Honors Theses and Capstones

Date Completed

Spring 2026

Abstract

Toxoplasma gondii (T. gondii) is an intracellular parasite chronically infecting approximately 31% of the global human population (Sengupta, 2025). While infections are typically asymptomatic in the majority of individuals, acute toxoplasmosis poses a genuine risk to nearly 25 million immunocompromised patients or developing fetuses in the United States at any given time (CDC, 2025; Strid, 2025). Currently, there is no medical technology capable of clearing the dormant cyst stage of the T. gondii life cycle. Anti-parasitic drugs can reduce symptoms of the deadly acute infection like parasite-induced fever, flu-like sickness, and nonspecific malaise, however they are limited in their capacity to suppress dormant bradyzoite cysts (Cleveland Clinic, 2026).

The pathogenicity of T. gondii depends on its ability to efficiently invade host cells, a process regulated in part by the ApiAP2 family of transcription factors (Zarringhalam, 2023). In 2016, one member of this AP2 family, AP2X-7, was identified as essential for parasite viability through a genome-wide CRISPR screen; AP2X-7 was also implicated in regulating invasion-associated gene expression (Sidik, 2016). AP2 factors are unique to T. gondii with no human analogs, therefore, this transcription factor family poses a gold standard for drug development with the goal of eliminating dormant bradyzoite cysts in humans and livestock tissues. Here, we investigate the role of AP2X-7 in host cell invasion using a conditional knockdown system employing a mini auxin-inducible degron (mAID) tag, which targets the fully functional AP2X-7 protein for degradation upon addition of indole-3-acetic acid (IAA).

We assessed invasion efficiency using a dual-color immunofluorescence assay (IFA) in addition to fluorescent microscopy to distinguish extracellular from intracellular parasites on confluent human foreskin fibroblast monolayers. A substantial portion of this project was dedicated to troubleshooting persistent background fluorescence that complicated image acquisition and analysis throughout the larger study. Variables including wash steps, bovine serum albumin (BSA) concentration, Triton X-100 detergent concentration, and primary antibody dilutions were systematically tested across multiple assay iterations in an effort to resolve this issue. While background fluorescence was never fully eliminated, significant progress was achieved and two biological replicates yielding interpretable invasion data were ultimately obtained.

Despite these technical challenges, results from both biological replicates demonstrated a striking reduction in invasion efficiency upon AP2X-7 knockdown, with IAA-treated knockdown parasites displaying greater than 80% reduction in invasion efficiency relative to the wild type controls. Plaque assays previously performed by the lab corroborated these findings, with no viable plaques forming in the knockdown condition following IAA treatment, suggesting that AP2X-7 depletion severely compromises parasite fitness. Future work will focus on obtaining additional biological replicates under further optimized imaging conditions, as well as Western blotting to assess secretion of the invasion-associated proteins following AP2X-7 knockdown. Genomic approaches including RNA sequencing and CUT&Tag chromatin profiling will be employed to map the broader AP2X-7 transcriptional network and identify downstream targets that may mediate its role in invasion, respectively. Together, the results presented here provide preliminary but compelling evidence that AP2X-7 plays an essential role in T. gondii host cell invasion; further characterization of its function may inform the development of novel anti-cystic therapeutics.

Document Type

Undergraduate Thesis

First Advisor

Vicki Jeffers

Second Advisor

Matt Waterman

College or School

COLSA

Department or Program

Molecular, Cellular, and Biomedical Sciences

Degree Name

Bachelor of Science

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