Actin monomers and filaments are reorganized into bundled structures at the leading edge of cells by bacterial pathogens to aid pathogen entry into host cells. Chlamydia trachomatis infection utilizes the translocated actin-recruiting phosphoprotein (Tarp), a secretion effector protein that alters the actin cytoskeleton to assist in internalizing the bacterium. A previous study on Tarp-bundling mechanics demonstrates that Tarp can form bundles with higher flexibility than bundles induced by other actin bundling proteins such as fascin in vitro. Understanding the bending stiffness, length and assembly rate dynamics of Tarp-induced bundles in vitro is critical for understanding the invasion mechanism of C. trachomatis, therefore simulating a cellular environment by observing Tarp in the presence of molecular crowders is vital. Trimethylamine N-oxide (TMAO) is a natural organic osmolyte which regulates osmotic stress and stabilizes protein structure by favoring the folded conformational state. Due to its ability to stabilize proteins in aqueous conditions, we hypothesize that TMAO will aid Tarp in restructuring actin filaments by increasing the bundling efficiency of Tarp and forming bundles with physiologically relevant mechanical strength. In this study, we investigate how TMAO affects Tarp's bundling efficiency and the mechanical properties of Tarp induced actin bundles using total internal reflection fluorescence (TIRF) microscopy, polymerization kinetics, and biophysical analysis. We show that moderate concentrations of TMAO increase the bundling efficiency of Tarp while forming more rigid bundles compared to Tarp-bundles in dilute buffer conditions. This in vitro model allows for characterizing Tarp-induced bundles in the presence of TMAO, giving insight on how the Chlamydia trachomatis bacterium triggers cytoskeletal rearrangements, beginning a complex infectious process in living cells.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd2020-2863 |
| Date | 15 August 2023 |
| Creators | Ariza, Brianna |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Electronic Theses and Dissertations, 2020- |
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