NYMC Student Theses and Dissertations

Corey Gaylets

Date of Award

5-19-2020

Document Type

Doctoral Dissertation - Open Access

Degree Name

Doctor of Philosophy

Department

Microbiology and Immunology

First Advisor

Dana Mordue, PhD

Second Advisor

Chandra Bakshi, PhD, DVM

Third Advisor

Mary Petzke, PhD

Abstract

Toxoplasma gondii is an obligate intracellular pathogen. It is considered the second deadliest foodborne pathogen in the world with up to 1/3 of the human population estimated to be currently infected. The majority of immune competent people may exhibit mild symptoms following T gondii infection. The acute stage of infection is often well controlled by cellular immunity in healthy individuals. However sterilizing immunity does not occur as the tachyzoite stage of the parasite responsible for acute infection differentiates to a slow growing bradyzoite stage contained in tissues cysts that can persist for the life of the host. However, if the host becomes immunocompromised, these parasites can differentiate back to tachyzoites that can cause pneumonia, severe central nervous system pathology and death. Current therapies are effective against the tachyzoite stage but not the chronic cyst stage and so infected patients are susceptible to recurrent disease should they become immunocompromised. Apicomplexa parasites, including T. gondii have a unique family of serine/threonine kinases referred to as FIKK kinases based on the conserved amino acids in its active site: region of amino acid in the protein’s active site consisting of a phenylalanine(F), and isoleucine(I), and two lysines(K). Plasmodium falciparum has 17 potential FIKK kinases while most Apicomplexa including T. gondii have a single FIKK kinase. The goal of the current study was to evaluate the importance of T. gondii’s sole FIKK kinase to parasite pathogenesis. Endogenous tagging of the TgFIKK kinase with YFP followed by live cell florescent microscopy during the parasite’s lytic cycle indicated the FIKK kinase localizes to the parasite posterior end, within the parasite basal complex, in both xi extracellular and intracellular parasites. It was also transiently found in the host cell cytosol. The FIKK kinase was deleted and complemented in order to study the function of the FIKK kinase in parasite pathogenesis. Studies comparing wild type, ΔFIKK parasites and FIKK complemented parasites indicate that the TgFIKK kinase is not critical for the parasites lytic cycle in human fibroblast (HFF) cells, including parasite replication. Instead, we show that the TgFIKK kinase is critical for the ability of the parasite to form cysts in vitro in cell culture and for the establishment of chronic infection in mice. The deletion of the FIKK gene prevents tachyzoite to bradyzoite differentiation. Thus, our current hypothesis is that the TgFIKK kinase may link changes in the parasite’s cell cycle at a proposed G1 checkpoint that serves as a gateway for parasite entry into the bradyzoite differentiation pathway.

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