AbstractsBiology & Animal Science

Inhibitory Effects of Securinine and Related Compounds on Toxoplasma gondii and Mechanistic Insights into Transcript-Specific Translational Repression in the Bradyzoite Developmental Stage

by Michael James Holmes

Institution: University of Windsor
Department: Chemistry and Biochemistry
Degree: PhD
Year: 2015
Keywords: Developmental regulation; Lactate dehydrogenase; Post-transcriptional gene regulation; Toxoplasma gondii; Toxoplasmosis; Translational repression
Record ID: 2058177
Full text PDF: http://scholar.uwindsor.ca/etd/5252



Toxoplasma gondii is an intracellular protozoan parasite that infects warm blooded animals the world over. In the host, the parasite exists as either rapidly growing tachyzoites or immunologically protected slowly dividing bradyzoites. While tachyzoites are generally cleared by the host's immune system, bradyzoites persist for life. If the immune system becomes compromised, bradyzoites reconvert to tachyzoites and cause severe illness. Bradyzoites develop in response to stressful stimuli, therefore discovering factors that modulate bradyzoite conversion as well as determining how Toxoplasma's stress response pathways operate are essential to understanding parasite disease. In this work, the plant alkaloid securinine as well as ten pyrrolidine derivatives were shown to have in vitro anti-Toxoplasma activity in the micromolar range. These compounds act in different capacities, either restricting parasite's invasion or by reducing the parasite's rate of replication. One of the hallmarks of the bradyzoite life stage is slowed replication. When tested, only securinine induced bradyzoite formation at comparable levels to treatment with alkaline media, suggesting that it initiates a stressful stimulus. When in the bradyzoite stage, Toxoplasma induces a global translational repression allowing for the remodeling of the transcriptome. However it remains unclear how certain transcripts are maintained throughout the long term translational repression seen in bradyzoites. Analysis of one such transcript, lactate dehydrogenase 1, showed a 40 nucleotide cis-acting element with its 5'UTR responsible for this activity. The formation of 16 nucleotide regulatory RNA hairpin is necessary for inducing sustained translational repression in bradyzoites, suggesting that it acts as a recognizable feature. Characterization of the transcript reveals that its poly(A) tail does not shorten in bradyzoites, indicating that it is likely targeted for long term storage instead of degraded under stress. Efforts were made to capture and analyse the protein complement interacting with the cis-acting element using RNA aptamer technologies, however they were unfruitful. Using extracellular parasites, it was determined that a rapid induction of stress is insufficient to selectively repress the transcript. This suggests that the reprogramming of the proteome may be necessary for the sustained translational repression of lactate dehydrogenase 1 in Toxoplasma bradyzoites.