CHARACTERIZATION OF NOVEL LEGIONELLA PNEUMOPHILA PHOSPHOINOSITIDE BINDING EFFECTOR PROTEINS
Date
2024-05
Authors
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Publisher
University of Delaware
Abstract
Legionella pneumophila is a Gram-negative, intracellular pathogen that infects
alveolar macrophages and causes Legionnaires’ disease, a debilitating pneumonia. L.
pneumophila manipulates host cell vesicular trafficking pathways to help construct a
self-contained replicative niche known as the Legionella Containing Vacuole (LCV).
Upon establishment of this LCV, L. pneumophila replicates freely inside of this
vacuole without detection from the host cell until eventual host cell death allowing
further infection of nearby macrophages. L. pneumophila employs its Icm/Dot type IV
secretion system to translocate over 300 effector proteins into the host cell. Many of
these effector proteins help construct the LCV and manipulate host vesicular pathways
through the binding to phosphoinositides (PIs). PIs serve as crucial two-tailed
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phospholipids that can be phosphorylated in key positions to control vesicular
trafficking. Our goal is to identify and characterize PI-binding effector proteins as
little is known about these proteins beyond their binding to PIs. Towards this goal, I
have two objectives. The first objective was to create HaloTagged fusion constructs of
three effector proteins hypothesized to bind to PIs. These fusion constructs will allow
us to not only confirm PI-binding, but also discover when and where these proteins are
secreted and if they bind to any other organelles that contribute to the establishment of
the LCV. The second objective was to identify the specific region or residues of a
PIbinding protein that allowed this binding to occur. This will help to elucidate the
unique and specific properties of L. pneumophila PI-binding proteins as opposed to
eukaryotic PI-binding proteins. Validating and characterizing the effector proteins that
bind to PIs is crucial to not only understanding how L. pneumophila evades host cell
detection, but also developing agents to specifically target and inhibit pathogenic
proteins that bind to PIs to allow host cells to degrade L. pneumophila.