Date of Award

2024

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Microbiology and Molecular Genetics

First Advisor

Christopher Huston

Abstract

Human babesiosis is a bloodborne, tick-transmitted zoonotic disease that has seen an increase in cases across the United States and Canada. Babesia duncani is the second most prevalent and most virulent cause of human babesiosis but little is known of its cellular biology, molecular pathogenesis, and molecular genetics. To remedy this and help accelerate research on Babesia duncani, we worked towards developing a functional Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system for Babesia duncani while identifying strong promoters to drive expression of genes, developing an optimized transfection protocol, and identifying suitable selectable markers. To identify strong promoters to drive expression of genes in transfections, reverse transcriptase (RT)-qPCR was performed on RNA extracted and synthesized into cDNA. To find a workable transfection protocol that could be optimized, established Babesia and Plasmodium transfection protocols from the literature were screened for their transfection efficacy (amount of nanoluciferase transcript produced) and impact on parasite viability following transfection by RT-qPCR readout. Once a workable transfection protocol had been identified, the protocol was optimized by varying the number of parasites and amount of DNA used per transfection. A CRISPR/Cas9 transfection was performed using the identified optimized transfection protocol. Lastly, to identify suitable selectable markers a novel 384-well high throughput compound screening format was developed. Babesia duncani were incubated for 48-hours with the compound of interest and lysed in the presence of propidium iodide before fluorescence was read 24-hours later. Histone 2B (H2B) promoter was identified as the strongest promoter of those tested. Enolase and elongation factor-1α (eF-1α) were the next strongest promoters but had roughly 3- and 5-fold less expression than H2B, respectively. Govindarajalu et al, 2019 Lyse-Reseal protocol gave the third highest amount of nanoluciferase transcript without negatively impacting parasite growth following transfection. It was selected as the protocol to be optimized, with results indicating increased number of parasites and increased amount of DNA used per transfection led to greater transfection efficiency. However the results also indicated there may also be an inherent limitation to the maximum efficiency obtainable with the protocol seen in the diminishing return when increasing the number of parasites or amount of DNA used. The results of CRISPR/Cas9 transfection were promising as a mCherry expressing Babesia duncani, the expected outcome, was observed 24-hours post transfection however no other ones were observed following that. Using the high throughput compound screening format a handful of compounds were tested, identifying potential novel selectable markers for Babesia duncani as well as avenues for drug development.

Language

en

Number of Pages

97 p.

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