Keywords
Site-directed mutagenesis; Chlamydia trachomatis; TARP effector; Tyrosine phosphorylation; Type III secretion system
Abstract
Chlamydia trachomatis is the leading cause of preventable blindness and the most common sexually transmitted bacterial infection (STI) worldwide. As an obligate intracellular bacterium, it relies entirely on the host’s cellular environment for survival and replication. Chlamydia utilizes a collection of bacterial proteins called effectors to hijack a multitude of host functions to promote chlamydial survival. A key chlamydial effector in this process is the Translocated Actin Recruiting Phosphoprotein (Tarp), which promotes bacterial entry by reorganizing the host cell’s actin cytoskeleton through C-terminal actin binding domains. While the Tarp effector is not essential for bacterial growth, a C. trachomatis deletion mutant (ΔtarP) is attenuated in both in vitro tissue culture and in vivo infection models. In addition to actin binding domains, Tarp contains six tandem tyrosine-rich repeats, each of which can be phosphorylated by host tyrosine kinases. The role of Tarp phosphorylation in infection is not well understood but appears to be distinct from its control of the actin cytoskeleton. This study aims to create a non-phosphorylatable version of Tarp by introducing point mutations at the tyrosine residues within these repeats. We hypothesize that mutating these residues will prevent phosphorylation and help us better understand Tarp’s role in Chlamydia pathogenesis. Using site-directed mutagenesis, we designed primers to introduce tyrosine-to-phenylalanine substitutions at all six repeat sites. The resulting mutant constructs were expressed in E. coli, with protein expression confirmed by western blotting. Tarp’s phosphorylated state will be evaluated using anti-phosphotyrosine antibodies. We anticipate that non-phosphorylatable Tarp mutants will ultimately help to reveal the functional consequences of phosphorylation on Chlamydia-host interactions, providing insights into its infection mechanisms and identifying potential therapeutic targets.
Thesis Completion Year
2025
Thesis Completion Semester
Spring
Thesis Chair
Jewett, Travis
College
College of Medicine
Department
Biomedical Sciences
Thesis Discipline
Biomedical Sciences
Language
English
Access Status
Campus Access
Length of Campus Access
5 years
Campus Location
Orlando (Main) Campus
STARS Citation
Wang, Victor, "Site-Directed Mutagenesis of the Chlamydia Tarp Effector" (2025). Honors Undergraduate Theses. 283.
https://stars.library.ucf.edu/hut2024/283
Rights Statement
