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

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