Author ORCID Identifier

https://orcid.org/0000-0001-8563-7593

Year of Publication

2021

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department/School/Program

Microbiology, Immunology, and Molecular Genetics

First Advisor

Dr. Kenneth A. Fields

Abstract

Chlamydia trachomatis is the causative agent of the most reported bacterial sexually transmitted disease in the United States. The establishment of an intracellular niche within mucosal epithelium is sufficient to drive immunopathology and disease sequela. As obligate intracellular bacteria, Chlamydia spp. have evolved numerous mechanisms for establishing an intracellular growth environment. The type III secretion system (T3SS) delivers effector proteins to the host cytosol and is essential for C. trachomatis invasion and development. The effectors TmeA, TmeB, and TarP, are all secreted during C. trachomatis invasion. TarP and TmeA have been associated with manipulation of actin networks and are essential for normal invasion levels. The functions of TarP are well established, whereas TmeA is less well characterized, and the role of TmeB is entirely unknown.

Recent progress in elucidating and characterizing these effectors has been bolstered by the development of techniques enabling basic genetic tractability of C. trachomatis L2. Florescence-reported allelic exchange mutagenesis (FRAEM) couples chromosomal gene deletion with the insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP); however, FRAEM-mediated deletion of Chlamydia trachomatis tmeA produces a polar effect on the downstream gene, tmeB, and negatively impacts its expression. Our laboratory has adapted FRAEM technology by employing a gfp-bla cassette flanked by loxP sites. Conditional expression of Cre recombinase in C. trachomatis tmeA null strain containing a floxed cassette resulted in the deletion of the marker cassette and restoration of tmeB expression.

The work presented here utilizes the novel marker-less C. trachomatis deletion mutant to determine the importance of TmeA and TmeB during development and identify their eukaryotic host targets. We leverage chlamydial genetics and proximity labeling to provide evidence that TmeA directly targets host N-WASP to promote Arp2/3-dependent actin polymerization. Our work also shows that TmeA and TarP influence separate yet synergistic pathways to accomplish chlamydial entry, while TmeB functions antagonistically to TmeA and inhibits Arp2/3-mediated actin polymerization.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2021.223

Funding Information

This study was supported by the National Institute of Allergy and Infectious Diseases F31 Grant (F31AI147417) in 2019-2021.

Available for download on Saturday, June 17, 2023

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