Knock-out of the bradyzoite marker P18 in Toxoplasma gondii :insights towards a functional characterization during neurotoxoplasmosis -

Abstract

Toxoplasma gondii is an apicomplexan protozoan parasite that infects all warm blooded animals including humans. T. gondii causes a severely morbid or fatal disease in fetus and immunocompromised patients. During its life cycle, T. gondii exhibits three morphologically infectious stages: tachyzoites, bradyzoites, and sporozoites. Tachyzoites are rapidly multiplying and responsible for the acute toxoplasmosis leading to tissue damage. Bradyzoites are slow-growing and responsible for the chronic neurotoxoplasmosis that often reactivates in immunocompromized patients. Lastly, sporozoites are the infective forms found in oocysts. The back and forth switch between tachyzoite and bradyzoite stages is a key modulator of the progression of toxoplasmosis between acute and chronic phases. However, this switch remains very poorly understood. Here, we are investigating the role of the bradyzoite marker p18 for which the gene sequence is annotated on www.toxoDB.org. Specifically, we have used the vector (P2854) containing the selectable marker cassette hypoxanthine-xanthine-guanine-phosphoribosyl-transferase (HXGPRT) and cloned the 5’ and 3’ flanking regions of p18. This vector was introduced by electroporation to the Pru Δku80 strain which favors its integration by double crossing over and homologous recombination. We have successfully generated and cloned the Pru Δku80Δp18 knock-out parasites and investigated their phenotype in vivo. Our results have shown that deleting p18 leads to the formation of more bradyzoite cysts in the brains of mice. However, these bradyzoites reactivate much later than the wild type strain. This result drove us to investigate the phenotype of the Pru Δku80Δp18 knock out during the acute phase of infection. We could clearly see a better survival rate of mice infected with the knock-out strain as compared to the wild type strain, furthermore, we have seen less parasites in all tested organs except in the brain where the amount of tachyzoites from both str