Abstract
Listeria monocytogenes is a gram-positive, facultative intracellular bacterial pathogen
responsible for listeriosis, a foodborne illness that can progress to septicaemia and/or
meningitis in the immunocompromised and elderly, or septic abortion in those who are
pregnant. The ability of L. monocytogenes to spread between cells of the intestinal epithelium,
liver, or placenta is important for disease. Cell-to-cell spread begins when bacteria enter
human cells and undergo actin-based motility (ABM). ABM involves L. monocytogenes
forming polymerised actin tails which propel bacteria to the host plasma membrane. Once
there, L. monocytogenes forms membranous protrusions from the host cell. These
protrusions are recognised by neighbouring human cells and internalised, resulting in cell-to-cell
spread of bacteria. The secreted virulence factor InlC directly promotes protrusion
formation. However, how L. monocytogenes shapes the host membrane into protrusions is
poorly understood. One possibility is that L. monocytogenes promotes protrusion formation
by co-opting one or more human BAR domain-containing proteins. Consisting of the N-BAR,
F-BAR, and I-BAR subfamilies, BAR domain-containing proteins are a class of eukaryotic
protein with the ability to induce and occasionally sense membrane curvature. These proteins
reshape the plasma membrane during various cellular processes, including formation of
filopodia, lamellipodia, and cellular migration. In this project, RNA interference (RNAi) was
used to investigate the roles of BAR domain-containing proteins in the cell-to-cell spread of L.
monocytogenes in the Caco2-BBE1 line of intestinal epithelial cells. It was first determined
which BAR domain-containing proteins could be detected and depleted in these cells with
small interfering RNAs (siRNAs). Afterwards, I investigated the effect of depletion of the I-BAR
domain-containing protein IRSp53 on protrusion formation and cell-to-cell spread of wild-type
(WT) L. monocytogenes and an isogenic mutant strain with an inlC gene deletion (ΔinlC).
In three independent experiments, siRNA-mediated depletion of IRSp53 decreased the
efficiency of protrusion formation and cell-to-cell spread of both WT and ΔinlC strains of L.
monocytogenes. These results suggest that host IRSp53 promotes cell-to-cell spread of L.
monocytogenes – at least partially – by enhancing protrusion formation independently of InlC.
Experiments were also performed to assess whether IRSp53 affects cell-to-cell spread by
influencing the internalisation of protrusions. Although not definitive, results did suggest a
potential role for IRSp53 in protrusion internalisation. Future experiments should concentrate
on controlling for potential siRNA off-target effects to provide conclusive evidence for IRSp53
in protrusion formation and cell-to-cell spread. Furthermore, experiments assessing
protrusion internalisation should be optimised to conclusively determine if IRSp53
contributes to protrusion internalisation.