ROP16-mediated activation of STAT6 facilitates encystment of type IIIToxoplasma gondiiin neurons

Abstract

Toxoplasma gondii establishes a long-lived latent infection in the central nervous system (CNS) of its hosts. Reactivation in immunocompromised individuals can lead to life threatening disease. Latent infection is driven by the ability of the parasite to convert from the acute-stage tachyzoite to the latent-stage bradyzoite which resides in long-lived intracellular cysts. While much work has focused on the parasitic factors that drive cyst development, the host factors that influence encystment are not well defined. Here we show that a polymorphic secreted parasite kinase (ROP16), that phosphorylates host cell proteins, mediates efficient encystment of T. gondii in stress-induced models of encystment and primary neuronal cell cultures (PNCs) in a strain-specific manner. Using short-hairpin RNA (shRNA) knockdowns in human foreskin fibroblasts (HFFs) and PNCs from transgenic mice, we determined that ROP16′s cyst enhancing abilities are mediated by phosphorylation of the host cell transcription factor STAT6. To test the role of STAT6 in vivo, we infected STAT6KO mice, finding that, compared to infected wild-type mice, infected STAT6KO mice have a decrease in cyst burden, but not overall parasite burden or dissemination to the CNS. Finally, we found a similar ROP16-dependent encystment defect in human pluripotent stem cell-derived neurons. Together, these findings identify a host cell factor (STAT6) that T. gondii manipulates in a strain-specific manner to generate a favorable encystment environment.

Mapping host-microbe transcriptional interactions by dual perturb-seq

“…Bradyzoite cysts can persist for the lifetime of the host and are essential in the T. gondii life cycle for transmission to the definitive felid host 57 ; moreover, they are clinically important as a source of recrudescent infection in immunocompromised individuals 58 . ROP16-dependent STAT6 signalling has previously been shown to facilitate bradyzoite cyst formation in vivo and in vitro, although the downstream host cell pathways involved remain unknown 59,60 . The findings here for SOS1 raised the question of whether sustained STAT6 signalling is necessary to maintain a permissive host cell environment for cyst formation, or whether only the initial ROP16-dependent burst of signalling is necessary to trigger cyst formation.…”

“…To determine whether SOS1 is required for efficient cyst formation, as is ROP16, we used a previously described high-content imaging and automated image analysis to quantify parasite differentiation from tachyzoites to bradyzoites in primary murine neurons 60 . Conversion of tachyzoite vacuoles to bradyzoite cysts was determined by staining of the cyst wall with Dolichos bisflorus agglutinin (DBA) (Figure 5C).…”

“…Cyst formation assays were carried out as previously described 60 . Briefly, primary neurons were cultured in 96-well, clear-bottom, blackwalled plates and infected with indicated T. gondii strains at a MOI of 0.1.…”