The role of endocytic components during the bacterial infections of non-phagocytic cells

Invasive and extracellular bacterial pathogens have developed strategies to usurp the endocytic machinery of their host cells. In my research, I studied the mechanism employed by two bacterial pathogens, Enteropathogenic E. coli (EPEC), and Francisella tularensis to usurp the endocytic machinery of the host cells. EPEC were found to control the internalization machinery for non-invasive functions, as they use clathrin to generate actin-rich structures that protrude from the cell surface known as pedestals, To elucidate the relationship between EPEC and the host cell clathrin-mediated endocytic machinery, I used a combination of experimental approaches, including fluorescent microscopy, immunoblotting and RNA interference, to examine the influence of an array of clathrin-associated accessory proteins on pedestal formation. Several key clathrin-associated components including CD2AP, Eps15 and epsin were localized at EPEC pedestals and were found to be necessary for pedestal formation, revealing a unique and differential strategy used by this extracellular pathogen to control clathrin endocytic machinery. Ubiquitylation is a post-translational modification process that regulates a broad spectrum of cellular signalling processes in eukaryotic cells, including endocytosis. Consequently, I also investigated the host cell ubiquitylation status during EPEC infections in vitro. I found that the overall abundance of ubiquitin-conjugated proteins was severely diminished. Suppression of ubiquitylation was attributed to an E. coli adherence factor (EAF) plasmid found in EPEC strain 2348/69 and was independent of the EPEC chromosome-encoded locus of enterocyte of effacement (LEE) and non-LEE (NLE) effectors. Finally, I studied the invasion mechanisms used by the intracellular pathogen Francisella tularensis. F. tularensis are highly infectious bacterial pathogens that cause tularemia. Using a F. tularensis surrogate (F. novicida) as a model, I demonstrated that this pathogen exploits both clathrin- and cholesterol- mediated endocytosis to gain entry into non-phagocytic cells. In conclusion, I have conducted a comprehensive analysis on how two different bacterial pathogens induce host subcellular alterations and demonstrated how similar endocytic proteins can function in very different manners when controlled by bacterial pathogens.