The Role of TolC in Francisella tularensis Virulence

Francisella tularensis is a Gram-negative, facultative intracellular pathogen and the causative agent of tularemia. F. tularensis is a Tier 1 agent of bioterrorism that is highly lethal via the pulmonary route of infection. F. tularensis invades host cells, escapes the phagosome, and replicates in the cytosol prior to being released upon host cell death. The molecular mechanisms behind the virulence of F. tularensis are largely unknown. Among the described virulence factors of F. tularensis is a functional type I secretion system (T1SS). The T1SS is important for the secretion of virulence factors from the bacterial cytoplasm to the extracellular environment. The F. tularensis T1SS consists of a periplasm-spanning outer membrane protein, TolC, which interacts with inner membrane adapter and transport proteins to form a contiguous channel. TolC has been shown to be important for the virulence of multiple F. tularensis subspecies. Infection of host cells with a F. tularensis live vaccine strain (LVS) ∆ tolC mutant leads to increased caspase-3 activation and host cell death compared to cells infected with the wildtype LVS. The LVS Δ tolC mutant also elicits increased secretion of proinflammatory cytokines from infected cells when compared to cells infected with the wildtype LVS. The work described here investigates the temporal induction of host cell apoptosis during LVS infection and the role that TolC plays in modulating this process. I show that the LVS delays activation of the intrinsic apoptotic pathway to allow for bacterial replication during infection and that TolC is necessary for this inhibition. Chromosomal deletion of tolC in the highly virulent, human pathogenic Schu S4 strain showed that TolC is necessary for virulence and inhibiting cell death during infection, demonstrating that TolC function is conserved across F. tularensis subspecies. Finally, I investigated the efficacy of the LVS ∆ tolC mutant strain as a live vaccine against tularemia. My results suggest that the ∆ tolC mutant strain may be a safer, more effective tularemia vaccine compared to the parental LVS. Taken together, my work characterizes the role of TolC as a major F. tularensis virulence factor aimed at suppressing innate immune responses during infection. | 139 pages