Cytolysin-mediated alterations of Listeria-containing vacuoles in macrophages

Abstract

Listeria monocytogenes (Lm) is a Gram-positive bacterium that causes the disease Listeriosis and affects primarily those individuals who cannot mount an effective immune response. Activated macrophages are necessary for acquiring resistance and clearance of Lm. An essential component for Lm virulence is the production of a pore-forming cytolysin, listeriolysin O (LLO). LLO is necessary for the distinct intracellular life-cycle of Lm. Bacteria are internalized by the host cell into a phagosome, or vacuole, from which they escape into the cytosol, grow, and spread to neighboring cells without lysing the primary host cells. Production of LLO is essential for efficient vacuolar escape into the cytoplasm. This thesis focused on (1) the LLO-mediated perforation of the Lm vacuole and (2) the host macrophage response to the Lm damaged vacuole.

First we determined that LLO causes small perforations in membranes of newly formed Lm vacuoles that persist for variable periods before expanding into larger membrane disruptions. Release of small molecules from vacuoles through these small perforations may afford Lm protection in an otherwise harsh environment, by preventing accumulation of ions or microbicidal compounds. Consistent with this mechanism, we showed using ratiometric fluorescence microscopy that LLO increases vacuolar pH and decreases [Ca²⁺]_vac early enough to inhibit Lm vacuole fusion with lysosomes.

We next analyzed the host macrophage responses to Lm infection and discovered that a host signaling molecule, Protein Kinase C epsilon (PKCϵ), is activated upon LLO-mediated vacuole perforation in macrophages. PKCϵ was not only activated by LLO-containing endosomes, but also by endosomes after osmotic lysis, indicating a new role for PKCϵ in the recognition of damaged compartments. siRNA knockdown of PKCϵ inhibited Lm escape from vacuoles, indicating the LLO-mediated activation of PKCϵ assists in bacterial escape.

In conclusion, this thesis has investigated the role of LLO beyond its mechanical role in escape of bacteria from the vacuole. Lm were shown to use LLO to form small perforations in the membrane to slow lysosome fusion and give the bacteria time to escape the vacuole. Lm were also shown to use LLO to exploit a host mechanism to aide in vacuolar escape.