A single-parasite transcriptional atlas of Toxoplasma Gondii reveals novel control of antigen expression

Abstract

Toxoplasma gondii, a protozoan parasite, undergoes a complex and poorly understood developmental process that is critical for establishing a chronic infection in its intermediate hosts. Here, we applied single-cell RNA-sequencing (scRNA-seq) on >5,400 Toxoplasma in both tachyzoite and bradyzoite stages using three widely studied strains to construct a comprehensive atlas of cell-cycle and asexual development, revealing hidden states and transcriptional factors associated with each developmental stage. Analysis of SAG1-related sequence (SRS) antigenic repertoire reveals a highly heterogeneous, sporadic expression pattern unexplained by measurement noise, cell cycle, or asexual development. Furthermore, we identified AP2IX-1 as a transcription factor that controls the switching from the ubiquitous SAG1 to rare surface antigens not previously observed in tachyzoites. In addition, comparative analysis between Toxoplasma and Plasmodium scRNA-seq results reveals concerted expression of gene sets, despite fundamental differences in cell division. Lastly, we built an interactive data-browser for visualization of our atlas resource.

The Growth of Eimeria tenella: Characterization and Application of Quantitative Methods to Assess Sporozoite Invasion and Endogenous Development in Cell Culture

“…Additionally, the MS identified a series of proteins directly related to invasion and development which are differentially expressed at specific time points in an in vitro time course. Results correlated with previous findings for some of these proteins: (i) presence of alternative types of surface antigen proteins (SAGs) between sporozoites and merozoites (Tabares et al, 2004 ); (ii) high abundance of microneme (MIC) proteins during invasion (Bumstead and Tomley, 2000 ); and (iii) overexpression of rhoptry (ROP) and dense granule (GRA) proteins during endogenous development (Oakes et al, 2013 ; Xue et al, 2020 ). These data further corroborate the suitability of the in vitro system to mimic endogenous development (Burrell et al, 2020 ) and are an important source of information to design specific interventions for the study of these relevant proteins.…”

An ex vivo model ofToxoplasmarecrudescence

“…These versatile tachyzoites also have the ability to re-develop into the dormant bradyzoite at any time within a host cell environment that is permissive for tissue cyst development. Adapting native strains to HFFculture can disrupt these pathways and may cause transcriptional confusion as recent single parasite RNA-seq studies have suggested (38).…”

Application of single-cell transcriptomics to kinetoplastid research

“…Additional filtering to remove all rRNA transcript counts from further analysis can also be performed. Studies of Plasmodium have also noted contaminating rRNA, which required removal prior to analysis (Poran et al, 2017;Müller et al, 2018;Reid et al, 2018;Waldman et al, 2020;Xue et al, 2020). Doublet rates vary between technologies and individual experiments.…”

“…Additionally, complete atlases provide a resource that can be mined for cell sub-populations, such as male and female Plasmodium falciparum gametes (Real et al, 2020). These, and other, scRNA-seq data sets have allowed apicomplexan parasitologists to study the dynamic gene expression change during developmental processes, such as the sexual commitment of Plasmodium species and T. gondii tachyzoite to bradyzoite differentiation (Waldman et al, 2020;Xue et al, 2020), and identify putative novel regulators. Additionally, cells from a query scRNA-seq sample can be mapped to these reference atlases to assess their developmental stage and unique gene expression differences (Howick et al, 2019;Real et al, 2020).…”