Abstract
Listeria monocytogenes is a gram-positive, food-borne, facultative intracellular pathogen. Infection with Listeria can result in a disease known as listeriosis and in individuals who are immunocompromised, elderly or pregnant, Listeria can cause invasive disease, including meningitis, encephalitis, septicaemia, or fetal infections. Invasive infections are thought to involve the ability of Listeria to traverse the intestinal cell barrier and spread via the blood to the liver, central nervous system, or placenta. The ability of Listeria to cause invasive disease therefore involves crossing of three tight barriers: the intestinal barrier, the blood-brain barrier and the fetoplacental barrier. Listeria has evolved a mechanism to disseminate through host tissues. The cell-to-cell spread of Listeria is essential for virulence and consists of the processes of actin-based motility, formation of protrusions, internalisation of protrusions and resolution of protrusions. Prior research has led to a detailed understanding of the molecular mechanisms of actin-based motility. By contrast, mechanisms of protrusion formation, internalisation and resolution are less well understood.
This project investigated the role of the host GTPase Dynamin 2 in the cell-to-cell spread of Listeria. Dynamin 2 may interact with other proteins or utilise its scission-based activity alone to play a role in either the formation, internalisation or resolution of Listeria protrusions. During my thesis work, I made three key findings. First, I found that Dynamin 2 is recruited to Listeria protrusions, suggesting that bacteria manipulate the function of this host GTPase. Secondly, I found that depletion of Dynamin 2 by RNAi restores normal cell-to-cell spread of a mutant strain of Listeria that is deleted for the inlC gene (∆inlC). This result indicates that Dynamin 2 limits spread of Listeria in the absence of Internalin C (InlC). Wild-type Listeria produces InlC and somehow removes the inhibition of spread that would otherwise be caused by Dynamin 2. Third, I found that Dynamin 2 associates with an amino-terminal region in Tuba, a human scaffolding protein previously found to control the cell-to-cell spread of Listeria. Interestingly, RNAi-mediated depletion of Tuba restores normal spread to the ∆inlC Listeria mutant strain, which is the same phenotype that I found is caused by depletion of Dynamin 2. My results on cell-to-cell spread therefore suggest that Dynamin 2 might act together with Tuba to control cell-to-cell spread of Listeria.
In addition to the results described, I performed experiments to assess the role of Dynamin 2 in protrusion formation, which is a key step in the cell-to-cell spread of Listeria. Results using RNAi-mediated depletion of Dynamin 2 were difficult to interpret, since I did not observe the expected defect in protrusion formation by the ∆inlC mutant Listeria strain. Experiments involving Dynasore, a chemical inhibitor of Dynamin 2 and other Dynamin family GTPase members, showed that this inhibitor increased the number of protrusions made by wild-type Listeria. These results raise the possibility that Dynamin 2 may either limit the production of protrusions and/or promote the internalisation of protrusions by neighbouring host cells. In summary, this thesis presents new information on the potential mechanisms involved in Listeria protrusion formation, internalisation and resolution.