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AP2X-1 is a negative regulator of Toxoplasma gondii sexual commitment

ABSTRACT: Toxoplasma gondii is a widespread protozoan with a complex life cycle, characterized by transitions between various hosts and developmental stages, each tailored to a specific niche within its host. However, the regulatory mechanisms governing these life cycle transitions are not well understood. In this study, we investigated the AP2 factor AP2X-1, which is expressed during the tachyzoite and bradyzoite stages but decreases in the mature merozoite stage. Knockout of ap2X-1 significantly impaired tachyzoite invasion and replication while increasing the frequency of bradyzoite differentiation. As a component associated with the HDAC3/MORC complex, loss of ap2X-1 led to the upregulation of bradyzoite- and sexual stage-specific genes. Single-cell sequencing revealed that ap2X-1 knockout strain exhibited a mixed population of tachyzoite-, bradyzoite-, merozoite-, and sporozoite-like parasites. CUT&Tag analysis revealed a substantial overlap between AP2X-1 and HDAC3/MORC complex at the promoters of bradyzoite- and sexual stage-specific genes. Additionally, ATAC-seq analysis demonstrated that AP2X-1 influences chromatin compaction and accessibility, suggesting that AP2X-1 may modulate the function of the HDAC3/MORC complex to facilitate the repression of bradyzoite differentiation and sexual commitment. Loss of ap2X-1 resulted in a significant attenuation of T. gondii virulence and decreased brain cyst formation in vivo. These findings identify AP2X-1 as a critical negative regulator of T. gondii sexual development.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Toxoplasma Gondii Me49

TISSUE(S): Tachyzoite

SUBMITTER: JinLei Wang

LAB HEAD: Jin-Lei Wang

PROVIDER: PXD065585 | Pride | 2025-06-29

REPOSITORIES: Pride

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Project description:Toxoplasma gondii, a protozoan parasite, causes toxoplasmosis, a widespread zoonotic and food-borne infection that poses significant risks, particularly in opportunistic and congenital cases. This obligate intracellular parasite is highly prevalent worldwide, largely due to its versatile life cycle, involving multiple hosts and transmission routes, and its ability to establish chronic infections. The presence of this neurotropic parasite in the brain poses a reactivation risk in immunocompromised individuals and might be associated with a higher likelihood of developing mental disorders. However, the role of the dormant bradyzoite stage in the pathophysiology of the disease is underexplored, mainly due to the lack of non-invasive detection methods and serologic tests targeting bradyzoite- or cyst-specific antigens. In this study, we performed an unbiased screening of the bradyzoite proteome and identified the Bradyzoite Serological Marker (BSM) as an additional serological biomarker, alongside the cyst-associated BCLA, to detect chronic stages in vivo. BSM and BCLA show high sensitivity and specificity in identifying cyst-bearing mice. However, in humans, these markers exhibit only moderate concordance, with a 30% positivity rate among individuals with prior immunity, suggesting variability in immune responses and complexities in diagnosing chronic toxoplasmosis. Importantly, bradyzoite serology helps differentiate recent from past infections by the teratogenic parasite Toxoplasma gondii, with BCLA improving the accuracy of pergestational infection diagnosis. Exploring the regulatory mechanisms controlling the expression of these markers revealed that the chromatin modifiers MORC and HDAC3 exert epistatic control over BFD1, a key regulator of bradyzoite development. While BFD1 governs a specific subset of bradyzoite markers, including BCLA, a sub-transcriptome comprising BSM relies on MORC/HDAC3 independently of BFD1. This complex gene regulation highlights the challenge in understanding Toxoplasma persistence, while offering new opportunities for improved serological diagnosis of congenital and chronic toxoplasmosis, particularly in individuals with mental health conditions or a risk of toxoplasmic reactivation.

Project description:Background: Considerable work has been carried out to understand the biology of the intermediate stages, the tachyzoite and bradyzoite, of Toxoplasma gondii in large part due to the accessible culturing methods for these stages. However, culturing methods for stages beyond the bradyzoite, including the merozoite and sexual stages, have not been developed hindering the ability to study a large portion of the parasite’s life cycle. We begin to unravel the molecular aspects of the merozoite stage focusing on gene expression. Results: To initiate this, we harvested merozoite parasites and hybridized mRNA to the Affymetrix Toxoplasma GeneChip. We analyzed the merozoite data in context of the life cycle by combining it with a previously published study that generated array data for the oocyst, tachyzoite, and bradyzoite stages (Fritz HM et al. PLoS One, 2012). Principal component analysis highlights the unique profile of the merozoite samples, placing them approximately half-way on a continuum between the tachyzoite/bradyzoite and oocyst samples. Prior studies have shown that antibodies to surface antigen p30 (SAG1) and many dense granule proteins do not label merozoites, and our microarray data confirms that these genes are not expressed at this stage. Also, the expression for many rhoptry and microneme proteins is drastically reduced while the expression for many surface antigens is increased at the merozoite stage. Gene Ontology and KEGG analysis reveals that genes involved in transcription/translation and many metabolic pathways are upregulated at the merozoite stage, highlighting unique growth requirements of this stage. We also show that an upstream promoter region of a merozoite specific gene is sufficient to control stage specific expression at the merozoite stage. Conclusion: The merozoite represents the first developmental stage within the gut of the definitive host. Determining the correct conditions that coax the parasite into the merozoite stage in vitro may allow the parasite to complete sexual development. The data presented here describe the global gene expression profile of merozoite stage and the creation of transgenic parasite strains that will be useful in unlocking how the parasite senses and responds to the felid gut environment to initiate coccidian development. The ToxoGeneChip microarray was used to measure both tachyzoite and merozoite mRNA expression in the type II TgNmBr1 strain.

			Action	DRS
	AP2X-1-2Ty_Rep1.mgf	Mgf
	AP2X-1-2Ty_Rep1.raw	Raw
	AP2X-1-2Ty_Rep2.mgf	Mgf
	AP2X-1-2Ty_Rep2.raw	Raw
	AP2X-1-2Ty_Rep3.mgf	Mgf

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AP2X-1 is a negative regulator of Toxoplasma gondii sexual commitment

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