17th International Congress on Toxoplasmosis

An ISWI-related chromatin remodeler tunes MORC DNA binding and insulates gene expression in a densely packed genome

Genomics

ATP-dependent nucleosome remodeling complexes, also known as chromatin remodelers, are specialized multiprotein machines involved in genome organization and accessibility. They alter the structure, composition and positioning of nucleosomes by histone displacement, rejection or incorporation of histone variants. These actions control the accessibility of regulatory regions such as promoters and enhancers for transcription factors, RNA and DNA polymerases as well as coactivators or repressors. Phylogenetic studies have shown that Toxoplasma has evolved two divergent ATP-dependent remodeler proteins of the ISWI family, TgSNF2h and TgSNF2L. These proteins differ not only in their internal structure, but also in their partnerships. They associate with different AP2 transcription factors and unique scaffold proteins, each characterized by specific folded domains. ChIP-seq data show significant colocalization of TgSNF2h and MORC throughout the genome. Depletion of TgSNF2h leads to a marked decrease in the binding of MORC to chromatin, but does not affect its binding to telomeres. When TgSNF2h is released from chromatin at highly active genes (approximately 572 genes), there is a corresponding decrease in chromatin accessibility near the transcription start site (TSS), as shown by ATAC-seq, which in turn triggers a marked drop in mRNA levels of these genes, many of which are essential for the tachyzoite lytic cycle (e.g., AMA1, MIC1, SAG1, and GRA15). In TgSNF2h-depleted zoites, release of MORC from chromatin leads to activation of the 1,017 genes associated with the sexual stages regulated by MORC. In this context, TgSNF2h functions as a chromatin remodeler that insulates neighboring genes by potentially blocking the influences of transcriptional activators and repressors. At highly transcribed loci, TgSNF2h and MORC appear to act as barriers to demarcate active chromatin regions typified by unique levels of acetylation or to polarize promoter regions. This action likely sets the stage for the establishment and maintenance of specific gene expression patterns. Together, TgSNF2h and MORC serve as a safeguard against the unintended spreading of transcriptionally active euchromatin from a gene active in tachyzoites to a neighboring developmental gene that is silenced. Thus, TgSNF2h, having an epistatic effect over MORC, plays a critical role in the partitioning of developmental genes in Toxoplasma.

Oral presentation

Forward genetic screen identifies SRS15 and IFT88 as critical determinants controlling Toxoplasma oocyst shedding in cats

Transmission of Toxoplasma gondii in nature is highly dependent on the parasite&quot;s sexual cycle, which occurs exclusively in felids. Infected cats shed high numbers of infectious oocysts, which are capable of causing toxoplasmosis outbreaks in both animals and people. The molecular pathways promoting sexual stage development in T. gondii are largely unknown. To identify essential genes that regulate oocyst formation we undertook a forward genetic signature-tag mutagenesis screen, using CRISPR/Cas9 to generate a library of T. gondii strains that each possess a unique barcode and are each deficient in a single gene predicted to be highly expressed in the parasite&quot;s sexual stages. Specifically, we used previous RNA-Seq datasets to identify 192 sexual stage-specific transcripts to test for their role promoting sexual competency. These included surface antigen genes related to 6-CYS proteins in Plasmodium, genes required to form flagella, a family of transcription factors collectively referred to as ApiAP2s, and a large family of secreted proteins expressed exclusively in merozoites (Families A-D). To identify which genes impacted oocyst formation, we pooled the 192 T. gondii knock-out (KO) strains, infected mice, then challenged cats with brain cysts to perform an input/output screen that identified sexual stage-specific genes critical for oocyst formation. Mouse brains were harvested 50 d.p.i. and split into an input sample and a cat inoculation sample. Mi-Seq analysis identified 2 genes, referred to as IFT88-, SRS15-KO that had reduced, or failed to produce oocysts in cats, respectively. When assayed individually through cats, IFT88- and SRS15-KO strains did not produce oocysts. Complementation of the IFT88 gene restored oocyst production. Cats vaccinated with either IFT88- or SRS15-KO strains were immune to homologous rechallenge, heterologous rechallenge experiments are ongoing and results will be presented. Our data demonstrate the potential efficacy of an attenuated oral vaccine to block oocyst shedding and reduce the transmission of Toxoplasma in food and water sources destined for human and animal consumption.

mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency

The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and dormant cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2α accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5&quot;-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence.

Integrative Analysis of AP2 Transcription Factor Dynamics and Binding Motifs in Toxoplasma gondii

The complex life cycle of Toxoplasma gondii encompasses two infectious phases in intermediate hosts: the rapidly replicating tachyzoite and the dormant bradyzoite, responsible for acute and chronic infections, respectively. The ability of the parasite to form tissue cysts, predominantly in the brain, eyes, heart, and kidneys, and to reactivate to cause acute disease underlies its significant pathogenicity, particularly in immunocompromised individuals such as HIV patients. Despite its prevalence, effective treatments targeting cyst formation and reactivation are lacking. Our study aims to unravel the mechanisms driving the formation and reactivation of these tissue cysts, focusing on the role of AP2 transcription factors (TFs), the major family of transcription factors in Apicomplexa. AP2 TFs are implicated in controlling the gene expression necessary for the parasite's lifecycle, pathogenicity, and developmental transitions. Therefore, interaction between AP2 TFs and their DNA binding sites is very important. We constructed an integrated single-cell RNA sequencing (scRNA-seq) atlas using published data from T. gondii Type II strains, ME49 and Pru that are typically used for bradyzoite models. This atlas delineates gene expression profiles across different cell cycle phases and environmental conditions, offering a detailed map of transcriptional changes associated with the parasite's development, and highlighting the functionality of specific genes and pathways. We then investigated this atlas using the binding dynamics of AP2 TFs. Utilizing Protein Binding Microarray (PBM) data, we performed motif scanning to align AP2 binding motifs with scRNA-seq data using the MAST algorithm from MEME. We focused on mapping the expression profiles of AP2 genes and their targets within the single-cell dataset. These data implicate AP2 in both gene activation and gene repression during regulation of cell cycle progression and tachyzoite to bradyzoite differentiation. Our research provides insights into the transcriptional control in T. gondii, emphasizing the critical role of AP2 TF motifs in its adaptive life cycle. The integrative approach of mapping AP2 factor dynamics and gene expression profiles opens new avenues for therapeutic interventions, targeting the developmental transitions of T. gondii, and potentially leading to more effective treatments for toxoplasmosis.

ToxoDB: Tools for Genomic-Scale Data Exploration, Analysis, Integration and Discovery

Biomedical research is increasingly driven by Big Data: genome sequences &amp; diversity data, all manner of multi-Omics datasets, etc. How can we effectively collect, store, maintain and integrate this information to ensure FAIR (Findable,Accessible, Interoperable, Reusable) data access, advancing biological understanding and defining targets for further study in the lab, field &amp; clinic? The Eukaryotic Pathogen &amp; Vector Genomics Resource (VEuPathDB.org) – including ToxoDB.org– provides a robust, sustainable data-mining resource, accessed by 1000s of researchers daily, and demonstrably expediting discovery &amp; translational research on diverse eukaryotic microbes (fungi &amp; protists). We will be available at Toxo XVII to demonstrate database functionality and discuss topics of community interest, e.g.: accessing &amp; interpreting information on genes, genomes, population diversity, comparative genomics, DNA/protein motifs, protein structures, interactomes, epigenetics, transcriptomes, proteomes, metabolomes, pathways, subcellular localization, phenotypic characterization, orthology-based functional inference, automated &amp; curated annotation, etc strategies for integrating &amp; interrogating diverse datasets … and analyzing &amp; sharing the results obtained assessing &amp; improving the quality and accuracy of available annotation … capturing expert knowledge from the community analyzing your own (or any public) datasets via the free, private, easy-to-use, cloud-based VEuPathDB Galaxy instance … and integrating/querying results in the context of other data in FungiDB identification and prioritization of new Toxoplasma datasets for integration, and development of new database features; prospects for sustainable coordination with other resources new datasets &amp; functionalities currently under development at VEuPathDB/FungiDB … what to expect over the coming years additional help &amp; assistance with database mining &amp; FAIR data access/sharing

Understanding the History of the Domestic Cycle of Toxoplasma gondii in South America

A growing body of evidence supports the significant role of Toxoplasma gondii genotype in determining disease severity. However, isolating and characterizing T. gondii strains infecting humans remains challenging. Clinical severity of toxoplasmosis varies geographically, with South America experiencing the highest burden, coinciding with the continent's remarkable T. gondii genetic diversity. In our study, we analyzed 156 T. gondii genomes and we provide the first direct estimates of T. gondii's mutation rate and generation time. Our findings offer insights into the epidemiology, ecology, and evolutionary history of South American T. gondii populations. We identified three distinct categories: (1) wild populations, transmitted through the sylvatic cycle involving wild felids and likely poorly adapted to domestic cats. This includes the "Amazonian" population, linked to various clinical forms in immunocompetent individuals, and the "Pan-American" population, isolated from wildlife and not yet associated with severe disease forms, (2) domestic intercontinental clonal lineages (types I, II, III, and Africa 1), introduced to the continent with the domestic cat and house mouse by European sailors. (3) hybrids of the first two categories, probably well adapted to domestic cats, and representing the majority of strains found in South America's domestic environment. We discuss how hybridization events and strong selection for a unique haplotype comprising only 0.16% of the T. gondii genome on chromosome 1a are likely driving the public health challenge associated with toxoplasmosis in South America.

Session I: Genomics

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