The effect of osmolytes on actin bundling and bundle mechanics by Chlamydia trachomatis Tarp

Brianna Ariza¹, Bryan Demosthene^1,2, Kaylyn Scanlon^2,3, Travis Jewett^2,3, Ellen H. Kang^1,4,5

1NanoScience Technology Center, University of Central Florida, United States
2Burnett School of Biomedical Science, University of Central Florida, United States
3Division of Immunity and Pathogenesis, College of Medicine, University of Central Florida, United States
4Department of Physics, University of Central Florida, United States
5Department of Material Science and Engineering, University of Central Florida, United States

Actin filaments are often reorganized into bundled structures at the leading edge of cells by bacterial pathogens to aid pathogen entry into host cells. Chlamydia trachomatis infection requires the translocated actin-recruiting phosphoprotein (Tarp), a secretion effector protein that alters the actin cytoskeleton to assist in internalizing the bacterium. Previous work showed Tarp’s ability to form bundles from monomers and pre-formed filaments that exhibited higher flexibility than those formed by common actin bundling proteins. Trimethylamine N-oxide (TMAO) is a natural organic osmolyte which regulates osmotic stress and stabilizes cellular structure. 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. In this study, we investigate how TMAO affects bundling efficiency and the mechanical properties of actin bundles formed by Tarp using total internal reflection fluorescence (TIRF) microscopy and biophysical analysis in an in vitro model. Our data suggests 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 model will help us better understand how Tarp, in the presence of TMAO, forms cytoskeletal rearrangements that contribute to the efficient internalization of C. trachomatis in host cells.