Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology
First Advisor
Kazmierczak, Barbara
Abstract
Acanthamoeba castellanii is a free-living amoeba (FLA) that causes untreatable, fatal infections of the central nervous system. FLA are ubiquitous in the environment, where they graze upon bacteria such as Pseudomonas aeruginosa. We interrogated P. aeruginosa—under evolutionary pressure to defend against amoebal grazing—as a producer of amoebicidal compounds that could serve as leads for new and urgently needed drugs to treat amoeba infections in humans. To accomplish this, we developed novel methods to quantify killing of A. castellanii trophozoites, pseudocysts, and cysts. Together, these high throughput, reproducible, and extensively validated assays provide a robust platform to measure viability of all Acanthamoeba life forms, the first in the field for pseudocysts and cysts. Using these assays, we tested the amoebicidal activity of all clinically relevant drug classes on both trophozoites and cysts. We additionally utilized these methods to demonstrate that P. aeruginosa, through complementary secreted molecules, uses a one-two punch to kill both the trophozoite and cyst forms of A. castellanii. We identified bacterial genes essential for killing by screening an arrayed P. aeruginosa transposon insertion mutant library for amoebicidal activity and surprisingly discovered that a shared pathway—fatty acid synthesis initiation—contributes to killing both trophozoites and cysts. Using an innovative biochemical pipeline, we discovered 22 candidate amoebicidal molecules. The structure of these compounds led to the identification of two clinically used drugs—an antibiotic and an antiemetic—that are lethal to A. castellanii in vitro. These studies provide essential tools to study FLA and advance our understanding of the chemical weapons P. aeruginosa uses to combat A. castellanii predation in the environment. These P. aeruginosa natural products have already led to the identification of two new, clinically available drugs with amoebicidal activity and are additional leads for novel therapeutic agents to treat devastating human infections by FLA.
Recommended Citation
Flynn, Carrie Anne, "Harnessing microbial warfare to identify products lethal to pathogenic free-living amoebae by Pseudomonas aeruginosa" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1276.
https://elischolar.library.yale.edu/gsas_dissertations/1276
