Vizcarra, E. A. | Search bioRxiv

bioRxiv2022

Engineering Toxoplasma transgenic tissue cysts.

Current approaches to find therapeutic solutions to treat and prevent reactivation of toxoplasmosis have suffered from limited accessibility to the relevant Toxoplasma stages and a lack of accurate in vitro developmental models. The loss of developmental competency in vitro that is exacerbated during the generation of transgenic tachyzoites is also a major impediment to understanding the molecular basis of bradyzoite recrudescence, which is the central parasite stage of reactivation. We have successfully modified Toxoplasma genes in the bradyzoite tissue cyst stage using ex vivo bradyzoite-based methods. Our new protocols validated the clonality of individual cysts and confirmed that single tissue cysts can robustly infect mice. We demonstrated these protocols by generating in vivo bradyzoites carrying a knockout of the Toxoplasma HXGPRT gene or the gene encoding the ApiAP2 transcription factor, AP2IX-9. Unexpectedly, the knockout of the AP2IX-9 gene in the Type II ME49EW strain eliminated one of the three developmental pathways initiated by the bradyzoite: host-dependent bradyzoite-to-bradyzoite replication. Our genetic protocols were further validated by producing in vivo bradyzoites lacking the bradyzoite-specific cyclin, TgCYC5. Interestingly, genetic ablation of TgCYC5 led to a large number of small cysts that formed from single mother cysts in mouse brain. Further study revealed the cause of the small-cyst phenotype in TgCYC5 knockout parasites was a disruption in the normal balance of bradyzoite subtypes that enhanced the bradyzoite-to-bradyzoite developmental pathway. These new data demonstrate the feasibility of generating transgenic parasites in a developmentally competent strain using ex vivo bradyzoite-based methods. Furthermore, by targeting the AP2IX-9 and TgCYC5 genes, whose transcripts are upregulated specifically in bradyzoites, we have clarified how these factors influence the pathways of tissue cyst recrudescence.

developmental biology↗

bioRxiv2020

An ex vivo model of Toxoplasma recrudescence

Toxoplasma has been a useful parasite model for decades because it is relatively easy to genetically modify and culture, however, attempts to generate and study the recrudescence of tissue cysts have come up short with lab-adapted strains generating low numbers of tissue cysts in vivo. Here we have established a new model of Toxoplasma recrudescence using bradyzoites from an unadapted Type II ME49 strain (ME49EW) isolated from murine brain tissue. Ex vivo bradyzoite infection of fibroblasts and astrocytes produced sequential tachyzoite growth stages; a fast-growing stage was followed by formation of a slower-growing stage. In astrocytes, but not in fibroblasts, bradyzoites also initiated a second recrudescent pathway involving bradyzoite to bradyzoite replication. Intraperitoneal infections of mice with either bradyzoites or the fast-growing tachyzoite stage efficiently disseminated to brain tissue leading to high numbers of tissue cysts, while infections with the slow-growing tachyzoite stage were largely retained in the peritoneum. Poor infection and cyst formation of slow-growing tachyzoites was reversible by serial tissue cyst passage, while the poor tissue cyst formation of lab-adapted tachyzoites was not reversible by these approaches. To distinguish strain developmental competency, we identified Toxoplasma genes highly expressed in ME49EW in vivo tissue cysts and developed a qPCR approach that differentiates immature from mature bradyzoites. In summary, the results presented describe a new ex vivo bradyzoite recrudescence model that fully captures the growth and developmental processes during toxoplasmosis reactivation in vivo opening the door to the further study of these important features of the Toxoplasma intermediate life cycle.

microbiology↗