Date of Award

8-1-2021

Publication Type

Thesis

Degree Name

M.Sc.

Department

Chemistry and Biochemistry

Keywords

CRISPRi, Liquid liquid phase separation, Membrane, Toxoplasma gondii

Supervisor

A. Swan

Supervisor

Y. Tong

Rights

info:eu-repo/semantics/openAccess

Abstract

Elucidating biomolecular interactions that underlie the cellular processes vital to the survival and infectivity of the parasitic protozoan Toxoplasma gondii is crucial for development of effective prophylactics and therapeutics against this resilient pathogen. In the first two parts of this three-part study, the significance of protein-protein, protein-nucleic acid, and protein-lipid interactions in T. gondii pathogenicity is explored. The first part of the study presents an overview of current knowledge on the topic of liquid-liquid phase separation, which is the condensation of macromolecules driven by multivalent interactions between protein and protein or protein and nucleic acids, and its multifaceted role in the proliferation and survival of pathogens such as T. gondii. The second part of the study investigates the influence of the T. gondii small ubiquitin-like modifier (TgSUMO) peptide on protein-lipid interaction using model membranes. In vitro studies with recombinant TgSUMO and giant unilamellar vesicles revealed the following: 1) the presence of weak, non-covalent interactions between TgSUMO and the artificial membrane, and 2) TgSUMO does not exhibit lipid bilayer phase preference and associates equally with liquid-ordered (lo) and liquid-disordered (ld) phases. The third part of the study describes the establishment of an inducible CRISPR interference system in T. gondii for gene function studies. Upon examining multiple factors that influence CRISPRi efficiency, it was found that induction of dCas9 transcription at 16 hours post-infection with 3.0 μg/mL anhydrotetracycline yielded a 15% reduction of gene expression, which is the highest knockdown efficiency observed in this study. This relatively low knockdown efficiency indicates that further optimization of the current CRISPRi system in T. gondii is necessary for it to be used for loss-of-function studies.

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