The identification and characterization of bacterial and host epithelial factors involved in the pathogenesis of invasive Francisella tularensis and extracellular enteropathogenic E. coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) and Francisella tularensis possess a toolkit of virulence factors that allow them to adapt to the host environment and prevail over the body’s natural defenses in different ways. For diarrheagenic EPEC, a type III secretion system is used to inject virulence factors directly into the host cell allowing the extracellular microbe to co-opt key host processes and generate motile ‘pedestal’-shaped structures at the site of intimate bacterial-host contact. Because many of the same principles that drive the biogenesis and movement of EPEC pedestals are paralleled at the leading edge of migrating cells, I used EPEC pedestals as a model of the leading edge and developed a strategy to identify pedestal proteins and tease out their biological functions. Using mass spectrometry-based proteomics of concentrated pedestal preparations, I identified 17 highly abundant novel proteins as well as 11 previously known pedestal proteins. One of the identified molecules, nexilin, was characterized in depth. Using EPEC and Listeria monocytogenes as bacterial models for actin-based dynamics, I revealed that nexilin is concentrated towards the rear of pedestals and Listeria comet tails when these actin-rich structures become motile. The use of siRNA-mediated knockdowns further suggested that depletion of nexilin results in unusually thin and short filamentous comet tails. Another pathogen that can colonize non-phagocytic cells is F. tularensis—the etiological agent of Tularemia. Here, I examined the internalization process and intracellular fate of Francisella in hepatocytes. To study the strategy that F. tularensis uses to invade epithelial cells I developed in vitro infection models and used those models to uncover clathrin, its associated endocytic components and cholesterol as key molecules needed for F. tularensis internalization. Finally, I elucidated the role of two Francisella virulence factors (IglC, PdpA) and showed that both PdpA and IglC are needed for the efficient invasion and intracellular growth of F. tularensis. Taken together, I have identified multiple novel targets co-opted by the extracellular EPEC and the intracellular pathogens L. monocytogenes and F. tularensis that play a central role in their pathogenesis such as the actin associated protein nexilin and clathrin endocytic components clathrin, epsin1, and Eps15.