Date of Award

Spring 5-31-2025

Document Type

Open Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Computational Sciences

First Advisor

Kourosh Zarringhalam

Second Advisor

Manoj T. Duraisingh

Abstract

Apicomplexa constitute a large phylum of single-celled, obligate intracellular protozoan parasites. Notably, Plasmodium spp. and Babesia spp. are apicomplexan parasites that infect red blood cells. Plasmodium species are the causative agent of malaria and are transmitted by Anopheles mosquitoes, affecting large human populations, whereas Babesia spp., transmitted through the bite of Ixodes ticks cause babesiosis.

In this dissertation, we investigate the essential genome of these parasites using high-throughput transposon mutagenesis. Identifying the essential genome is key to finding new drug targets and understanding resistance mechanisms, a crucial pursuit given the rising resistance to frontline antimalarial drugs and the challenges in treating babesiosis. We have developed analytical frameworks and Bayesian methodologies to quantify and predict the essentiality and the fitness of protein-coding genes, transcript variants, and lncRNAs in P. knowlesi and B. divergens. These organisms serve as ideal representatives of the Plasmodium vivax and Babesia clades, respectively, offering robust in vitro systems for genetic studies. Highly saturated transposon mutagenesis libraries enable identification of essential domains within larger, non-essential genes. We introduce a robust mathematical framework to systematically assess truncatability, offering high-resolution analysis of essentiality of protein domains. Our analysis not only confirmed previously known truncatable genes such as PTEX150 but also uncovered novel candidates. Furthermore, by employing Hidden Markov models, we systematically evaluate insertional bias based on sequence composition, ensuring rigorous data quality control.

Leveraging our highly saturated mutagenesis libraries, we conducted drug perturbation experiments to uncover resistance mechanisms to both antimalarial and antibabesial drugs. We present a robust computational framework for analyzing perturbation data, which enables the identification of drug resistance genes through differential insertion analysis. Analysis of these data revealed both previously validated and novel genes implicated in drug resistance. Interestingly, our findings reveal that mitochondrial metabolism genes NDH2 and RFK are enriched under drug pressure in P. knowlesi, and that RFK inhibition by roseoflavin shows an antagonistic effect on DHA activity, pointing to a potential mitochondrial-based resistance mechanism to artemisinin.

The thesis is organized as follows. In chapter one, we review the background on Plasmodium spp., Babesia spp., transposon mutagenesis, and current essentialomes in Apicomplexan parasites. Chapter two details the application of our mathematical models and computational approaches to P. knowlesi, revealing its essential genome and identifying novel resistance genes and synergistic mechanisms associated with the frontline antimalarial drug DHA and the investigational compound GNF179. In chapter three, we present optimized protocols for generating transposon mutagenesis libraries in B. divergens and apply our models to construct a preliminary essentialome for this organism. Chapter four provides a comparative analysis of the essentialomes across major Apicomplexans, including P. falciparum, P. berghei, P. knowlesi, and T. gondii. We close the thesis with concluding remarks and future directions. Overall, this thesis delivers a novel computational toolbox for transposon-based essentialome analysis in Apicomplexan parasites, paving the way for innovative therapeutic strategies and applications in diverse organisms.

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