ORCID ID

0000-0001-8547-1765

Graduation Date

Spring 5-8-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Programs

Immunology, Pathology & Infectious Disease

First Advisor

Paul H. Davis

Abstract

Toxoplasma gondii is a globally-distributed obligate intracellular parasite and the causative agent of toxoplasmosis in humans, a biphasic disease often culminating in lifelong chronic infection. Though often mild, toxoplasmosis can lead to severe clinical presentation in immunocompromised patients and developing fetuses. Current therapeutic options are severely limited, with a combinatorial pyrimethamine/sulfadiazine regimen acting as the current standard of care for toxoplasmosis; however, both of these compounds can elicit serious adverse reactions in patients, and, additionally, are only effective against the acute stage of infection. To address these limitations, screenings of several analogs derived from the marine natural product marinopyrrole A identified a series of small-molecule inhibitors against T. gondii tachyzoites: RL002, RL003, and RL125. The most potent molecule, RL003, showed an in vitro IC50 of 0.096 µM and corresponding low toxicity to non-tumorigenic immortalized human cell lines, with a selectivity of ≥20X for parasites over any susceptible host cell line. RL003 also potently inhibited encysted bradyzoites in vitro, with an IC50 of 0.245 µM. Further screening of RL003 demonstrated additional activity against other pathogens, including a number of gram-positive and gram-negative bacteria, such as Mycobacterium bovis BCG, Naegleria fowleri, and Acanthamoeba castellanii. Forward genetic screening of mutagenized compound-resistant T. gondii clones suggested a role of parasite-specific cGMP-dependent protein kinase (PKG) as a mediator of RL003 activity, with subsequent observations of impaired invasion, motility, and egress supporting this finding. Identification of apoptosis-like morphological changes by scanning electron microscopy (SEM) followed by differential expression analysis by both RNA-seq and RT-qPCR provided evidence for downstream upregulation of programmed cell death pathways in compound-treated parasites. Taken together, these data support the role of RL003 as a novel and pathogen-selective antiparasitic compound.

Available for download on Saturday, April 29, 2023

Included in

Parasitology Commons

Share

COinS