMD 18,19 . However, a recent study in P . falciparum demonstrated that NMD is not essential and does not target nonsense transcripts 20 , suggesting a potentially novel degradation mechanism. In Toxoplasma , mis-spliced transcripts generated by TgCdc5 depletion not only contribute to DEGs, but also undergo translation, resulting in the production of non-functional, misfolded proteins. These misfolded protein aggregates exert stress on the parasite, prompting a transition from the highly replicating tachyzoite to the latent bradyzoite stage, evidenced by the increased expression of transcripts and proteins needed for bradyzoite development. The spontaneous conversion from tachyzoite to bradyzoites in RH after the loss of TgCdc5 was unexpected, given that RH strain parasites are widely considered non-cystogenic. However, these bradyzoites were unable to develop into mature cysts over time or reactivate to tachyzoites once IAA was removed, indicating that under the enormous stress of splicing error, the parasite's attempt to survive by converting into dormant forms is ultimately unsuccessful, possibly due to the lack of functional proteins necessary for bradyzoite development. The depletion of TgCdc5 results in the complete protection of mice against a lethal dose of infective tachyzoites, highlighting its indispensable role in parasite survival within the host. While immunity acquired upon initial parasite exposure confers protection against subsequent lethal infections in mice, it does not provide full protection for female mice during pregnancy. However, this raises the question of how TgCdc5-depleted parasites generate a protective immune response. There are several potential explanations. Firstly, the high number of parasites in the initial exposure may be sufficient to generate protective immunity before they are cleared by immune cells. Secondly, the initial immune response may have heightened after the parasite challenge, offering protective immunity. Thirdly, exposure to a high number of tachyzoites and short-sustained infection due to tachyzoite to bradyzoite conversion may have stimulated a reasonable immune response, which could have increased after the parasite challenge, providing protective immunity. The fact that the immune response generated by the TgCdc5-depletion strategy conferred partial protection in pregnant mice suggests the need for additional parasite boosters and subsequent depletion of TgCdc5 by IAA before challenging. Nonetheless, these observations underscore the host-protective role of the TgCdc5-depletion strategy against lethal toxoplasmosis. Given the high global burden of disease and the lack of effective drugs, developing vaccines against T . gondii infection in humans is a high priority. The live-attenuated vaccine is considered good and can offer better protection than other types of vaccines 67–69 . A Toxoplasma strain with low virulence and no ability to form a bradyzoite cyst will be the right candidate 67–70 . Studies in rodent models showed that only a few live attenuated T . gondii strains used for immunization conferred protective immunity and significantly reduced the tissue cyst burden after the challenge 71–75 . Considering this, it is tempting to suggest that the IAA-mediated TgCdc5-depletion approach in the RH strain may be a good vaccine strategy for several reasons: 1. It induces protective immunity upon being challenged with more than a lethal dose of T . gondii and provides complete protection to mouse (natural host); 2. This parasite strain does not form latent bradyzoite, an immune protective stage, and is also refractory to current therapeutics; 3. It partially protects maternal-fetal transmission; however, additional parasite boosting followed by IAA treatment may improve the outcome of vertical transmission; 4. This approach may also protect against other highly prevalent low-virulent strains (type II and III). Overall, this study establishes the indispensable role of the splicing factor Cdc5 in preserving transcriptional homeostasis, which is essential for the survival of intron-rich Toxoplasma parasites. Erroneous splicing resulting from TgCdc5 loss significantly affects key parasite functions and yet triggers a protective immune response in the mouse host. Declarations Ethics statement The institutional ethics committee of National Institute of Animal Biotechnology has approved using laboratory research protocols (IBSC/Feb2023/NIAB/AD001) and animals (IAEC/NIAB/2024/09/ASD). Consent to participate Not applicable Competing interests The authors declare no competing financial or personal interests. Authors' contributions Poonam Kashyap: Data curation, investigation, analysis and writing manuscript. Kalyani Aswale: Data curation and analysis. Abhijit S. Deshmukh: Conceptualization, funding acquisition, investigation and writing manuscript. Funding This work was supported by an NIAB core grant (C022) for ASD. Data and materials availability RNA-sequencing data of Wild-type and knockdown (TgCdc5) Toxoplasma gondii parasites have been submitted to NCBI, BioProject: PRJNA1133933. Differential splicing analysis data of wild-type and mutant parasites have been submitted to NCBI, BioProject: PRJNA1126029. Proteomic data has been submitted to MassIVE - Data ID_MassIVE - MSV000095119. The illustration in this study was created using BioRender (https://biorender.com/). 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SupplementaryTable6.xlsx Supplementary Table 6 Differential splicing analysis performed using RNA-seq data. SupplementaryTable7.xlsx Supplementary Table 7 Details of downregulated and upregulated proteins identified by mass spectrometry. Cite Share Download PDF Status: Published Journal Publication published 22 Apr, 2025 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4811664","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":337782144,"identity":"bd844ef4-2ab6-4edd-bc0d-bd1427051b37","order_by":0,"name":"Abhijit Deshmukh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYDACCR6GAxIGDHIMzAxsEBFm4rQYGJOmBQgMEhsYYFoIAf7ZvQcPWBT8Sd/OzvzsAUPFPbsGdt4D+C25cy4B5LDcnc1s5gYMZ4qTG5j5EvBbcyPHAKxlw2EGMwnGtoRkBmYeA7w65KFa0g0Os38jTosBVEuCwWEesC12BLUY3jkD0mJsuOEwT7lBwpmEBDZCWuRu9xh/lvgjJ29w/vi2Bx8qEuz5+c/g1wICzBIwVgIDQ2IbQfVAwPgBiWNPjI5RMApGwSgYWQAADCA+TMtwixMAAAAASUVORK5CYII=","orcid":"","institution":"BRIC-National Institute of Animal Biotechnology","correspondingAuthor":true,"prefix":"","firstName":"Abhijit","middleName":"","lastName":"Deshmukh","suffix":""},{"id":337782145,"identity":"2c491515-24a2-4ec5-a714-b3f1df186f3d","order_by":1,"name":"Kalyani Aswale","email":"","orcid":"","institution":"National Institute of Animal Biotechnology","correspondingAuthor":false,"prefix":"","firstName":"Kalyani","middleName":"","lastName":"Aswale","suffix":""}],"badges":[],"createdAt":"2024-07-27 07:25:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4811664/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4811664/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-025-58805-3","type":"published","date":"2025-04-22T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62240632,"identity":"c706cdde-f99d-4c35-944d-61e76acf41be","added_by":"auto","created_at":"2024-08-12 03:10:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":294655,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe compositional dynamics of the spliceosome machinery in apicomplexans.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e The percentage of the genes with introns (0, 1-5, 6-10, 11-15, and \u0026gt;16) in six model apicomplexans (\u003cem\u003eT\u003c/em\u003e.\u003cem\u003e gondii\u003c/em\u003e, \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ecaninum\u003c/em\u003e \u003cem\u003eT\u003c/em\u003e.\u003cem\u003e annulata\u003c/em\u003e, \u003cem\u003eB\u003c/em\u003e.\u003cem\u003e microti\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e.\u003cem\u003e falciparum\u003c/em\u003e, and \u003cem\u003eC\u003c/em\u003e.\u003cem\u003e parvum\u003c/em\u003e) and compared to the relatively low intron content of \u003cem\u003eS\u003c/em\u003e.\u003cem\u003e cerevisiae\u003c/em\u003e, yeast. \u003cstrong\u003eB.\u003c/strong\u003e The spliceosomal assembly, catalytic activation, and disassembly pathway. The ordered interactions of the small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/ U6, and U5 are shown. The Prp19/Cdc5 complex required for splicing in both yeast and humans is indicated. \u003cstrong\u003eC.\u003c/strong\u003e The conservation of spliceosomal proteins across the apicomplexans (mentioned above) by BLAST search on VEuPathDB using protein sequences of \u003cem\u003eH\u003c/em\u003e. \u003cem\u003esapiens\u003c/em\u003e and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e. Summary of spliceosomal proteins, indicating localization in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e based on hyperLOPIT\u003csup\u003e76\u003c/sup\u003e and fitness score based on a genome-wide CRISPR fitness screen\u003csup\u003e77\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/4098403681b984d628265641.png"},{"id":62240312,"identity":"87f5d8c9-8a2e-44eb-9420-7f9ae3d9dc09","added_by":"auto","created_at":"2024-08-12 03:02:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1478776,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eT\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003egondii\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Cdc5 (TgCdc5) is a \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebona-fide\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003espliceosomal protein.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Schematic depicting TgCdc5: Myb domain (2-54 aa) and SANT domain (55-108 aa).\u003cstrong\u003e B-D.\u003c/strong\u003e The expression and localization of TgCdc5. Coomassie blue-stained SDS-PAGE gel of recombinant full-length TgCdc5-His protein (B), Western blot analysis of TgCdc5 expression levels in tachyzoites and bradyzoites (C). Immunofluorescence analysis in RH/ME49 parasites with α-TgCdc5 antibody. Scale bar, 5µm (D). Loading controls: TgAldolase (TgALD) - parasite proteins, TgSAG1 and TgBAG1, tachyzoite- and bradyzoite stage-specific markers, respectively. Tz: tachyzoite and Bz: bradyzoite. \u003cstrong\u003eE.\u003c/strong\u003e Selected TgCdc5 interactors identified by mass spectrometry analysis of isolated TgCdc5 complexes. The table summarizes selected spliceosomal proteins predicted to localize to the nucleus. \u003cstrong\u003eF.\u003c/strong\u003e Schematic of TgPrp19: Ubox (4-57 aa) and WD40 repeat (245-281 aa).\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;\u003cstrong\u003eG-I.\u003c/strong\u003eThe expression and localization of the TgCdc5 interactor and conserved spliceosome protein TgPrp19. Coomassie blue-stained SDS-PAGE gel of recombinant full-length TgPrp19-His protein (G), TgPrp19 expression and localization in tachyzoites and bradyzoites analysed by Western blotting (H) and immunofluorescence (I). Scale bar, 5µm. \u003cstrong\u003eJ and K.\u003c/strong\u003e TgCdc5 forms a complex with TgPrp19. TgCdc5/TgPrp19 complexes were immunoisolated from the soluble fraction (input). Beads with α-TgCdc5 antibodies precipitated complexes were probed with α-Prp19 antibodies and vice-versa to confirm the interaction between TgCdc5 and TgPrp19. \u003cstrong\u003eL.\u003c/strong\u003e IFA images of tachyzoites. Parasites were co-stained with α-TgCdc5/α-rabbit IgG Fluor 488 and α-TgPrp19/α-mouse IgG Fluor 594. Scale bar, 5µm. \u003cstrong\u003eM.\u003c/strong\u003e Microscale thermophoresis (MST) analysis of 5’ cyanine labeled U1, U2, U4, U5, and U6 snRNAs with TgCdc5. \u003cstrong\u003eN,O.\u003c/strong\u003e \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e Cdc5 and Prp19 functionally complement yeast (\u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e) counterparts. The strain of S. cerevisiae Δcef1, or ΔPrp19, transformed with pYES3 (TRP1) plasmid containing TgCdc5 or TgPrp19 gene or pYES3 plasmid. The transformants were patched to agar plates lacking tryptophan (-Trp) and containing FOA (-Trp+FOA). FOA-resistant colonies were streaked on -Trp+FOA agar medium.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/678dbd5b00cb9f85287683e5.png"},{"id":62240316,"identity":"6627bcb8-2da1-4e9e-8320-b5d4303b558b","added_by":"auto","created_at":"2024-08-12 03:02:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":524390,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDepletion of TgCdc5 impairs RNA splicing and arrest parasite replication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Strategy for tagging of TgCdc5 protein in RH-TIR1-3FLAG parental line. A mAID-3HA tag and HXGPRT selection cassette were integrated into the C-terminus of the TgCdc5 protein by CRISPR/Cas9-mediated homologous recombination. \u003cstrong\u003eB.\u003c/strong\u003e Diagnostic PCR from genomic DNA of RH-TIR1 and TgCdc5-mAID-HA parasites to check the 3′ integration of mAID-3HA in the gene locus. \u003cstrong\u003eC.\u003c/strong\u003e The expression of TgCdc5-mAID-HA in the RH-TIR1 transfected parasites determined by Western blotting using α-HA antibodies. \u003cstrong\u003eD.\u003c/strong\u003e Western blot analysis of expression of TgCdc5-mAID-HA expression levels in tachyzoites with or without IAA as indicated time-point using anti-HA antibodies. \u003cstrong\u003eE.\u003c/strong\u003e Immunofluorescence analysis of TgCdc5-mAID-HA grown with IAA or vehicle for 1 h. Scale bar, 5µm. \u003cstrong\u003eF.\u003c/strong\u003e Schematic diagram of the dual reporter gene under TgTub promoter. The dual luciferase reporter encodes a firefly luciferase (Fluc) and a Renilla luciferase (Rluc), fused in-frame via an exon1–intron–exon2 of TgPrp19 gene. This region of TgPrp19 contains an in-frame three translation stop codons TAG in the intronic region. Upon transfection of this reporter, in the case of normal splicing, the stop codon will be spliced out, which results in the generation of Fluc-Rluc fusion protein. In contrast, when the mRNA splicing is inhibited by TgCdc5 depletion, the stop codon will be reserved in the RNA product, leading to the expression of Fluc alone. \u003cstrong\u003eG.\u003c/strong\u003e TgCdc5-mAID-HA parasites were transfected with reporter plasmid DNA and cultured in the presence of IAA or vehicle. Dual luciferase assay was carried out to at indicated time points to measure the luminescence intensity of Fluc and Rluc, and then the Rluc/Fluc ratio was calculated. The relative Rluc/Fluc ratio is shown. \u003cstrong\u003eH.\u003c/strong\u003e qRT-PCR showing mRNA levels of the RLuc gene. Gene expression was normalized to FLuc levels. \u003cstrong\u003eI.\u003c/strong\u003eSchematic diagram of the primer pairs that detect spliced and unspliced mRNAs (top panel). qRT-PCR was used to determine the spliced to unspliced mRNA ratio for Prp19 (mini-gene) in control or TgCdc5L-knockdown parasites (bottom panel). \u003cstrong\u003eJ.\u003c/strong\u003e Percent parasite stained with Trypan blue in control or TgCdc5L-knockdown parasites. \u003cstrong\u003eK.\u003c/strong\u003e Parasite replication. TgCdc5-mAID-HA parasites grown in HFF monolayers for 18 h with IAA or vehicle. Fifty random vacuoles were counted to determine the number of parasites per vacuole, which was then plotted as a percentage of the total number of vacuoles. \u003cstrong\u003eL.\u003c/strong\u003eTgCdc5-mAID-HA parasites were grown with IAA or vehicle for 8 h. The Number of parasites with defective morphology was counted and plotted as a percentage of normal and abnormal parasites. \u003cstrong\u003eM.\u003c/strong\u003e Crystal violet stained images of plaques formed by RH-TIR1 and TgCdc5-mAID-HA parasites on HFF monolayer treated with IAA or vehicle. Bottom panel showing quantification of plaque numbers and plaque areas from three independent experiments (Mean ± SD). \u003cstrong\u003eN.\u003c/strong\u003e Rescue experiment. TgCdc5-mAID-HA parasites were grown on HFF monolayers with IAA or vehicle at the indicated time points, media were replaced with normal medium, and parasite growth was determined by plaque assays. \u003cstrong\u003eO.\u003c/strong\u003e Parasite invasion. The graph shows the percent invasion of RH-TIR1 and TgCdc5-mAID-HA parasites on HFF monolayer. \u003cstrong\u003eP.\u003c/strong\u003e Parasite egress. The graph shows the percent egress of RH-TIR1 and TgCdc5-mAID-HA parasites cultured with IAA or vehicle followed by calcium ionophore treatment. The intact or collapsed vacuoles were visualized and counted by IFA analysis. The graph representation is Mean ± SD (\u003cem\u003en\u003c/em\u003e=3 replicates). Student’s t-test was used to calculate the p-value: *\u0026lt;0.05; *** \u0026lt;0.001; ****\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/46c190d23fae692d5dabdcd5.png"},{"id":62240629,"identity":"fd64b1e4-21f8-4acb-a44b-fa2fc9040a1b","added_by":"auto","created_at":"2024-08-12 03:10:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":196082,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eknockdown of TgCdc5 affects global gene expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Pie chart showing the overall percentage distribution of upregulated, downregulated, and unchanged transcripts in the TgCdc5 knockdown parasites. \u003cstrong\u003eB.\u003c/strong\u003e \u003cem\u003ek\u003c/em\u003e-means clustering of 2000 transcripts. A total of four clusters (A, B, C, and D) were defined. The colour key ranges from -2 to +2 (green to red). \u003cstrong\u003eC.\u003c/strong\u003e Volcano plot showing differentially expressed genes in TgCdc5-mAID-HA parasites with IAA or vehicle. \u003cstrong\u003eD.\u003c/strong\u003e Percentage of the upregulated and downregulated genes with introns (0, 1-4, 5-8, and \u0026gt;9) in the TgCdc5 knockdown parasites. \u003cstrong\u003eE.\u003c/strong\u003e Validation of altered gene expression induced by TgCdc5 knockdown in a subset of genes including \u003cem\u003eTub1, Rbp1, Rps3, MCM4, Nhe2, Sec62, TDCP, Rab7, AMA1, \u003c/em\u003eand \u003cem\u003eSRS40E\u003c/em\u003eqRT-PCR. Gene expression was normalized to \u003cem\u003eSRS40E\u003c/em\u003e levels. Data are shown as mean ± S.D. (\u003cem\u003en\u003c/em\u003e=2 replicates) \u003cstrong\u003eF.\u003c/strong\u003e Schematic diagram of the primer pairs that detect spliced and unspliced mRNAs (top panel). qRT-PCR was used to determine the spliced to unspliced mRNA ratio for \u003cem\u003eTub1, Rbp1, Rps3, MCM4, Nhe2, Sec62, TDCP, Rab7, \u003c/em\u003eand \u003cem\u003eAMA1 \u003c/em\u003ein control or TgCdc5-knockdown parasites (bottom panel). \u003cstrong\u003eG,H.\u003c/strong\u003e Gene ontology analysis of downregulated (G), and upregulated (H) expressed genes with respect to cellular components, biological processes, and molecular functions.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/8cc019ddf802198c1519b043.png"},{"id":62241240,"identity":"6802384c-a9f7-45c1-bcd6-43ee716742e9","added_by":"auto","created_at":"2024-08-12 03:18:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":190354,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of TgCdc5 significantly impacts alternative splicing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA,B. \u003c/strong\u003ePie chart showing the percent distribution of alternative splicing (AS) events (A) and percentage of genes with the type of AS event (B) in the TgCdc5 knockdown parasites. \u003cstrong\u003eC-F.\u003c/strong\u003e Representative genes with particular AS events, namely intron retention (C), Exon skipping (D), and Alternative 5’/3 splice site (E,F), are shown. \u003cstrong\u003eG.\u003c/strong\u003e Relationship between differentially spliced (all AS types) and differentially expressed genes in TgCdc5 depleted parasites. Pie chart showing the overlap between differentially spliced and differentially expressed genes.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/41fe62d93cc1afeec949ecff.png"},{"id":62240335,"identity":"d8d6157f-ca71-403d-a37b-64e3cf65fb7c","added_by":"auto","created_at":"2024-08-12 03:02:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1137898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTgCdc5 knockdown affects progression and fitness of parasite\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Schematics showing different phases of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e tachyzoite cell cycle. \u003cstrong\u003eB.\u003c/strong\u003e Freshly infected TgCdc5-mAID-HA parasites were grown for indicated hours and co-stained with α-TgCentrin1 (red, centrosome marker), α-TgMorn1 (green, basal complex and centrosome marker) and DAPI (blue). \u003cstrong\u003eC,D.\u003c/strong\u003e 1 h invaded TgCdc5-mAID-HA parasites were grown in a vehicle or IAA for indicated hours and co-stained with α-TgCentrin1 and α-TgMorn1. Fifty parasites were randomly selected and counted for duplicated (green column) or single centrosomes (blue column). \u003cstrong\u003eE,F.\u003c/strong\u003e 4 h invaded TgCdc5-mAID-HA parasites were grown in a vehicle or IAA for indicated hours and co-stained with α-TgCentrin1 and α-TgMorn1. Fifty parasites were randomly selected and counted for duplicated (green column) or single centrocone (blue column). \u003cstrong\u003eG,H.\u003c/strong\u003e TgCdc5-mAID-HA tachyzoites treated with vehicle or IAA for indicated hours were immunofluorescently labelled (red fluorescence - α-HA/α-rabbit IgG Fluor 594), and DNA strand breaks were visualized by in situ TUNEL labelling (green fluorescence): scale bar, 5µm. Mean percentages ± SEM of TUNEL-positive TgCdc50mAID-HA parasites as determined by IFA; \u003cem\u003en\u003c/em\u003e=3. \u003cstrong\u003eI.\u003c/strong\u003eVehicle or IAA-treated TgCdc5-mAID-HA parasites were collected and analyzed by FACS for annexin V-FITC/-propidium iodide (PI) staining.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/c53c0a179d4926c5638f1402.png"},{"id":62240338,"identity":"330f7410-18d0-4ad9-81d2-6da0c563e8b8","added_by":"auto","created_at":"2024-08-12 03:02:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2566745,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTgCdc5 depletion induces bradyzoite formation mediated by protein aggregates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Representative IFA images of TgCdc5-mAID-HA tachyzoites treated with either vehicle, IAA, or CHX for indicated hours. For both groups, a 15-minute puromycin pulse (to label newly synthesized proteins) was given prior to parasite fixation. Parasites were co-stained with α-HA/α-rabbit IgG Fluor 594 and α-Puromycin/α-mouse IgG Fluor 488. Scale bar, 5µm. \u003cstrong\u003eB,C.\u003c/strong\u003eProteoStat staining is used to detect intracellular protein aggregates in Vehicle or IAA-treated TgCdc5-mAID-HA parasites. Representative IFA images of tachyzoites showing parasites were co-stained with α-HA (magenta) and ProteoStat Aggresome detection reagent (red) and DAPI (blue): scale bar, 5µm. Fifty parasites were randomly selected and counted for ProteoStat-negative (green column) or ProteoStat-positive (blue column). \u003cstrong\u003eD and F.\u003c/strong\u003e Representative immunofluorescent microscopy images of RH strain Cdc5-mAID-HA parasites after 1- or 6-day treatment with 500 µM IAA (D) or alkaline medium for stress-induced bradyzoite formation (E). BAG1 was used as a bradyzoite marker, and Rhodamine-conjugated \u003cem\u003eDolichos biflorus\u003c/em\u003e lectin (DBL) was used as a marker of the cyst wall. Scale bar = 5 µm. \u003cstrong\u003eE.\u003c/strong\u003e Quantification of DBA positive vacuoles as indicated. \u003cstrong\u003eG.\u003c/strong\u003e RH Cdc5-mAID-HA parasites were treated for 6 days with 500 µM IAA to induce bradyzoite formation; subsequently, parasites were harvested and allowed to recover in a vehicle or IAA for 15 days before being evaluated for parasite growth by plaque assay. \u003cstrong\u003eH.\u003c/strong\u003e Pie chart showing distribution of upregulated, downregulated, and unchanged proteins in the TgCdc5 knockdown parasites. \u003cstrong\u003eI.\u003c/strong\u003ePercentage of the upregulated and downregulated proteins with introns (0, 1-4, 5-8, and \u0026gt;9) in the TgCdc5 knockdown parasites. \u003cstrong\u003eJ.\u003c/strong\u003e GO analysis of common downregulated genes (mRNA and protein) with respect biological processes.\u003c/p\u003e","description":"","filename":"Fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/c6cbab4108b2b732fca7bd8b.png"},{"id":62240325,"identity":"1371cb30-c070-478a-89f1-e5228721f367","added_by":"auto","created_at":"2024-08-12 03:02:00","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":905559,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTgCdc5 is essential for parasite survival in mouse host\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B.\u003c/strong\u003e Experimental design of \u003cem\u003ein vivo\u003c/em\u003e test of TgCdc5 essentiality. Three groups \u003cem\u003en\u003c/em\u003e=10 mice per group. BALB/c mice were injected with 50 TgCdc5-mAID-HA tachyzoites intraperitoneally (i.p.) and treated with IAA or vehicle from day 2 to 15. On day 6, two mice were sacrificed, and peritoneal exudate cells (PECs) were collected for IF microscopy. PECs were co-stained with α-HA/α-mouse IgG Fluor 488 and α-TgIMC1/α-rabbit IgG Fluor 594. Scale bar, 5µm. \u003cstrong\u003eC.\u003c/strong\u003e Survival curve of mice. The Gehan-Breslow-Wilcoxon test was used to compare differences between the survival curves, p \u0026lt; 0.0001 (IAA vs control). \u003cstrong\u003eD.\u003c/strong\u003e Mean body weight ± SD of mice. \u003cstrong\u003eE.\u003c/strong\u003e Experimental design of mouse challenge experiment. Two IAA groups of 8 male and female mice and two without IAA groups (-IAA) of 3 male and female mice. BALB/c mice were injected with 5x10\u003csup\u003e3 \u003c/sup\u003eTgCdc5-mAID-HA tachyzoites i.p. and treated with IAA or vehicle from day 2 to 15. On day 16, 2 mice from each group (+IAA) were sacrificed, and organs (heart and brain) were collected to detect bradyzoite cysts by microscopy and \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e DNA by qRT-PCR. On day 21, serum from all mice were collected for IFA. From day 22-25, male and female mice mating was carried out. On day 27, 3 mice, each male/female (1 group each), were injected with 5x10\u003csup\u003e3\u003c/sup\u003e TgCdc5-mAID-HA tachyzoites. On day 60, 1 female and male mice were sacrificed, and sera and organs (heart and brain) were collected to detect anti-\u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e antibodies, bradyzoite cyst by microscopy, and \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e DNA by qRT-PCR.\u003c/p\u003e","description":"","filename":"Fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/adf5d34f1ca3424a72a75954.png"},{"id":62240332,"identity":"9539dace-10c3-4942-b0ea-9dd689b739dd","added_by":"auto","created_at":"2024-08-12 03:02:01","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":230330,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIllustration depicting the impact of inefficient splicing due to TgCdc5 depletion in the parasite and infected host. \u003c/strong\u003e\u003cem\u003eToxoplasma\u003c/em\u003e's genome is rich in introns, making efficient splicing crucial. The splicing factor Cdc5 is essential in maintaining the transcriptome integrity necessary for the parasite's survival. Depletion of TgCdc5 leads to alternative splicing and widespread gene expression changes, causing catastrophic effects on the parasites and, concomitantly, triggers unproductive bradyzoite formation from tachyzoites, likely due to misfolded protein aggregates caused by erroneous transcripts. When TgCdc5 is depleted upon high parasite infection in mice, these mice generate a protective immune response, protecting them from future infection and providing partial protection during pregnancy.\u003c/p\u003e","description":"","filename":"Fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/3c83a0fc042340f264adc818.png"},{"id":81179373,"identity":"ca5af359-ecac-44bd-b9a2-2d764c0a6b84","added_by":"auto","created_at":"2025-04-23 07:08:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9191235,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/b3eb3122-47ac-49e2-8fda-04e68c982328.pdf"},{"id":62241587,"identity":"f95d4022-b65f-49ff-9789-9bd0f9d83f56","added_by":"auto","created_at":"2024-08-12 03:26:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":340941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetails of primers and yeast strains used in this study.\u003c/p\u003e","description":"","filename":"SupplementaryTable1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/392c14cdddc5e91ff96f65c7.pdf"},{"id":62240314,"identity":"ea11b7da-b7fa-425a-ba1d-8b2b6d9f4851","added_by":"auto","created_at":"2024-08-12 03:02:00","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24238,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn-silico\u003c/em\u003e identification of spliceosomal proteins in apicomplexans.\u003c/p\u003e","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/a471d7c11c5e14ecfcf700ba.xlsx"},{"id":62240322,"identity":"eb7ccd25-e1ff-49ef-96c5-806608497c94","added_by":"auto","created_at":"2024-08-12 03:02:00","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIntron distribution in apicomplexans.\u003c/p\u003e","description":"","filename":"SupplementaryTable3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/7ff1d0c3d210d828472dca93.pdf"},{"id":62240328,"identity":"25520572-9cb9-4df8-bcc3-158810b6abcc","added_by":"auto","created_at":"2024-08-12 03:02:00","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":44838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetails of TgCdc5 immunocoprecipitated proteins identified by mass spectrometry.\u003c/p\u003e","description":"","filename":"SupplementaryTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/7c751860db7bf6ee699578d6.xlsx"},{"id":62240327,"identity":"c067e4cd-8b31-4f0b-b44f-b4c2625c7ec9","added_by":"auto","created_at":"2024-08-12 03:02:00","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1088539,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetails of downregulated and upregulated genes determined by RNA-seq.\u003c/p\u003e","description":"","filename":"SupplementaryTable5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/69998f4fb975cb4f6431d4bd.xlsx"},{"id":62240330,"identity":"4fa8c7e7-6a3c-4a66-b6f0-61531d4ee5f6","added_by":"auto","created_at":"2024-08-12 03:02:00","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1066020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferential splicing analysis performed using RNA-seq data.\u003c/p\u003e","description":"","filename":"SupplementaryTable6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/88aa94f4cba8f42292a03821.xlsx"},{"id":62240633,"identity":"556338c4-4ad2-415f-913f-80962fbec410","added_by":"auto","created_at":"2024-08-12 03:10:01","extension":"xlsx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":365365,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Table 7\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetails of downregulated and upregulated proteins identified by mass spectrometry.\u003c/p\u003e","description":"","filename":"SupplementaryTable7.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4811664/v1/ba6508510d7316d42e9ecd8b.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Depletion of splicing factor Cdc5 in Toxoplasma disrupts transcriptome integrity, induces stress-driven abortive bradyzoite formation, and triggers host protective immunity","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eToxoplasma gondii\u003c/em\u003e is a member of the phylum Apicomplexa, a diverse group of several human (e.g., \u003cem\u003ePlasmodium\u003c/em\u003e, \u003cem\u003eCryptosporidium\u003c/em\u003e) and animal (e.g., \u003cem\u003eNeospora\u003c/em\u003e, \u003cem\u003eTheileria\u003c/em\u003e, \u003cem\u003eBabesia\u003c/em\u003e) pathogens\u003csup\u003e1\u003c/sup\u003e. \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e causes lifelong chronic infections in warm-blooded animals, including humans, and severe disease in fetuses and immunocompromised individuals\u003csup\u003e2\u003c/sup\u003e. The life cycle of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e is complex, involving sexual replication in feline definitive hosts and asexual replication in other intermediate hosts with three infective stages: tachyzoites, bradyzoites, and sporozoites\u003csup\u003e3\u003c/sup\u003e. During infection, parasites disseminate as tachyzoites, causing acute disease, and then differentiate into bradyzoite cysts, leading to a long-lived latent infection\u003csup\u003e4\u003c/sup\u003e. \u003cem\u003eToxoplasma\u003c/em\u003e displays distinct gene expression programs during stage transitions to provide the required diverse protein repertoire, potentially controlled through RNA-based regulatory mechanisms\u003csup\u003e5\u0026ndash;7\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMost parasites in the Apicomplexan genome have genes with a high number of introns\u003csup\u003e8\u0026ndash;10\u003c/sup\u003e. \u003cem\u003eToxoplasma\u003c/em\u003e has over 75% of genes with introns compared to 5% in yeast\u003csup\u003e6\u003c/sup\u003e. Apicomplexan introns follow the standard 5\u0026prime; GU-AG 3\u0026prime; splice junction with some nucleotide variations at the branch point and are primarily excised by the U2-type spliceosome\u003csup\u003e6,7,11\u003c/sup\u003e. Recent studies suggest that the RNA splicing mechanism is largely conserved in apicomplexans, and the few identified splicing factors acquired divergent features\u003csup\u003e6,7\u003c/sup\u003e. Regardless, the presence of intron-rich genes in these species is intriguing, given the reductive mode of evolution observed in diverse parasite clans\u003csup\u003e7\u003c/sup\u003e. Introns generally regulate the expression of genes at several levels\u003csup\u003e12\u003c/sup\u003e, including mRNA export, stability, and translation efficiency, indicating intron selection in evolution in apicomplexans, particularly Toxoplasma, aids in different developmental stages and thrives in diverse host species.\u003c/p\u003e \u003cp\u003ePre-mRNA splicing is an important step during eukaryotic gene expression. The spliceosome, a multisubunit complex of five small nuclear RNPs (snRNPs) and several non-snRNP proteins, catalyzes RNA splicing by coordinating the removal of introns and joining adjacent exons to create mRNA\u003csup\u003e13\u0026ndash;15\u003c/sup\u003e. In higher eukaryotes, most pre-mRNAs undergo alternative splicing (AS), producing different mRNA isoforms, contributing to expanding the proteome and also serving regulatory functions\u003csup\u003e16,17\u003c/sup\u003e. In contrast, recent studies suggest that alternative splicing is less prevalent in apicomplexans\u003csup\u003e7\u003c/sup\u003e, and AS events do not produce multiple alternative proteins but lead to the generation of aberrant or noncoding transcripts\u003csup\u003e7\u003c/sup\u003e. Disrupting conserved regulators of AS has shown lethal effects in apicomplexans\u003csup\u003e7\u003c/sup\u003e, indicating a biological role for some observed alternative splicing. In addition to protein diversity, AS can also generate non-functional products by introducing toxic exons or transcripts with a premature stop codon (PTC) that might undergo nonsense-mediated decay (NMD)\u003csup\u003e18,19\u003c/sup\u003e. A recent study in \u003cem\u003eP\u003c/em\u003e. \u003cem\u003efalciparum\u003c/em\u003e showed NMD is not essential and reported no change in the nonsense transcripts levels after deleting two core NMD proteins, indicating the regulatory role of the erroneous transcripts in gene expression\u003csup\u003e20\u003c/sup\u003e. The observed discrepancy between transcripts and protein levels in apicomplexan parasites highlights posttranscriptional gene regulation, mainly RNA splicing. The regulation of gene expression through RNA splicing is well-documented in model eukaryotes; however, studies on \u003cem\u003eToxoplasma\u003c/em\u003e splicing are limited, resulting in a poor understanding of this regulatory mechanism.\u003c/p\u003e \u003cp\u003eIn addition to the snRNPs, several non-snRNP splicing proteins play critical roles in the splicing process. Among those is cell division cycle 5 (Cdc5), a highly conserved splicing factor in animals, plants, and fungi\u003csup\u003e21\u003c/sup\u003e. Cdc5 is an essential splicing factor required for catalytic activation of the spliceosome by forming a heteromeric protein complex known as the \u003cem\u003eNineteen Complex\u003c/em\u003e (NTC) in \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e, yeast\u003csup\u003e22\u003c/sup\u003e, and Prp19/Cdc5L complex in humans\u003csup\u003e23\u003c/sup\u003e. The protein composition of human and yeast complexes differs; however, they share the subunits Prp19, Cdc5L/Cef1, Spf27/Snt309, and Prl1/Prp46 (human/yeast)\u003csup\u003e22,24,25\u003c/sup\u003e. In addition to splicing, Cdc5 participates in diverse molecular processes, such as cell cycle\u003csup\u003e26\u0026ndash;28\u003c/sup\u003e, DNA repair\u003csup\u003e29\u003c/sup\u003e, and RNA transcription\u003csup\u003e30\u003c/sup\u003e. In yeast and humans, mutation or knockdown of Cdc5 leads to the accumulation of retained introns in partially spliced pre-mRNAs, causing G2/M cell cycle arrest\u003csup\u003e26\u003c/sup\u003e. In plants (\u003cem\u003eArabidopsis\u003c/em\u003e), Cdc5 is required for development and immunity to bacterial infection\u003csup\u003e31,32\u003c/sup\u003e; however, its role in splicing is unclear. Despite Cdc5's pleiotropic function in model organisms, its role in splicing or other related processes in apicomplexans has yet to be studied. Considering the importance of RNA splicing for apicomplexans, a potential new regulatory role of Cdc5 in gene expression or parasite-centric processes cannot be discounted.\u003c/p\u003e \u003cp\u003eHere, we report why RNA splicing is crucial for the intron-rich \u003cem\u003eToxoplasma\u003c/em\u003e, show how depleting the core splicing factor Cdc5 affects various parasite processes, and offer protection in the mouse host. TgCdc5 is part of a large spliceosomal complex of snRNPs and non-snRNP with several unidentified proteins in \u003cem\u003eToxoplasma\u003c/em\u003e. We demonstrate that TgCdc5 is an essential pre-mRNA splicing factor, and its depletion results in significant alternative splicing and dysregulated gene expression associated with various parasite functions, including unproductive differentiation into slow-growing bradyzoite cyst in non-cystogenic strain. Finally, we demonstrate that TgCdc5 is essential for \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e survival in mice, as depleting TgCdc5 provides complete protection against a lethal dose of tachyzoites, and interestingly, the protective immune response generated against them offers total protection against future infections and partial protection during pregnancy.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eParasite culture\u003c/h2\u003e \u003cp\u003e \u003cem\u003eT. gondii\u003c/em\u003e strains RH, ME49, RH-TIR1-3FLAG were maintained in confluent monolayers of human foreskin fibroblast (HFFs, ATCC) cells in DMEM containing 10% foetal bovine serum, 10 \u0026micro;g/ml gentamicin, 1% penicillin-streptomycin, and 2 mM L-glutamine at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. The stage conversion from tachyzoite to bradyzoite was carried out by incubating the ME49 tachyzoites in bradyzoite induction medium (RPMI pH 8.2) at 37\u0026deg;C for 5 days without CO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e33\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCloning, expression, and protein purification\u003c/h2\u003e \u003cp\u003eA synthetic DNA encoding \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e Cdc5\u003csup\u003e1\u0026ndash;2664\u003c/sup\u003e and Prp19\u003csup\u003e1\u0026ndash;1545\u003c/sup\u003e (superscript number denotes nucleotide coordinates) were purchased from Life Technologies (USA). The synthetic TgCdc5 gene containing C-terminal His\u003csub\u003e6\u003c/sub\u003e was codon-optimized for expression in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e and was initially cloned into a pMS vector between \u003cem\u003eNdeI\u003c/em\u003e-\u003cem\u003eBamH\u003c/em\u003eI sites, which was subsequently subcloned into the pET-21a vector (Novagen, USA). The synthetic TgPrp19 gene containing C-terminal His\u003csub\u003e6\u003c/sub\u003e was initially cloned into the pMA-T vector between \u003cem\u003eNde\u003c/em\u003eI-\u003cem\u003eXho\u003c/em\u003eI sites, which was further subcloned into the pET-28a vector. The recombinant TgCdc5-His (~\u0026thinsp;100 kDa) TgMorn1-His (~\u0026thinsp;41 kDa) proteins were expressed in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e BL21 Rosetta and purified on a Ni\u003csup\u003e2+\u003c/sup\u003e-NTA agarose resin column (Qiagen). TgPrp19 protein was purified from the inclusion bodies. The inclusion bodies were solubilized in 6 M GuHCl and the TgPrp19-His (~\u0026thinsp;57 kDa) protein was purified over Ni\u003csup\u003e2+\u003c/sup\u003e-NTA agarose under denaturing condition\u003csup\u003e34\u003c/sup\u003e. The protein was then greatly diluted and refolded in the refolding buffer (50 mM Tris-HCl pH8.5, 9.6 mM NaCl, 0.4 mM KCl, 1 mM EDTA, 0.5% Triton X-100, 1 mM DTT). The purified proteins were dialyzed in 1X PBS and stored at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePolyclonal antibody raising\u003c/h2\u003e \u003cp\u003eMouse and/or rabbit polyclonal antibodies to recombinant TgCdc5, TgPrp19 and TgMorn1 were generated by primary injection with 30 \u0026micro;g (mouse) or 60 \u0026micro;g (rabbit) of purified recombinant protein in Freund's complete adjuvant (Sigma) followed by four boosts of 10 \u0026micro;g (mouse) or 25 \u0026micro;g (rabbit) each in Freund's incomplete adjuvant (Sigma) at 2-week intervals. Serum was collected after day 60 post-immunization. Polyclonal antibodies for Tg- IMC1\u003csup\u003e35\u003c/sup\u003e, SAG1\u003csup\u003e34\u003c/sup\u003e, BAG1\u003csup\u003e36\u003c/sup\u003e, CST1\u003csup\u003e34\u003c/sup\u003e, and Aldolase (ALD)\u003csup\u003e36\u003c/sup\u003e antibodies were used from previous studies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImmunoblotting\u003c/h2\u003e \u003cp\u003eFilter-purified 10\u003csup\u003e5\u003c/sup\u003e parasites were suspended in SDS-PAGE sample buffer, boiled for 10 min, and run on a single lane of an 8 or 10% polyacrylamide gel. The gel was then transferred to a 0.2 \u0026micro;m PVDF membrane using a Trans-Blot System (BioRad) for 2 h at 150 V. The membrane was blocked in 5% (w/v) non-fat milk in PBS for 60 min and probed with a primary antibody (α-TgCdc5/TgPrp19/TgSAG1/TgBAG1/TgALD-1:1000; αHA-1:5,000) in PBS overnight at 4\u0026deg;C. The membrane was washed 3x with PBS plus Tween-20 detergent (PBST; 0.1% Tween-20) and probed with HRP-conjugated α-rabbit IgG or α-mouse IgG antibodies (Invitrogen). The blot was developed using the SuperSignal West Pico PLUS (ThermoScientific) and visualized on a ChemiDoc Imager.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence (IF) staining\u003c/h2\u003e \u003cp\u003e \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e RH/ME49/TgCdc5-mAID-HA parasites infected HFFs were fixed with 4% paraformaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS for 15 min at room temperature (RT), and blocked with 5% (w/v) BSA in PBS for 60 min at RT. Samples were first incubated with the primary antibody (α- TgCdc5/TgPrp19-1:100 and α-TgCentrin/Morn1/IMC1/BAG1//HA-1:1,000) at RT for 60 min, washed 5x with PBS, and then incubated with fluorescent secondary antibodies (Alexa Fluor 488/594 1:1000) along with 4',6-diamidino-2-phenylindole (DAPI; 300 nM) and \u003cem\u003eDolichos biflorus\u003c/em\u003e agglutinin (DBA) as appropriate at RT for 60 min. Samples were then washed 5x with PBS and coverslips were mounted on a glass slide using Vectashield medium (Vector Laboratories). IF staining was visualised using a Leica confocal microscope with a 100X oil immersion objective. Images were processed using las x software (Leica Microsystems).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunoprecipitation (IP)\u003c/h2\u003e \u003cp\u003eThe IP was performed using Crosslink IP Kit (ThermoScientific). TgCdc5/TgPrp19 antibodies (5 \u0026micro;g) were crosslinked to protein A/G agarose beads by using disuccinmidyl suberate. Parasite protein lysate was prepared from 2 x 10\u003csup\u003e8\u003c/sup\u003e RH parasites using IP lysis buffer supplemented with complete protease inhibitor cocktail (Sigma). The parasite protein lysate diluted with IP buffer was first pre-cleared by mixing it with protein A/G agarose. Supernatant was added to the TgCdc5/TgPrp19 antibody-linked beads and mixture was incubated for 10 h at 4\u0026deg;C. The beads were collected and washed 3x with IP buffer to remove any unbound proteins. TgCdc5/TgPrp19 associated proteins were eluted and samples were prepared in SDS-PAGE sample buffer. The eluates were separated by 10% SDS-PAGE for Western blot analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eLC-MS/MS and interactome analyses\u003c/h2\u003e \u003cp\u003eTgCdc5 IP elute protein concentration was determined using BCA method and further used for LC-MS sample preparation according to the standard protocol. IP elutes was reduced (100 mM DTT for 1 h at 55\u0026deg;C) and alkylated (400 mM Iodoacetamide for 1 h at 25\u0026deg;C in the dark). One \u0026micro;g of proteomics grade trypsin (NEB) was then added per sample and digestion was carried out overnight at 37\u0026deg;C. Digested peptides were desalted and purified using C18 spin column (ThermoScientific). Total 40 \u0026micro;l elute was then dried using a speed vacuum and stored at -80\u0026deg;C. For analysis, the samples were redissolved in 0.1% formic acid and 5 \u0026micro;L of each digest was run by LC-MS/MS using a 3 h gradient on a 75\u0026micro;m x 50cm C18 column (PepMap RSLC C18) feeding into a Q-Exactive HF mass spectrometer (Orbitrap).\u003c/p\u003e \u003cp\u003eMS/MS data was analyzed using Proteome Discoverer 2.2 software package (Thermo) against \"UniProt-proteome_TgGT1_UP000005641\" with 8450 entries and a common lab contaminants database having 244 common contaminants proteins. Sequest HT searches were performed with fragment ion mass tolerance of 1 Da and precursor mass tolerance of 20 ppm. Cysteine carbamidomethylation due to iodoacetamide was set as the default modification, and methionine oxidation was set as a differential/variable modification. Sequest HT-identified proteins were validated by the percolator validation algorithm and grouped according to the significance of peptide evidence. Identified proteins with a minimum of two unique peptides (min #unique peptide\u0026thinsp;=\u0026thinsp;\u0026gt;\u0026thinsp;2) and False Discovery Rate (FDR) equal to or less than 1% were considered along with percolator validation, which separates the identified PSMs into high-, medium-, and low-confidence identifications. Protein groups with no unique peptides (with similar peptides) that cannot be classified based on unique peptides were grouped by applying the strict parsimony principle parameter of protein grouping in the consensus workflow. After the contaminants were removed, the identified proteins list was moved to Excel and further analyzed using ToxoDB tools\u003csup\u003e37\u003c/sup\u003e. The resulting proteins were normalized by the proteins obtained in the pre-immune sera IP. The proteins having high abundances in common with those of pre-immune sera were omitted/not considered the interactome hits.\u003c/p\u003e \u003cp\u003eFor the whole proteome analysis for the TgCdc5 mAID-HA vehicle or IAA treated parasites were suspended in IP lysis buffer containing complete protease inhibitor cocktail and incubated on ice for 20 min with intermittent mixing. Subsequently, the protein-containing supernatant was collected by centrifugation and subjected to reduction, alkylation, trypsin digestion, and peptide purification as per the protocol described above. Label-free quantification was performed to analyze the whole cell proteome of TgCdc5-depleted parasites (+\u0026thinsp;IAA) compared with the vehicle-treated parasites (-IAA) for comparative analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMicroscale thermophoresis (MST) assay\u003c/h2\u003e \u003cp\u003eMST experiments were performed with a Monolith NT.115 (Nanotemper). The 50 nM of each 5' cyanine-labeled U1/U2/U4/U5/U6 snRNA (Supplementary Table\u0026nbsp;1) was titrated against the serial dilution of TgCdc5 protein. The reaction mixtures were incubated at 25\u0026deg;C for 15 min and then loaded into glass capillaries (Nanotemper). The fluorescence intensity for each reaction was measured at 23\u0026deg;C using 15% LED power and 40% MST power. The fluorescence values were analyzed using the Affinity Analysis software version 2.3 (Nano Temper) to determine the binding affinity (dissociation constant: K\u003csub\u003eD\u003c/sub\u003e) between TgCdc5 and snRNA variants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eYeast complementation\u003c/h2\u003e \u003cp\u003eFor complementation assays, \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae Δcef1\u003c/em\u003e, \u003cem\u003eand Δprp19\u003c/em\u003e chromosomal copy deletion mutant strains (Supplementary Table\u0026nbsp;1) carrying the wild-type copy in a plasmid with \u003cem\u003eURA\u003c/em\u003e marker were utilized\u003csup\u003e38\u003c/sup\u003e. Full-length \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii cdc5\u003c/em\u003e, and \u003cem\u003eprp19\u003c/em\u003e genes were cloned into pYES3/CT vector between \u003cem\u003eBam\u003c/em\u003eI-\u003cem\u003eEcoR\u003c/em\u003eI and \u003cem\u003eBamH\u003c/em\u003eI-\u003cem\u003eXho\u003c/em\u003eI, respectively. The \u003cem\u003ecef1\u003c/em\u003e and \u003cem\u003eprp19\u003c/em\u003e, yeast mutant stains were transformed with respective plasmid carrying respective \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e gene or empty plasmid. Single Trp\u003csup\u003e+\u003c/sup\u003e transformants were patched to agar plates lacking tryptophan (-Trp) and then patched on agar medium containing FOA (-Trp\u0026thinsp;+\u0026thinsp;FOA). FOA-resistant colonies were picked and streaked on -Trp\u0026thinsp;+\u0026thinsp;FOA agar medium.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of auxin-inducible TgCdc5-mAID-HA transgenic parasites\u003c/h2\u003e \u003cp\u003eTgCdc5-mAID-3HA transgenic parasites were generated by CRISPR/Cas9-mediated site-specific gene editing using the \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e line RH TIR1-3FLAG\u003csup\u003e39\u003c/sup\u003e. A TgCdc5 CRISPR/Cas9 plasmid with a specific guide RNA targeting the 3\u0026rsquo; end of Cdc5 was generated from the pSAG1::Cas9-U6::sgUPRT plasmid (Addgene, #54467). The PCR fragment containing the mAID-3HA tag and the HXGPRT selection was amplified from the plasmid pTUB1:YFP-mAID-3HA with 40 bp of homology with the 3\u0026prime; end of Cdc5 to facilitate direct insertion of the PCR fragment and double homologous recombination. Fifteen \u0026micro;g each of TgCdc5-mAID-3HA amplicon and pSAG1::Cas9-U6::sgCdc5 were transfected into 10\u003csup\u003e7\u003c/sup\u003e RH TIR1-3FLAG parasites by electroporation using the Gene Pulser Xcell Total System (Biorad, #1652660). Parasites were drug-selected using mycophenolic acid (25 \u0026micro;g/ml) and xanthene (50 \u0026micro;g/ml) for three growth cycles and subsequently cloning out by serial dilution. Endogenous tagging of TgCdc5-mAID-HA was verified using sequencing, diagnostic PCR, immunoblotting, and IF staining. The auxin-inducible degradation of TgCdc5-mAID-HA was tested by culturing the parasites in a medium containing 500 \u0026micro;M indole-3-acetic acid (IAA) (Sigma), followed by immunoblotting and IF staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eLuciferase splicing assay\u003c/h2\u003e \u003cp\u003eA dual luciferase reporter gene system was employed for the in vivo splicing assay. An intron-containing TgPrp19 minigene (522bp) with exon1 (172bp)-intron1 (225bp)-exon2 (125bp) was introduced between \u003cem\u003eBgl\u003c/em\u003eII and \u003cem\u003eAvr\u003c/em\u003eII restriction sites in plasmid pTUB1:YFP-mAID-3HA (Addgene #87259) replacing YFP coding sequence. Next, the renilla luciferase (Rluc) gene was amplified from the pmirGLO plasmid (Promega #E1330) and cloned in frame and tandem with TgPrp19 minigene (exon I-intron I-exon II) between \u003cem\u003eAvr\u003c/em\u003eII and \u003cem\u003eNde\u003c/em\u003eI restriction sites. For control, the firefly luciferase (Fluc) gene was amplified from the pmirGLO plasmid and cloned (from the pmirGLO plasmid, Promega) between \u003cem\u003eBgl\u003c/em\u003eII and \u003cem\u003eAvr\u003c/em\u003eII restriction sites in plasmid pTUB1:YFP-mAID-3HA replacing YFP coding sequence. Around 10 \u0026micro;g of each reporter plasmid construct were used to co-transfect 10\u003csup\u003e7\u003c/sup\u003e TgCdc5-mAID-HA parasites. Transfected parasites were immediately transferred to a new flask containing a confluent HFF monolayer and incubated for 24 hours before supplementing with vehicle or IAA and then harvested at different time points as indicated. The luciferase activity was determined using the Dual-Luciferase Reporter Assay System (Promega, # E1910).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePlaque assay\u003c/h2\u003e \u003cp\u003eFreshly egressed 100 TgCdc5-mAID-HA parasites were inoculated on HFF monolayers. After 24 h, the medium was removed, and parasites were grown in the presence 500 \u0026micro;M IAA or an equivalent volume of MeOH vehicle for 5 days prior to fixation with 4% paraformaldehyde in PBS and stained with 1% crystal violet for 20 min. A number of plaques were measured from 50 random fields. The plaque areas were quantified using ImageJ. A similar procedure was followed for the TgCdc5-mAID-HA rescue experiment; however, the IAA-containing medium was replaced with a normal medium at the indicated time points.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular replication assay\u003c/h2\u003e \u003cp\u003eHFF monolayers on glass coverslips were inoculated with freshly egressed parasites. After 14 h, the medium was removed, and parasites were grown in the presence 500 \u0026micro;M IAA or MeOH vehicle for 18 h fixation with 4% paraformaldehyde in PBS and stained with α-TgIMC1 (1:2,000) and DAPI to detect individual parasites. Results are shown as the mean and the standard deviation of the number of parasites per vacuole determined by counting the parasites from 50 random vacuoles in triplicate from three independent biological replicates. Morphologically defective parasites were counted by following a similar procedure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eInvasion assay\u003c/h2\u003e \u003cp\u003eFreshly egressed TgCdc5-mAID-HA parasites 10\u003csup\u003e3\u003c/sup\u003e were incubated with a medium containing 500 \u0026micro;M IAA or MeOH vehicle at 25\u003csup\u003eo\u003c/sup\u003eC for 30 min. Subsequently, these parasites were allowed to infect HFFs grown on glass coverslips in a 6-well plate for 30 min at 37\u0026deg;C before fixation with 4% paraformaldehyde. Standard non-permeabilizing IFA was performed by staining extracellular parasites with rabbit α-TgIMC1 (1:2,000) antibody, followed by permeabilization with PBS/Triton and subsequent staining of invaded parasites with mouse α-TgIMC1 (1:2,000). HFFs were stained with AF-conjugated secondary antibodies (anti-mouse IgG AF 488 and anti-rabbit IgG AF 568) and DAPI. For each experiment, at least 50 parasites were counted, each time distinguishing non-invaded (green) from invaded (red) parasites from three independent biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEgress assay\u003c/h2\u003e \u003cp\u003eFreshly egressed TgCdc5-mAID-HA parasites were inoculated on HFF monolayers grown on glass coverslips. After 30 h, the medium was replaced with DMEM containing 500 \u0026micro;M IAA or MeOH vehicle, and cells were further incubated for 8 h. Parasite egress was initiated by adding 3 \u0026micro;M calcium ionophore A23187 to infected HFFs for 2 min. Cells were fixed, permeabilized, and stained with α-TgGRA2 (1:1000) and α-TgIMC1 (1:2000). The number of egressed versus non-egressed vacuoles was calculated by counting 150 vacuoles in triplicate from three independent biological replicates. Vacuoles containing\u0026thinsp;\u0026gt;\u0026thinsp;2 parasites were considered intact.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing and transcriptome analyses\u003c/h2\u003e \u003cp\u003eTgCdc5-mAID-HA parasites (5 x 10\u003csup\u003e7\u003c/sup\u003e) were harvested from infected HFFs grown in DMEM containing 500 \u0026micro;M IAA or MeOH vehicle for 8 h. Total RNA was isolated using the RNeasy Plus mini kit (Qiagen) and quantified using a Qubit fluorometer (Thermofisher #Q33238). RIN values of the samples were determined using tape station 4150 and HS RNA screen tape. The cDNA libraries were prepared using a TruSeq Stranded Total RNA kit (Illumina #15032618, Illumina #20020596). Final libraries were quantified using a Qubit 4 Fluorometer (Thermofisher #Q33238), and the samples were subjected to sequencing of paired-end reads on the Illumina NovaSeq 6000 platform.\u003c/p\u003e \u003cp\u003eThe raw fastq reads of the sample were quality assessed using FastQC, and summarization was performed using MultiQC. The processed reads from the Illumina seq were mapped against the \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e reference genome (GCF_000006565.2) using STAR v2.7.9. The rRNA and tRNA features were removed from the GTF file of the \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e. The alignment file (BAM) from individual samples was quantified using feature Counts v. 2.0.1 to obtain transcript counts. These transcript counts were used as inputs to DESeq2 for differential expression estimation, keeping a threshold of statistical significance of \u0026lt;\u0026thinsp;0.05 p-value adjustment using the Benjamini and Hochberg method\u003csup\u003e40\u003c/sup\u003e. The resulting abundance counts were imported into R using the Bioconductor package \u0026lsquo;tximport\u0026rsquo;. The 'regularized log' transformation in DESeq2 was used for principal component and clustering analyses. An adjusted p-value (FDR) threshold\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05 and log2 fold change of ∓\u0026thinsp;2.0 were used for statistical estimation of gene expression. The k-Means clustering was done using iDEP.96, with the count file provided as an input (default parameters: most variable genes to include- 2000; number of clusters- 4; normalize by gene-mean center).\u003c/p\u003e \u003cp\u003eFor Gene Ontology (GO) annotation\u003csup\u003e41\u003c/sup\u003e, the protein sequences were extracted based on the DESeq2 result (Adjusted p-value\u0026thinsp;\u0026lt;\u0026thinsp;=\u0026thinsp;0.05 and Log Fold Change\u0026thinsp;∓\u0026thinsp;2) and were subjected to the ToxoDB database\u003csup\u003e37\u003c/sup\u003e to generate GO terms utilizing the integrated GO tool. A threshold of p-value 0.05 and q-value less than 0.25 were set to define statistically significant GO terms, which were further used to generate GO plots in GraphPad prism.\u003c/p\u003e \u003cp\u003eTwo types of splicing analyses were performed i) Exon usage analysis by aligning the reads against the reference genome \u0026ldquo;\u003cem\u003eToxoplasma\u003c/em\u003e_\u003cem\u003egondii\u003c/em\u003e_TGA4.fna\u0026rdquo; and indexed using STAR\u003csup\u003e42\u003c/sup\u003e v2.7.9a. The generated bam files were used to perform Differential exon usage analysis using the \u0026ldquo;DEXSeq\u0026rdquo; package in R and ii) Splicing event analysis\u0026rdquo; using ASpli (v.1.5.1), a Bioconductor computational suite using the default commands as previously described\u003csup\u003e43\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation and quantitative RT-PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated using the RNeasy Plus mini kit (Qiagen) followed by reverse transcriptase PCR using SuperScript III First-strand Synthesis System (Invitrogen) to produce cDNA following the manufacturer\u0026rsquo;s protocol. The qRT-PCR was performed on the 7500 ABI apparatus (Applied Biosystems) using cDNA, gene-specific forward and reverse primers with SYBR green PCR Master Mix (Applied Biosystems). The levels of unspliced and spliced transcripts of indicated genes were determined using primers targeting specific exon-intron and exon-exon junctions, respectively. The transcript level of the intron-less gene SRS40E is expected to be unaltered (based on RNA-seq data) and used as a normalizing control. Duplicate reactions were performed for each sample using the following cycle conditions: 95\u0026deg;C, 15 min followed by 40 cycles of 94\u0026deg;C, 30 s; 55\u0026deg;C, 40 s and 68\u0026deg;C, 50 s. Relative transcript levels were analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. The obtained transcript values were used to calculate splicing efficiency as the ratio of mRNA (spliced)/pre-mRNA (unspliced) and compared for vehicle and IAA-treated parasites.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eApoptosis assay\u003c/h2\u003e \u003cp\u003eParasite apoptosis was determined using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences # 556547). Briefly, filter-purified TgCdc5-mAID-HA parasites from the vehicle or IAA-treated at indicated time points were washed two times with cold PBS, centrifuged, and parasite pellets were resuspended in 100 \u0026micro;l 1\u0026times; Annexin V binding buffer. Subsequently, 5\u0026micro;l of PE-Annexin V was added to the parasite suspension, mixed gently, and incubated in the dark for 30 minutes at room temperature. Later, 400 \u0026micro;l of 1\u0026times; Annexin V binding buffer was added to each sample tube, and samples were analyzed immediately using a FACSCalibur flow cytometer (BD Biosciences). Flow cytometric data was analyzed using FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eTUNEL assay\u003c/h2\u003e \u003cp\u003eDNA fragmentation in the intracellular parasites was detected using the One-step TUNEL In Situ Apoptosis Kit (Elab Science #E-CK-A321). The assay uses the IFA-based terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) method. Briefly, HFF monolayer-infected TgCdc5-mAID-HA parasites were grown for 12 h. Later, the medium was replaced with DMEM containing 500 \u0026micro;M IAA or MeOH vehicle, and cells were further incubated for 8 h. Cells were fixed, permeabilized, equilibrated with TdT buffer at 37\u0026deg;C for 30 min, stained with labeling solution containing TdT enzyme at 37\u0026deg;C for 60 min. For the positive control, cells were treated with DNaseI enzyme prior to the TdT equilibration step. For each experiment, 100 parasites were counted from three independent biological replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003ePuromycin incorporation assay\u003c/h2\u003e \u003cp\u003eThe parasite's nascent RNA synthesis levels were determined using puromycin labeling\u003csup\u003e44\u003c/sup\u003e. Parasites were grown on HFF monolayers in the presence of a vehicle or IAA for the indicated time points. Prior to paraformaldehyde fixation, cells were treated with 10 \u0026micro;g/ml puromycin for 30 min and then subjected to IFA. The complete block of protein synthesis in the control was achieved by treating the infected cells with 100 mg/ml cycloheximide (CHX) for 2 h before adding puromycin and followed by standard IFA. An anti-puromycin antibody was used to stain the newly synthesized proteins in the parasites and HA to visualize the parasite TgCdc5 protein.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eProtein aggregation assay\u003c/h2\u003e \u003cp\u003e Parasite protein aggregates were determined using the PROTEOSTAT Aggresome detection kit (Enzo Life Sciences #ENZ-51035-0025) according to the manufacturer\u0026rsquo;s instructions. Parasites were grown on HFF monolayers in the presence of a vehicle or IAA, which was fixed and permeabilized. Proteostat reagent was used to stain the protein aggregates and the HA to visualize the TgCdc5 protein.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eMouse infection\u003c/h2\u003e \u003cp\u003eThe auxin-inducible TgCdc5-mAID-HA protein degradation in mice was performed as described previously\u003csup\u003e45,46\u003c/sup\u003e with some modifications. A 30-day survival experiment was performed using six-week BALB/c male mice with three groups 10 mice/group. Mice from two groups were injected intraperitoneally (i.p.) with 50 tachyzoites of RH TgCdc5-mAID-HA. On the second day of post-infection (pi), 1 of 2 groups was given IAA, and the other group was administered an equivalent volume of MeOH vehicle in the drinking water containing 5% sucrose. Third group was no infection control with IAA. The IAA was administered for 14 days (2 days pi to 15 days pi) in two ways: i) in drinking water (0.5 mg/ml) and ii) by oral gavage (12.5 mg/mL). All mice were weighed and monitored daily. To test the TgCdc5 protein depletion, on day 6 pi, 2 mice each were sacrificed by CO\u003csub\u003e2\u003c/sub\u003e asphyxiation, and the peritoneal exudate cells (PECs) were collected and examined by IF staining using α-HA and α-TgIMC1 antibodies. All survived mice were sacrificed on day 30 and brain samples were processed to examine bradyzoite cyst. The relative weight loss of the mouse was calculated based on the initial body weight on the day of infection.\u003c/p\u003e \u003cp\u003e \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e challenge experiment - A 60-day survival experiment was performed using six-week BALB/c mice - two IAA groups of 8 male and female mice and two without IAA groups (-IAA) of 3 male and female mice. Mice were injected with 5x10\u003csup\u003e3\u003c/sup\u003e TgCdc5-mAID-HA tachyzoites i.p. and treated with IAA or vehicle from day 2 to 15. On day 16, 2 mice from each group (+\u0026thinsp;IAA) were sacrificed by CO\u003csub\u003e2\u003c/sub\u003e asphyxiation, and heart and brain tissues were collected. Tissue samples were processed to detect bradyzoite cysts by microscopy. Genomic DNA was isolated by using a tissue DNA extraction kit (QIAmp, Qiagen), and qRT-PCR was performed for the \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e 529 repeat region. On day 21, serum from all mice was collected, and IgG-IFA was performed for tachyzoite and bradyzoite stages, as mentioned previously. From day 22\u0026ndash;25, male and female mice mating was carried out. Further, male and female mice were segregated into 2 groups each (3 mice male or female/group). On day 27, 3 mice from 1 group each were injected with 5x10\u003csup\u003e3\u003c/sup\u003e TgCdc5-mAID-HA tachyzoites i.p, and mice were observed for another 33 days. Female mice in the control group delivered 12 pups on 21 days post-mating, and only 1 of 3 female mice injected with tachyzoites had a normal pregnancy with 2 pups. On day 60, 1 female and one male mouse were sacrificed by CO\u003csub\u003e2\u003c/sub\u003e asphyxiation. Sera and heart and brain tissues were collected to detect anti-\u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e antibodies, bradyzoite cyst, and \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e DNA.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eData analyses\u003c/h2\u003e \u003cp\u003eAll data analyses, including graph preparation and statistics, were performed using GraphPad Prism 9.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of intron-containing genes and spliceosomal proteins in apicomplexans\u003c/h2\u003e \u003cp\u003eAlveolates are reported to have high number of introns; however, the genes with intron numbers have not been analyzed in Apicomplexa. Using VEuPathDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://veupathdb.org/veupathdb/app/\u003c/span\u003e\u003cspan address=\"https://veupathdb.org/veupathdb/app/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e47\u003c/sup\u003e, we determined the percentage of the genes with introns in the six apicomplexans (\u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e, \u003cem\u003eNeospora caninum\u003c/em\u003e, \u003cem\u003eTheileria annulata\u003c/em\u003e, \u003cem\u003eBabesia microti\u003c/em\u003e, \u003cem\u003ePlasmodium falciparum\u003c/em\u003e, and \u003cem\u003eCryptosporidium parvum\u003c/em\u003e) and compared to the relatively low intron content of \u003cem\u003eS. cerevisiae\u003c/em\u003e. This genome-wide comparison of introns revealed moderate to high intron densities (1\u0026ndash;5 introns/gene) in apicomplexans, except \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eparvum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Tables\u0026nbsp;2 and 3). Compared to other apicomplexans, \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e and \u003cem\u003eN\u003c/em\u003e. \u003cem\u003ecaninum\u003c/em\u003e have a wide range of exon distribution, with ~\u0026thinsp;25% of genes being intronless and ~\u0026thinsp;5% containing\u0026thinsp;\u0026gt;\u0026thinsp;16 exons/gene. \u003cem\u003eT\u003c/em\u003e. \u003cem\u003eannulata\u003c/em\u003e and \u003cem\u003eB\u003c/em\u003e. \u003cem\u003emicroti\u003c/em\u003e show similar exon distribution with ~\u0026thinsp;29% of genes without intron and ~\u0026thinsp;0.7% of genes containing\u0026thinsp;\u0026gt;\u0026thinsp;16 exons/gene. While \u003cem\u003eP\u003c/em\u003e. \u003cem\u003efalciparum\u003c/em\u003e contains a relatively similar percentage of genes with no introns (~\u0026thinsp;46%) and 2\u0026ndash;5 introns (~\u0026thinsp;44%), the exon distribution of \u003cem\u003eC\u003c/em\u003e. \u003cem\u003eparvum\u003c/em\u003e is comparable to \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Tables\u0026nbsp;2 and 3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 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 searches\u003csup\u003e48\u003c/sup\u003e of six apicomplexan genomes using amino acid sequences of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e and \u003cem\u003eH\u003c/em\u003e. \u003cem\u003esapiens\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). This analysis revealed that nearly all primary splicing factors are present in apicomplexan, similar to model organisms.\u003c/p\u003e \u003cp\u003e \u003cb\u003eT\u003c/b\u003e. \u003cb\u003egondii\u003c/b\u003e \u003cb\u003eCdc5 is a conserved splicing factor of large spliceosomal complex\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe identified \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e Cdc5 (TgME49_275480), a 888-aa protein contains conserved nucleic acid binding Myb-domain and SANT domain in the N-terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). To gain insight into the expression, and localization of TgCdc5, full-length TgCdc5-His protein of ~\u0026thinsp;100 kDa was purified (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and used to generate specific anti-TgCdc5 antibodies. TgCdc5 is robustly expressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) in both the asexual stages and localized in the nucleus of the tachyzoite and the perinuclear to cytoplasm in the bradyzoite stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). 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 \u003cem\u003eToxoplasma\u003c/em\u003e, we immunoprecipitated TgCdc5 protein from freshly lysed tachyzoites and identified co-purifying proteins by mass spectrometry (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). With the cut-off of two unique peptides, 146 proteins (Supplementary Table\u0026nbsp;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 BLASTP\u003csup\u003e48\u003c/sup\u003e and HHpred\u003csup\u003e49\u003c/sup\u003e to identify and name these proteins following the human/yeast nomenclature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Based on the coverage and peptide score, TgPrp19 (TgME49_320210), a conserved splicing factor containing Ubox and WD40 repeat (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF), was the major protein co-purified with TgCdc5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Before confirming their interaction, we first bacterially expressed full-length TgPrp19-His\u003csub\u003e6\u003c/sub\u003e protein (~\u0026thinsp;56 kDa) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG) and generated anti-TgPrp19 polyclonal antibodies. Similar to TgCdc5, TgPrp19 is also highly expressed in both the asexual stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH) and localized predominantly in the nucleus of the tachyzoite and bradyzoite stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI), suggesting an interaction of these proteins. Further, the reciprocal pull-down experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ,K) and co-localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eL) studies confirmed the strong interaction between TgCdc5 and TgPrp19.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCdc5 family members associate with the spliceosome throughout the entire splicing reaction\u003csup\u003e50\u003c/sup\u003e. Given that TgCdc5 contains nucleic acid binding domain\u003csup\u003e51\u003c/sup\u003e 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 (K\u003csub\u003eD\u003c/sub\u003e=440 nM) and U6 (K\u003csub\u003eD\u003c/sub\u003e=481 nM) compared to U5 (K\u003csub\u003eD\u003c/sub\u003e=1 \u0026micro;M), U1 (K\u003csub\u003eD\u003c/sub\u003e=1.5 \u0026micro;M), and U4 (K\u003csub\u003eD\u003c/sub\u003e=3.7 \u0026micro;M) snRNAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eM). These results confirm the specificity of TgCdc5 with U2 and U6 snRNAs.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, 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 \u003cem\u003eTgCdc5\u003c/em\u003e, and \u003cem\u003eTgPrp19\u003c/em\u003e genes into a yeast 2\u0026micro; \u003cem\u003eTRP1\u003c/em\u003e pYES3 plasmid, and found that 2\u0026micro; \u003cem\u003eTgCdc5\u003c/em\u003e and \u003cem\u003eTgPrp19\u003c/em\u003e supported the growth of \u003cem\u003eΔcef1\u003c/em\u003e, and \u003cem\u003eΔprp19\u003c/em\u003e cells, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eN,O). These results demonstrate that \u003cem\u003eToxoplasma\u003c/em\u003e encodes the biologically active Cdc5 protein, the core spliceosomal factor.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eTgCdc5 is an essential pre-mRNA splicing factor\u003c/h2\u003e \u003cp\u003eTo 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) with the addition of IAA. The resulting TgCdc5-mAID-HA strain was confirmed by diagnostic PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) and western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Western blot (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and IF (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE) staining using an anti-HA antibody revealed that TgCdc5-mAID-HA protein was completely depleted in \u0026lt;\u0026thinsp;1 h after adding IAA in the culture medium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine how TgCdc5 affects gene splicing, we generated a reporter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eG) in the TgCdc5 depleted parasites consistent with reduced mRNA level of Rluc (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). 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 \u0026lt;\u0026thinsp;15% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ), suggesting a specific effect of TgCdc5 on pre-mRNA splicing.\u003c/p\u003e \u003cp\u003eNext, we analyzed the effect of TgCdc5 depletion on parasite-specific processes. The Cdc5-depleted parasites showed the complete arrest of parasite replication (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eK) and severe morphological defects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eL). A parasite growth assay revealed that TgCdc5-depleted parasites produced no visible plaques (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). To determine whether parasites can recuperate from a transient loss of TgCdc5, we performed a plaque assay using six different IAA treatment conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eN). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eN), no plaques were observed when treated for 2/4/8/12/24 h IAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eN). Furthermore, TgCdc5 depletion significantly reduced the parasite's invasion efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eO); however, the parasites' ability to egress upon inducing egress using calcium ionophore showed a marginal difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eP). Together, these data show that TgCdc5 is essential for \u003cem\u003eToxoplasma\u003c/em\u003e proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eTgCdc5 loss perturbs global gene expression through dysregulation of RNA splicing\u003c/h2\u003e \u003cp\u003eTo assess the global impact of the severe phenotypic defect obtained after TgCdc5 loss, we performed RNA-seq on TgCdc5 (+\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Supplementary Table\u0026nbsp;5). Of 1125 DEGs identified, 394 were upregulated, and 731 were downregulated in TgCdc5 depleted parasites (IAA-treated) compared with vehicle-treated parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The k-means clustering of the differentially expressed genes (showed four clusters (A, B, C, and D) with similar expression profiles (n\u0026thinsp;=\u0026thinsp;2) in IAA-treated and vehicle-treated parasites (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The genes that showed significantly different gene expressions (up or down) relevant to the study are shown in the volcano plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The host cell invasion genes RON2, RON3, RON4, and RON5 were found to be downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), which was consistent with compromised invasion for TgCdc5-depleted parasites. Other downregulated genes were PAN domain-containing genes involved in protein ubiquitination/proteolysis\u0026rsquo;, PFK domain domain-containing genes responsible for a decreased rate of protein synthesis, and Golgi enzyme CD39 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Surprisingly, we found a few bradyzoites inducing Apetala\u0026thinsp;\u0026minus;\u0026thinsp;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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Genes with more intron count were more affected, conceivably due to reduced splicing efficiency.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eImpaired RNA splicing may directly reduce mature mRNA; therefore, based on RNA-seq data, we selected 9 downregulated candidate genes (\u003cem\u003eTub1\u003c/em\u003e, \u003cem\u003eRbp1\u003c/em\u003e, \u003cem\u003eRps3\u003c/em\u003e, \u003cem\u003eMCM4\u003c/em\u003e, \u003cem\u003eNhe2\u003c/em\u003e, \u003cem\u003eSec62\u003c/em\u003e, \u003cem\u003eTDCP\u003c/em\u003e, \u003cem\u003eRab7\u003c/em\u003e, and \u003cem\u003eAMA1\u003c/em\u003e) and performed qRT-PCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). 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, \u003cem\u003eSrs40E\u003c/em\u003e remained unchanged by TgCdc5 knockdown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). 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\u0026thinsp;~\u0026thinsp;1.5-to-2-fold decrease in the splicing efficiency of the \u003cem\u003eTub1, Rbp1, Rps3, MCM4, Nhe2, Sec62, TDCP, Rab7, and AMA1\u003c/em\u003e downregulated genes tested (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eTo understand the processes in which the differentially expressed genes might be involved, we conducted gene ontology (GO) enrichment analyses (Supplementary Table\u0026nbsp;5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). 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.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTgCdc5 modulates alternative splicing of genes\u003c/h3\u003e\n\u003cp\u003eIn 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\u0026thinsp;=\u0026thinsp;20987) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Supplementary Table\u0026nbsp;6) corresponding to 3604 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) for TgCdc5-depleted parasites compared to wild-type parasites. The AS events were detected in 54.8% (n\u0026thinsp;=\u0026thinsp;3604) of exon-containing transcripts (n\u0026thinsp;=\u0026thinsp;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\u0026rsquo; splice site (Alt5\u0026rsquo;SS), alternative 3\u0026rsquo; splice site (Alt3\u0026rsquo;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\u0026rsquo;SS/Alt3\u0026rsquo;SS (0.02% events; 0.09%), and other unclassified events (17.09% events; 32.4% genes). Figures\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-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\u0026thinsp;=\u0026thinsp;1125). The AS events were detected in 29.66% of DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), whereas 70.34% of DEGs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) did not have AS events (AS independent).\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eTgCdc5 depletion leads to the cell cycle arrest and DNA damage in the parasites\u003c/h2\u003e \u003cp\u003eGO 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)\u003csup\u003e52\u003c/sup\u003e and MORN1 (centrocone - mitosis marker)\u003csup\u003e53\u003c/sup\u003e. Centrosome duplication marked the S phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,B), whereas centrocone duplication indicated the M phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA,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 (~\u0026thinsp;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 (~\u0026thinsp;80%) of TgCdc5-depleted parasites (+\u0026thinsp;IAA) contained a single centrosome and did not progress to S phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC,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 (~\u0026thinsp;80%) of TgCdc5-depleted parasites (+\u0026thinsp;IAA) contained single centrocone (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE,F). Together, these results suggest that TgCdc5 is essential for all cell cycle stages, not only for mitosis, as reported for human Cdc5.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDNA fragmentation, a hallmark of DNA damage was tested using a TUNEL assay. Direct labeling of DNA breaks confirmed a significant increase (~\u0026thinsp;80) in TgCdc5-depleted parasites (+\u0026thinsp;IAA) compared to vehicle-treated parasites (~\u0026thinsp;10%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG,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 (~\u0026thinsp;32%) was observed after 12 h of TgCdc5 depletion (+\u0026thinsp;IAA) compared to vehicle treatment (~\u0026thinsp;12%), suggesting apoptosis-like cell death in \u003cem\u003eToxoplasma\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eDepletion of TgCdc5 generates protein aggregates that triggers bradyzoite induction program\u003c/h2\u003e \u003cp\u003eIn metazoans, NMD, an mRNA quality control process, removes erroneous transcripts\u003csup\u003e18\u003c/sup\u003e. However, recently, in \u003cem\u003eP\u003c/em\u003e. \u003cem\u003efalciparum\u003c/em\u003e, disruption of core NMD proteins showed no degradation of nonsense transcripts\u003csup\u003e20\u003c/sup\u003e, 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB,C) in a significant number of parasites (~\u0026thinsp;22%), suggesting that translation of erroneous transcript generates non-functional misfolded protein aggresomes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCellular and environmental stresses are known to develop latent bradyzoites from rapidly growing tachyzoites in \u003cem\u003eToxoplasma\u003c/em\u003e\u003csup\u003e4\u003c/sup\u003e. 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 (+\u0026thinsp;IAA), confirmed by irregular morphology of the parasites and loss of parasites in the vacuoles, and only a few (~\u0026thinsp;20%) vacuoles showed standard morphology of the parasites with evidence of bradyzoite formation as determined by BAG1 staining upon IAA-induced TgCdc5 depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Importantly, those\u0026thinsp;~\u0026thinsp;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\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026ge;\u003c/span\u003e\u0026thinsp;6), suggesting these vacuoles containing bradyzoites were transitioning to cyst or will remain immature cysts (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, 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 CO\u003csub\u003e2\u003c/sub\u003e depletion) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). 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.\u003c/p\u003e \u003cp\u003eAfter 6 days of IAA-induced TgCdc5 depletion, these partially DBA-positive vacuoles showed loosely packed and misshaped bradyzoites (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). 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.\u003c/p\u003e \u003cp\u003eFurthermore, 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 \u0026minus;\u0026thinsp;636) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH). A comparative analysis of vehicle and IAA-treated parasites showed that more proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH) had reduced (n\u0026thinsp;=\u0026thinsp;424) than elevated expression (n\u0026thinsp;=\u0026thinsp;212). Downregulated genes contain more introns than upregulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI), consistent with RNA-seq data. Of 636 differentially expressed proteins (Supplementary Table\u0026nbsp;7), 75 (~\u0026thinsp;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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). TgCdc5-mediated RNA splicing deregulation affects \u003cem\u003eToxoplasma\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). As expected for lowered invasion, rhoptry proteins and kinases showed a significant reduction in the expression, which lowers parasite survival in the host (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ). 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.\u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eKnockdown of TgCdc5 protects mice from lethal toxoplasmosis as well as induces protective immunity\u003c/h2\u003e \u003cp\u003eTgCdc5 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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) 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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB), confirming effective in vivo depletion of TgCdc5 protein\u0026thinsp;\u0026lt;\u0026thinsp;6 days. By day 11 post-infection, all mice receiving the vehicle control treatment died from lethal toxoplasmosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC), whereas IAA treatment rescued all mice from fatal infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The vehicle treatment group showed severe morbidity and complete mortality compared to the IAA treatment group (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). 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 \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e replication since stopping IAA treatment following day 15 did not result in morbidity (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), mortality (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). We did not observe \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e 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 (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;50), potentially leading to effective elimination by the host's immune cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo address this, we performed a similar experiment involving two groups of 8 male and female mice injected with 5x10\u003csup\u003e3\u003c/sup\u003e TgCdc5-mAID-HA tachyzoites (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). After 15 days of IAA treatment, we found no mortality in mice and also could not detect bradyzoite cysts (not shown) and \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e 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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE) but not against bradyzoite antigens (not shown), indicating that anti-\u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e antibodies were generated in response to the high dose of parasites. From day 22\u0026ndash;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 5x10\u003csup\u003e3\u003c/sup\u003e 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 ~\u0026thinsp;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.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Until day 60, we found no mortality in mice and could not detect bradyzoite cysts and \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e DNA in the sacrificed mouse; however, the sera of all mice were immunoreactive for tachyzoite (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePre-mRNA splicing is a crucial step in eukaryotic gene expression. This study focuses on the role of the essential pre-mRNA splicing factor Cdc5 in maintaining transcriptional homeostasis of the intron-rich genome of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e. Despite high intron densities, the splicing mechanism in \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e appears to be largely conserved. Like model eukaryotes, TgCdc5 is part of a large spliceosomal complex involving both proteins and RNA. TgCdc5 is an essential splicing factor, and its depletion generates erroneous splicing, leading to significant alternative splicing events with widespread defects in gene expression for various parasite processes, including DNA replication, cell cycle, DNA damage, invasion, egress, protein degradation, and bradyzoite differentiation in non-cystogenic RH strain. Notably, TgCdc5 is essential for parasite survival in mice, as depleting TgCdc5 provides full protection against a lethal dose of tachyzoites. Interestingly, the immune response generated during this first exposure offers complete protection against future Toxoplasma infections and partially protects vertical transmission.\u003c/p\u003e \u003cp\u003eThe genes of \u003cem\u003eToxoplasma\u003c/em\u003e, \u003cem\u003ePlasmodium\u003c/em\u003e, and \u003cem\u003eTheileria\u003c/em\u003e show remarkable conservation of intron positions, suggesting that shared introns are predominantly ancestral and play an essential role in gene expression\u003csup\u003e9\u003c/sup\u003e. \u003cem\u003ePlasmodium\u003c/em\u003e and \u003cem\u003eTheileria\u003c/em\u003e ancestors experienced significant intron loss, while \u003cem\u003eToxoplasma\u003c/em\u003e showed no intron loss or gain\u003csup\u003e9\u003c/sup\u003e. Given the high energetic burden of intron-splicing, it's intriguing to consider what advantage apicomplexans gain from having genomes rich in introns. Introns play a crucial role in regulating gene expression by influencing mRNA export, stability, and translation efficiency\u003csup\u003e12,54\u0026ndash;56\u003c/sup\u003e. Additionally, introns can affect transcriptional output by regulating the promoter-proximal chromatin profiles\u003csup\u003e57,58\u003c/sup\u003e and RNA Pol II occupancy\u003csup\u003e59\u003c/sup\u003e. Some introns also contain non-coding RNAs, whose processing from introns can speed up or slow down the rate of gene expression\u003csup\u003e60\u0026ndash;62\u003c/sup\u003e. Consequently, it is tempting to speculate that the complex gene architecture in apicomplexans, particularly \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e, allows for more intricate RNA processing and gene regulation, possibly through alternative splicing or non-coding RNAs. This molecular adaptation of intron selection in evolution may hold the key to \u003cem\u003eToxoplasma\u003c/em\u003e's success in its complex life cycle and ability to thrive in diverse host species.\u003c/p\u003e \u003cp\u003eThe immunoaffinity purification of TgCdc5 has enabled the identification of ~\u0026thinsp;80 spliceosomal proteins, including eight core proteins. Based on the coverage and peptide score, TgPrp19, TgSpf27, and TgPrl1 were identified as the major core proteins co-purified with TgCdc5. Particularly, the presence of Prp19, Cdc5, Spf27, and Prl1 is consistent across humans, yeast, \u003cem\u003eTrypanosoma\u003c/em\u003e parasite, and \u003cem\u003eToxoplasma\u003c/em\u003e complexes, indicating that these four proteins form the conserved core of the complex\u003csup\u003e63\u0026ndash;65\u003c/sup\u003e. Given the diverse phylogenetic lineages of \u003cem\u003eToxoplasma\u003c/em\u003e, \u003cem\u003eTrypanosoma\u003c/em\u003e, and model organisms from yeast to humans (Alveolata, Excavata, and Opishokonta, respectively), it is evident that the spliceosome's composition and mechanism are highly conserved over evolutionary time. Besides, \u003cem\u003eToxoplasma\u003c/em\u003e Cdc5 has retained a conserved role in the catalytic phase of splicing, as evidenced by its strong interaction with U2, U6, and U5 snRNAs.\u003c/p\u003e \u003cp\u003eA loss-of-function mutation in the \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e Cef1 (Cdc5 homolog) leads to slower growth, thermosensitivity, and cell cycle arrest in the G2/M phase\u003csup\u003e21\u003c/sup\u003e. Similarly, knockdown of human Cdc5L causes mitotic arrest, chromosome misalignments, and DNA damage, ultimately resulting in mitotic catastrophe\u003csup\u003e27\u003c/sup\u003e. These cell cycle arrests in yeast\u003csup\u003e66\u003c/sup\u003e and humans\u003csup\u003e27\u003c/sup\u003e are associated with defective splicing in cytoskeleton genes crucial for microtubule stability during mitosis. Likewise, TgCdc5 regulates the expression (RNA and protein) and splicing efficiency of cell cycle-specific ApiAP2 factors and a set of genes involved in maintaining the cytoskeleton; however, unlike yeast and humans, its depletion causes stage-independent cell cycle arrest. In addition to parasite replication arrest, depletion of TgCdc5 causes reduced parasite invasion, egress, ER-Golgi vesicular transport, translation fidelity, chaperone-mediated protein folding, ubiquitin-mediated protein degradation, redox balance, and induces severe DNA damage, ultimately leading to death, similar to apoptosis. The impact of TgCdc5 depletion on myriad parasite processes highlights the necessity of pre-mRNA splicing of genes involved in these processes, as every two out of three genes require splicing in Toxoplasma owing to the intron-rich genome.\u003c/p\u003e \u003cp\u003eErroneous splicing often generates transcripts containing PTC, which are usually targeted for degradation by NMD\u003csup\u003e18,19\u003c/sup\u003e. However, a recent study in \u003cem\u003eP\u003c/em\u003e. \u003cem\u003efalciparum\u003c/em\u003e demonstrated that NMD is not essential and does not target nonsense transcripts\u003csup\u003e20\u003c/sup\u003e, suggesting a potentially novel degradation mechanism. In \u003cem\u003eToxoplasma\u003c/em\u003e, mis-spliced transcripts generated by TgCdc5 depletion not only contribute to DEGs, but also undergo translation, resulting in the production of non-functional, misfolded proteins. These misfolded protein aggregates exert stress on the parasite, prompting a transition from the highly replicating tachyzoite to the latent bradyzoite stage, evidenced by the increased expression of transcripts and proteins needed for bradyzoite development. The spontaneous conversion from tachyzoite to bradyzoites in RH after the loss of TgCdc5 was unexpected, given that RH strain parasites are widely considered non-cystogenic. However, these bradyzoites were unable to develop into mature cysts over time or reactivate to tachyzoites once IAA was removed, indicating that under the enormous stress of splicing error, the parasite's attempt to survive by converting into dormant forms is ultimately unsuccessful, possibly due to the lack of functional proteins necessary for bradyzoite development.\u003c/p\u003e \u003cp\u003eThe depletion of TgCdc5 results in the complete protection of mice against a lethal dose of infective tachyzoites, highlighting its indispensable role in parasite survival within the host. While immunity acquired upon initial parasite exposure confers protection against subsequent lethal infections in mice, it does not provide full protection for female mice during pregnancy. However, this raises the question of how TgCdc5-depleted parasites generate a protective immune response. There are several potential explanations. Firstly, the high number of parasites in the initial exposure may be sufficient to generate protective immunity before they are cleared by immune cells. Secondly, the initial immune response may have heightened after the parasite challenge, offering protective immunity. Thirdly, exposure to a high number of tachyzoites and short-sustained infection due to tachyzoite to bradyzoite conversion may have stimulated a reasonable immune response, which could have increased after the parasite challenge, providing protective immunity. The fact that the immune response generated by the TgCdc5-depletion strategy conferred partial protection in pregnant mice suggests the need for additional parasite boosters and subsequent depletion of TgCdc5 by IAA before challenging. Nonetheless, these observations underscore the host-protective role of the TgCdc5-depletion strategy against lethal toxoplasmosis.\u003c/p\u003e \u003cp\u003eGiven the high global burden of disease and the lack of effective drugs, developing vaccines against \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e infection in humans is a high priority. The live-attenuated vaccine is considered good and can offer better protection than other types of vaccines\u003csup\u003e67\u0026ndash;69\u003c/sup\u003e. A \u003cem\u003eToxoplasma\u003c/em\u003e strain with low virulence and no ability to form a bradyzoite cyst will be the right candidate\u003csup\u003e67\u0026ndash;70\u003c/sup\u003e. Studies in rodent models showed that only a few live attenuated \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e strains used for immunization conferred protective immunity and significantly reduced the tissue cyst burden after the challenge\u003csup\u003e71\u0026ndash;75\u003c/sup\u003e. Considering this, it is tempting to suggest that the IAA-mediated TgCdc5-depletion approach in the RH strain may be a good vaccine strategy for several reasons: 1. It induces protective immunity upon being challenged with more than a lethal dose of \u003cem\u003eT\u003c/em\u003e. \u003cem\u003egondii\u003c/em\u003e and provides complete protection to mouse (natural host); 2. This parasite strain does not form latent bradyzoite, an immune protective stage, and is also refractory to current therapeutics; 3. It partially protects maternal-fetal transmission; however, additional parasite boosting followed by IAA treatment may improve the outcome of vertical transmission; 4. This approach may also protect against other highly prevalent low-virulent strains (type II and III).\u003c/p\u003e \u003cp\u003eOverall, this study establishes the indispensable role of the splicing factor Cdc5 in preserving transcriptional homeostasis, which is essential for the survival of intron-rich \u003cem\u003eToxoplasma\u003c/em\u003e parasites. Erroneous splicing resulting from TgCdc5 loss significantly affects key parasite functions and yet triggers a protective immune response in the mouse host.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe institutional ethics committee of National Institute of Animal Biotechnology has approved using laboratory research protocols (IBSC/Feb2023/NIAB/AD001) and animals (IAEC/NIAB/2024/09/ASD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial or personal interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePoonam Kashyap: Data curation, investigation, analysis and writing manuscript. Kalyani Aswale: Data curation and analysis. Abhijit S. Deshmukh: Conceptualization, funding acquisition, investigation and writing manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by an NIAB core grant (C022) for ASD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-sequencing data of Wild-type and knockdown (TgCdc5) Toxoplasma gondii parasites have been submitted to NCBI, BioProject: PRJNA1133933. Differential splicing analysis data of wild-type and mutant parasites have been submitted to NCBI, BioProject: PRJNA1126029. Proteomic data has been submitted to MassIVE - Data ID_MassIVE - MSV000095119. The illustration in this study was created using BioRender (https://biorender.com/). The data that support the findings of this study are available on request from the corresponding author. The following reagents were obtained through the NIH Biodefense and Emerging Infections Research Sources Repository, NIAID, NIH: Toxoplasma gondii, RH-88, NR-223; Toxoplasma gondii, ME49 (B7 Clone), NR-20729; Toxoplasma gondii, RH TIR1-3FLAG, NR-51145.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is funded by the NIAB core grant (C0022) to ASD. We thank Prof. Kathleen L. Gould, Vanderbilt University School of Medicine, USA, for the yeast mutant strains. Prof. Marc-Jan Gubbels, Boston College, Chestnut Hill, MA, USA for TgMorn1 plasmid. PK and KA acknowledge UGC and DBT, respectively, for graduate studies fellowships provided by the Government of India.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Adl SM, \u003cem\u003eet al.\u003c/em\u003e Diversity, nomenclature, and taxonomy of protists. \u003cem\u003eSyst Biol\u003c/em\u003e \u003cb\u003e56\u003c/b\u003e, 684\u0026ndash;689 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. \u003cem\u003eInt J Parasitol\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 1217\u0026ndash;1258 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Dubey JP, Lindsay DS, Speer CA. 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Here, we demonstrate the role of pre-mRNA splicing factor Cdc5 in maintaining transcriptome integrity by harmonizing interaction with spliceosomal proteins and snRNAs in \u003cem\u003eToxoplasma\u003c/em\u003e. TgCdc5 is an essential splicing factor, and its depletion generates significant alternative splicing with widespread changes in gene expression demonstrated by RNA-seq and proteomic studies. Loss of TgCdc5 leads to catastrophic effects on the parasites, concomitantly triggering a switch from rapidly replicating tachyzoite to dormant bradyzoite cysts in many parasites, likely due to the formation of misfolded protein aggregates caused by the translation of erroneous transcripts. However, these dormant state parasites could not survive due to lacking functional proteins for bradyzoite development. Remarkably, the knockdown of TgCdc5 in vivo protects mice from lethal infection, and the immune response generated during initial parasite exposure completely protects these mice from future infection and offers partial protection in vertical transmission. Overall, this study unveils a novel role of TgCdc5-mediated pre-mRNA splicing in governing \u003cem\u003eToxoplasma\u003c/em\u003e stage conversion, providing new insights into developmental stage gene regulation.\u003c/p\u003e","manuscriptTitle":"Depletion of splicing factor Cdc5 in Toxoplasma disrupts transcriptome integrity, induces stress-driven abortive bradyzoite formation, and triggers host protective immunity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-12 03:01:55","doi":"10.21203/rs.3.rs-4811664/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b87fec9f-4fa9-4949-a2cb-1670b0ef9202","owner":[],"postedDate":"August 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":35781557,"name":"Biological sciences/Microbiology/Parasitology/Parasite biology"},{"id":35781558,"name":"Biological sciences/Molecular biology/RNA metabolism/RNA splicing"}],"tags":[],"updatedAt":"2025-04-23T07:07:50+00:00","versionOfRecord":{"articleIdentity":"rs-4811664","link":"https://doi.org/10.1038/s41467-025-58805-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2025-04-22 04:00:00","publishedOnDateReadable":"April 22nd, 2025"},"versionCreatedAt":"2024-08-12 03:01:55","video":"","vorDoi":"10.1038/s41467-025-58805-3","vorDoiUrl":"https://doi.org/10.1038/s41467-025-58805-3","workflowStages":[]},"version":"v1","identity":"rs-4811664","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4811664","identity":"rs-4811664","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}