Date of Award
2025
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Cellular, Molecular and Biomedical Sciences
First Advisor
John Salogiannis
Abstract
Toxoplasmosis is one of the most prevalent foodborne illnesses with nearly one third of the world being infected by Toxoplasma gondii (T. gondii). These parasites have complex life cycles that are accompanied by distinct morphological and genetic changes that allow them to infect nearly all nucleated cell types across species, however the exact molecular mechanisms governing these process remains incompletely understood. Little risk exists for immunocompetent individuals infected with T. gondii, as the host immune system drives the parasites into a dormant, non-replicative state, called bradyzoites, that reside in muscle and neuronal cells. However, individuals who are immunocompromised are at a heightened risk for reactivation of the parasites into the infective and highly replicative form called tachyzoites, which can spread and divide leading to serious adverse health events including death. Additionally pregnant woman who become infected with T. gondii are at risks for parasites crossing the placenta and infecting the unborn fetus, leading to serious developmental complications, and even death. Vaccines and anti-parasitic agents exist to treat toxoplasmosis however, due to an increase in drug resistant strains as well as toxic side effects, there is a need for better treatment options. Recent studies have identified conserved bromodomain containing proteins in T. gondii (TgBDP1) that are integral for proper host cell invasion and parasite survival. These findings suggest that this protein may be good targets for drug development. Bromodomains are evolutionarily conserved proteins that bind to acetylated lysine’s on histones, thus playing a key role in transcription regulation. Additionally, bromodomains are validated targets of small molecule inhibitors to treat disease by blocking their interaction with acetylated lysine’s on histone proteins. A Gap in knowledge that is stalling the design of affective inhibitors is that the specific histone ligands targeted by TgBDP1 remain poorly characterized. Previous work in the Glass lab has outlined the specific histone ligands recognized by the homologous TgBDP1 protein in Plasmodium falciparum, PfBDP1, and we hypothesize that the bromodomain of TgBDP1 will recognize similar histone ligands. The goal of my research was to identify the binding specificity of the TgBDP1 bromodomain via isothermal calorimetry to understand the molecular mechanisms driving toxoplasmosis. Rosetta DE3 PlysS Competent E. coli cells were transformed with a Pets28+(a) vector harboring the DNA sequence of the His6x-TgBDP1-BRD (residues 383-602). The recombinant protein was isolated and subject to ITC binding experiments. We found that TgBDP1 displays overlapping binding specificity with PfBDP1 towards a subset of histone peptides carrying multiple acetylation modifications while demonstrating unique binding affinity. For example, the TgBDP1-BRD has a 4-fold stronger affinity toward the tetra-acetylated histone H4 peptide as compared to PfBDP1. In order to develop highly specific and efficacious inhibitors, outlining the acetyl histone peptides recognized by the TgBDP1 BRD is the key first step in understanding the molecular mechanisms driving epigenetic regulation and thus expression of genes pertaining to toxoplasmosis pathology. Our results are the first to outline specific histone peptides recognized by the TgBDP1-BRD.
Language
en
Number of Pages
75 p.
Recommended Citation
Lignos, James, "Elucidating The Histone Ligand Binding Specificity Of Toxoplasma Gondii Bromodomain Containing Protein 1" (2025). Graduate College Dissertations and Theses. 2144.
https://scholarworks.uvm.edu/graddis/2144