17th International Congress on Toxoplasmosis

Single-cell RNA sequencing and immunohistochemical analysis of reactivating bradyzoites in vivo.

Stage Conversion & Developmental Biology

Reactivation of chronic Toxoplasma infection is a critical element in disease pathology and is commonly associated with HIV comorbidity and the occurrence of toxoplasmic encephalitis. Reactivation of latent infection during pregnancy can result in congenital transmission and life-threatening disease in the neonate, whereas reactivation in the eye can cause severe ocular disease. Although a clinically relevant and critical component of the Toxoplasma life cycle, very little is known about the reactivation process. To map the process in situ, we synchronously dysregulated the immune system of chronically infected mice and analyzed parasite transcriptional changes using single-cell RNA sequencing. We also performed immunohistochemistry on the brains of these same mice to correlate morphological changes in the cysts with changes in gene expression at different time points. Analysis of gene expression in individual parasites before reactivation revealed significant heterogeneity in cell cycle progression and the expression of canonical bradyzoite-specific genes. This heterogeneity was unexpected based on what has been previously captured using in vitro models. Immunohistochemistry and transcriptional profiling of encysted parasites during the first five days after stimulating reactivation show that the transition from bradyzoites-to-tachyzoites occurs mainly after significant structural changes to cysts, demonstrating that within-cyst transcriptional changes are likely only the beginning of the bradyzoite-to-tachyzoite transition. This work represents the first step in mapping the reactivation process in vivo. It suggests that the process may be more dynamic and complex than previously recognized, involving heterogeneous starting populations of encysted parasites and intermediate transitional stages

Oral presentation

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A Genetic Screen Identifies Factors Required for Toxoplasma Differentiation into Latent Forms

Toxoplasma differentiates from acute stage tachyzoites into latent bradyzoite forms in the muscle and central nervous system. How Toxoplasma senses the tissue environment and undergoes differentiation, at the molecular level, in poorly understood. We undertook a whole genome CRISPR screen in RH strain to determine genes required for utilization of different carbon sources and serendipitously discovered that switching to glutamine, from glucose, triggers a differentiation-like program, highlighting 60 genes involved in this process. We show that glutamine, as the sole carbon source, also triggers differentiation in cytogenic strains and relies on the transcription factor BFD1. We then generated a CRISPR sub-library of these 60 genes and tested their role in differentiation in cystogenic strains in vitro and in mice. Individual mutants show that several genes involved in central carbon metabolism are required for differentiation in vitro and in vivo as well as genes involved in regulation of RNA. We also identified orthologs of the "GID" E3 ubiquitin ligase complex, which regulates translational repression in during embryogenesis in Drosophila. By genetic tagging and immunoprecipitation, we show that Toxoplasma also has a GID complex, whilst gene knockouts of GID components show impaired differentiation. Current work is centered around identifying TgGID ubiquitinated substrates. This work, therefore, identifies new molecular players and biological processes, including ubiquitination, required for Toxoplasma to differentiate into latent forms

Translation initiation factor eIF1.2 is a crucial early regulator of Toxoplasma bradyzoite cyst formation

The bradyzoite form of Toxoplasma gondii poses a therapeutic challenge since there is no available effective drug or vaccine for humans. Although differentiation of T. gondii tachyzoites to bradyzoites is a pivotal event during infection, our understanding of this process remains limited to a few differentiation factors that are sparsely linked. Using a chemical mutagenesis screen, we identified the translation initiation factor eIF1.2 as a critical player in T. gondii differentiation. A F97L mutation in eIF1.2 or complete knockout of eIF1.2 (∆eIF1.2) markedly impaired bradyzoite cyst formation in vitro and in vivo. Utilizing single-molecule scanning technology, we demonstrated that the T. gondii eIF1.2 F97L mutation alters the scanning process of the yeast ribosome preinitiation complex on a model yeast mRNA. RNA sequencing and ribosome profiling experiments unveiled that ∆eIF1.2 parasites exhibit defects in upregulating differentiation markers, such as BAG1, LDH2, and regulators, including BFD1 and BFD2/ROCY1, during stress-induced differentiation. Forced expression of BFD1 or BFD2 effectively restored differentiation in ∆eIF1.2 parasites. Our findings indicate that eIF1.2 regulates the translation of key differentiation factors that are essential for establishing chronic infection. Notably, non-tissue cyst-forming apicomplexan parasites only possess one eIF1, whereas most tissue cyst-forming apicomplexans like T. gondii have two eIF1 paralogs, eIF1.1 and eIF1.2. This work sets the stage for further elucidating the specific regulatory role of eIF1.2 in the expression of differentiation-related genes.

In vitro reactivation of chronic-stage Toxoplasma gondii parasites

In the intracellular apicomplexan parasite Toxoplasma gondii, differentiation from proliferative to chronic, cyst-forming life stages facilitates stress tolerance, immune evasion, and environmental persistence. Differentiation is accompanied by global changes in parasite gene expression and regulation, but tissue cysts retain the capacity to reactivate and resume rapid growth in immunocompromised hosts, which largely drives the morbidity associated with Toxoplasma infection. Our lab previously demonstrated that BFD1, a Myb-like transcription factor required for differentiation, is upregulated under stress via a positive feedback loop involving one of its targets, the RNA-binding protein BFD2. However, the molecular pathways underlying reactivation, including the role of the BFD1-BFD2 circuit, remain poorly understood. We established a genetic model to study Toxoplasma reactivation in vitro by introducing an additional, conditionally stabilizable copy of BFD2 (DD-BFD2) into differentiation-competent ME49 parasites. Similar to conditional BFD1 stabilization (DD-BFD1) in Δbfd1 parasites, this new strain reliably differentiates and expresses canonical markers of early and late bradyzoite development. Using stage-specific fluorescent reporters, we demonstrate that DD-BFD2 stabilization induces bradyzoite reporter expression to a higher magnitude and with greater heterogeneity compared to DD-BFD1 stabilization, suggesting that DD-BFD2 parasites explore a broader range of cellular states during differentiation. Critically, DD-BFD2 destabilization in chronic-stage parasites produces a subpopulation of cysts that remain differentiated over time, consistent with long-term maintenance of the endogenous BFD1-BFD2 positive feedback loop. Under these conditions, we simultaneously observe the emergence of a rapidly growing tachyzoite population distinct from mature cysts. Conversely, DD-BFD1 destabilization in Δbfd1 cysts leads to a rapid switch in stage-specific reporter expression. These observations highlight the role of heterogeneity in bradyzoite development and imply that reactivating parasites may originate from an as-yet-undefined parasite subpopulation.

Single-cell analysis of Toxoplasma cat stages identifies multiple developmental trajectories.

Toxoplasma gondii"s sexual development is limited to the epithelial cells of the small intestine of felids. Sexual development is a key to genetic diversity and this process is crucial to dissemination, as a single felid can shed millions of environmentally stable oocysts. Due to challenges associated with cultivating presexual and ultimately sexual stages, there is still much to learn about how this developmental program is triggered and executed by the parasite in response to infection of the feline host. Prior studies (Hehl et al., 2015 and Behnke et al,. 2014) demonstrated clear differences in the transcriptomes of asexual and sexual stages using bulk transcriptomics, and metabolic differences in the feline host play a key role in triggering the transition into sexual stages (Martorelli di Genova et al., 2019). Important recent work (Artunas et al., 2024, among others) has identified multiple parasite transcription factors as critical suppressors of presexual development, permitting the study of presexual stages and opening ways to using genetics to drive the full sexual cycle in vitro. To further investigate the sexual developmental phase of Toxoplasma and identify previously unknown regulators of the distinct phases of sexual development, we have conducted multiple single-cell transcriptomics analyses of Toxoplasma parasites isolated from the small intestines of infected cats. To do this we used flow cytometry to sort fluorescent reporter strains of T. gondii VEG from scraped small intestine samples and obtained single-cell transcriptomic data from 16,966 cells across two experiments. The data made it possible to classify, characterize, and parse each cell at the transcriptome level, which led to the identification of rare cell populations, including some that we hypothesize are macrogametes and microgametes. Candidate genes emerging from this study have now been targeted in a CRISPR-CAS9 screen and we are using Perturb-seq and loss of function assays on feline enteroepithelial stages to determine which factors are required for sexual development and/or the regulation of sexual stage gene networks. We aim to identify previously unknown drivers of differentiation, especially those that mediate gametogenesis. Our single-cell data also extends what is known about gene expression changes throughout sexual development and should be useful to those in the field working towards inducing gametogenesis, mating, and oocyst formation in feline-free conditions.

Characterizing the impact of mRNA regulatory features on translation efficiency in Toxoplasma gondii

Protein expression can be regulated through cis and trans factors acting on mRNA and translation machinery. This regulation allows cells to fine-tune expression of specific factors in response to environmental cues. The transcriptome of Toxoplasma gondii represents a challenge to canonical models of translational regulation in eukaryotes as its 5′ UTRs are several times longer than most other characterized species and the vast majority of 5′ UTRs harbor at least one, if not several, upstream AUGs. We hypothesize that these unusual features may be maintained in Toxoplasma because they confer regulatory information important for gene expression across the parasite life cycle. To understand how the parasite regulates translation across both its tachyzoite and bradyzoite stages, we performed high-resolution ribosome profiling on Toxoplasma and human host cells. Bioinformatic analyses of these data have characterized general transcript features that contribute to translation efficiency differently in Toxoplasma and human cells. Additionally, we identified a cohort of translationally-regulated factors whose expression changes between tachyzoites and bradyzoites. Based on these data, we aim to dissect the relative contributions of mRNA regulatory features to translation efficiency in the parasite. Using quantitative reporter systems, we are examining the sufficiency of endogenous 5′ UTRs to regulate both steady-state protein expression and changes under stress. Mutational analyses will reveal how specific sequence features, such as upstream open reading frames, impact translation efficiency. Together, this work expands our molecular framework for gene regulation in Toxoplasma.

Session VI: Stage Conversion

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