Listeria monocytogenes pathogenesis: Part I: The role of flagella mediated motility Part II: The function of the metalloprotease propeptide

Abstract

Listeria monocytogenes is a food borne, intracellular bacterial pathogen. Successful infection requires completion of two steps: penetration of the intestinal epithelium and escape from the phagocytic vacuole. This dissertation examines the role of flagella mediated motility in host cell invasion, and the function of the metalloprotease (Mpl) propeptide in mediating the maturation of a bacterial phospholipase required for efficient escape from the phagocytic vacuole.

We examined the contribution of flagella to L. monocytogenes pathogenesis. We observed that flagella mediated motility enhance the bacterial rate of invasion. To determine if flagella are adhesins, we performed adhesion and invasion assays with flagellated motile and non-motile bacteria, and non-flagellated bacteria. Flagellated but non-motile bacteria did not adhere to or invade human epithelial cells more efficiently than non-flagellated bacteria. These results indicate that flagella do not function as adhesins to host cells. Instead, motility is important for host cell invasion. Moreover, in vivo motile bacteria out competed non-motile bacteria in the colonization of the mouse intestines and liver at early time points after oral infection, suggesting that flagella-mediated motility enhances L. monocytogenes infectivity soon after bacterial ingestion.

Mpl regulates the activity and compartmentalization of a bacterial phospholipase C (PC-PLC). Mpl is secreted as an inactive proprotein. In related proteases, the propeptide can serve as a folding catalyst (either in cis or in trans), influence protein compartmentalization, participate in intracellular trafficking, or decrease folding kinetics. We investigated the role of the Mpl propeptide by monitoring the behavior of Mpl synthesized in absence of its propeptide (Mpl?pro) and of two Mpl mutants with unstable propeptides. All three mutants mediated PC-PLC activation in vitro but were not functional in infected cells. This defect was not rescued by providing the propeptide in trans to the mpl?pro mutant. We also determined that PC-PLC co-purified with wild-type Mpl, Mpl?pro, and the Mpl propeptide indicating that the propeptide is not required for Mpl / PC-PLC interaction. However, the mutant Mpl species were aberrantly secreted in the cytosol of infected cells. These data indicate that the propeptide of Mpl maintains Mpl bacteria-associated, and that localization is essential to Mpl function during infection.