A transcriptional network required for Toxoplasma gondii tissue cyst formation is dispensable for long-term persistence

Abstract

Cyst formation is a key feature of the T. gondii life cycle but the genetic networks that drive this process are not yet fully characterized. To identify new components of this network, we compared T. gondii to its nearest extant relative Hammondia hammondi given the critical differences between these species in the timing and efficiency of cyst formation. Using transcriptional data from critical developmental and pH exposure time points from both species, we identified the gene TGVEG_311100, which we named Regulator of Cystogenesis 1 (ROCY1), as being both necessary and sufficient for cyst formation in T. gondii. Compared to WT parasites, TGVEGΔROCY1 parasites formed significantly fewer tissue cysts in response to alkaline pH stress in vitro and cysts were nearly undetectable in mouse brains for up to 9 weeks post-infection. Overexpression of tagged ROCY1 in WT parasites was sufficient to induce cyst formation in vitro in both WT and ROCY1-deficient parasites, demonstrating that ROCY1 is both necessary and sufficient for cyst formation. Moreover this induction of cyst formation required at least 1 of 3 predicted CCCH Zinc finger domains. Mice chronically infected with ΔROCY1 parasites had detectable tachyzoites in the brain for up to 37 days post-infection (while mice infected with WT parasites did not), and CNS transcriptional analyses at day 30 post-infection throughout the chronic phase of infection revealed inflammatory signatures consistent with acute infection in ΔROCY1 parasites compared to WT. Despite our inability to detect brain cysts in infected mice, both WT and ΔROCY1 knockout parasites reactivated after dexamethasone treatment with similar timing and magnitude for up to 5 months post infection, challenging the paradigm that long term parasite persistence in the CNS requires cyst formation. These data identify a new regulator of cyst formation in T. gondii that is both necessary and sufficient for cyst formation, and whose function relies on its conserved nucleic acid binding motif.

The latent stage ofToxoplasma gondiiis targeted by the immune response and host protective

“…6f). These datain addition to a recent study showing higher tachyzoite burdens in CBA mice infected with a type III strain (VEG) of T. gondii lacking BFD2 14 highlight that across mouse models of varying resistance to TE, the inability of T. gondii to form cysts is associated with increased levels of tachyzoite replication and associated pathology in the CNS.…”

“…For example, the Sarcocystidae family of apicomplexan parasites (which includes Toxoplasma gondii) converts from the tachyzoite stage to the slow growing bradyzoite stage which forms long-lived tissue cysts found predominantly in neurons 6,[9][10][11] . The molecular basis for this switch is mediated by the transcription factor BFD1 and the RNA binding protein BFD2 that enforce and maintain the bradyzoite transcriptional program [12][13][14] . This latent stage facilitates oral transmission and has been considered important for immune evasion, making it a major contributor to the evolutionary success of this parasite 15 .…”

Histone variant H2B.Z acetylation is necessary for maintenance ofToxoplasma gondiibiological fitness

“…Although a massive analysis of gene expression is necessary to detect specific changes, part of the study shows that the expression of Rop5 and Rop18 does not occur at the transcriptional level, but rather posttranscriptionally, affecting their translation. This regulation could be given by the presence of non-coding RNAs (ncRNAs) and there is increasing evidence for a significant role for pos-transcriptional mechanisms [65,66]. Future studies should take this possibility into account, considering a possible role of H2B.Z acetylation, to analyze ncRNAs expression in T. gondii.…”

Advances towards the complete in vitro life cycle of Toxoplasma gondii

“…BFD1 was therefore termed “the master regulator” 52 . Following this seminal work, the regulatory network of BFD1 has been studied in more detail, and two laboratories have now independently provided evidence for a zinc finger motif-containing protein (ToxoDB ID TGME49_311100/TGVEG_311100) as a critical secondary effector of BFD1, which they named ‘BFD2’ 54 or ‘Regulator of Cystogenesis 1’ (ROCY1) 55 , respectively. A positive feedback loop was proposed, in which the novel factor promotes the translation of BFD1 transcripts under stress conditions and which in turn further increases transcription of BFD2/ROCY1 , thereby reinforcing the fate commitment 54 .…”