Identification and characterization of Francisella tularensis proteins required for invasion and escape into non-phagocytic epithelial cells

The potential bioterror agent Francisella tularensis subspecies tularensis (F. tularensis) is an intracellular human pathogen and the causative agent of tularemia. As an invasive pathogen, Francisella invades host cells; occupies and escapes membrane bound vacuoles; replicates in the host cytosol; then initiates their release to infect other cells. Tularemia begins when bacteria invade the phagocytic and non-phagocytic cells of the host. While non-phagocytic cell colonization contributes significantly to disease, the process is poorly understood. In this thesis, I identify and characterize key proteins in the invasion and vacuole escape stages of the non-phagocytic cell infection process. In chapter 2, I evaluated Francisella in vitro cell culture infection models present in the literature side-by-side with the model our lab developed using the murine surrogate of F. tularensis, F. tularensis subspecies novicida (F. novicida) and murine cultured hepatocytes. I found compared to other models, our model most accurately reflected colonization levels seen in vivo. In chapter 3 and chapter 4, I investigated bacterial proteins involved in invasion and vacuole escape. I screened a F. novicida transposon mutant library using our infection model for microbes deficient in bacterial replication. Using bioinformatics, I searched for invasion-deficient transposon mutants inactivated in Francisella surface proteins to screen for proteins that could interact with the host cell surface. I then tested their ability to cause tularemia-induced mortality in mice. I showed that bacteria inactivated in two genes caused no disease in mice and protected mice as live-vaccines against a wild-type F. novicida challenge. One gene I identified as Francisella infectivity potentiator A (FipA). I presented evidence that FipA enables bacteria to escape the vacuole using both florescence and electron microscopy. The next gene I identified, characterized, and named Francisella virulence factor A (FvfA). I demonstrated that FvfA is a bacterial surface-exposed ligand that exploits host clathrin-mediated endocytosis for entry using functional assays and dot blots. Lastly, I crystallized FvfA and compared FvfA to its structural homolog, E. coli RcsF. Taken together, I described two virulence factors, FipA and FvfA, that are critical for the initial stages of the Francisella non-phagocytic cell infection process and consequently, tularemia-effected death.