Scott Roscoe

Date of Award

1-1-2022

Publication Type

Dissertation

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

DEAD-box protein, Mass spectrometry, Poly(A) binding protein, RNP granules, Toxoplasma gondii

Supervisor

A. Swan

Supervisor

P. Vacratsis

Rights

info:eu-repo/semantics/openAccess

Abstract

Toxoplasma gondii is an obligate intracellular parasite capable of infecting mammals, birds, reptiles, and fish. T. gondii only undergoes sexual reproduction in a feline host. In all other organisms the parasite reproduces asexually, either as fast growing tachyzoites or slow growing bradyzoites. Bradyzoites form latent cysts inside the host cell that can lay dormant for years and convert back to tachyzoites when the host’s immune system becomes weakened. Tachyzoites rapidly replicate in the host cell, eventually causing it to lyse. While extracellular, tachyzoites repress their own translation by phosphorylating the eukaryotic initiation factor eIF2α and form microscopically visible aggregates of non-translating RNA and associated proteins. These aggregates are generally called RNP granules.

In this work poly(A) binding proteins (PABP) in T. gondii were identified and used to mark RNP granules. Visualizing RNP granule formation throughout the process of parasite egress from the host cell revealed that RNP granules are formed prior to host cell lysis. RNP granules were also found not to require polymerized microtubules to form, or to localize to any particular position in the parasites. Interestingly, RNP granules were not found in bradyzoites or under nutrient starvation, despite reported eIF2α phosphorylated. Both conditions can be thought of as gradual stressors, suggesting that RNP granule formation requires rapid translational repression and/or a sudden increase in non-translating RNA and not just eIF2α phosphorylation. Finally, it was observed that after 5 minutes of extracellular exposure, the number of RNP granules in parasites decreased, with a corresponding decrease in plaque formation when these parasites were reintroduced to host cells.

To follow up on these observations, a method for the isolation and analysis of RNP granules from extracellular tachyzoites was developed. By using paraformaldehyde cross-linking, RNP granules could be ‘frozen’ in their conformation at a given time post egress. These cross-linked RNP granules were then isolated by differential centrifugation and their protein components were identified using mass spectrometry. RNP granules from freshly lysed parasites were compared to those of parasites which had been extracellular for a prolonged period. It was found that RNP granules from freshly lysed parasites lacked many of the proteins found in more mature granules and contained no unique components.

Finally, the localization of the DEAD-box helicase TgHoDI into RNP granules and the effect of TgHoDI on RNP granule formation and parasite fitness were analyzed. Colocalization between TgHoDI and mRNA in tachyzoites outside a host cell was examined using RNA-FISH. It was found that the decrease in RNP granules after 5 min occurred in granules that did not contain TgHoDI. Analysis of a TgHoDI knock out strain showed no change in the formation of RNP granules and no decrease in parasite fitness. Together, these results suggest that TgHoDI containing granules are not sites for mRNA degradation. Overall, this work reveals that there are multiple types of RNP granules in extracellular T. gondii, and establishes new tools for their analysis and characterization.

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