This item is non-discoverable
Investigation of a Legionella pneumophila effector protein that manipulates host autophagy
Date
2022
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
Many intracellular bacteria engage with the host cell’s autophagy pathway. The autophagy pathway is designed to isolate and degrade misfolded proteins, damaged organelles, or foreign material. Autophagy can be an obstacle for intracellular bacteria, as it sequesters them into a vacuole before delivering them to the lysosome for degradation (xenophagy). Or it can be a useful tool for the bacteria to acquire nutrients from the host. While some intracellular pathogens actively evade and inhibit the autophagy pathway, others induce it or upregulate it. ☐ The facultative intracellular, Gram-negative pathogen Legionella pneumophila is a particularly interesting case-study in the relationship between bacterium intracellular lifecycle and autophagy. L. pneumophila infection can develop into Pontiac Fever, or the more serious Legionnaires’ disease. Upon human inhalation, the bacterium typically infects the alveolar macrophages that inhabit the lung. Within the macrophage, the bacterium can release over 350 effector proteins using a Type 4 Secretion System. These effector proteins have an array of functions within the host, including the development of a nutrient-rich replication vacuole that escapes the phagosomal-degradation pathway designed to dispose of intracellular bacteria. Within this undetectable vacuole, the bacterium will replicate until the host has been effectively colonized. ☐ Several of the bacterium’s effector proteins have been characterized as proteins that function to specifically inhibit the autophagy pathway. However, as more effector proteins are being investigated, a number of proteins have been identified that stimulate autophagy. One of those recently identified effector proteins is Lpg2411. Although first identified in 2009, interest in the effector did not peak until 2019, when it was identified in a screen for effectors that bind phosphoinositides, special lipids imbedded in eukaryotic cell membranes. It was discovered that Lpg2411 binds specifically to the phosphoinositide species indicative of the autophagosome membrane, the compartment that transports substrate for the autophagy pathway. Furthermore, an autophagic flux assay indicated that Lpg2411 increases autophagy within the host cell. ☐ In this study, Lpg2411 was explored as an L. pneumophila effector protein that upregulates the autophagy pathway. In chapter 2, an anti-Lpg2411 antibody was developed that would allow for the detection of endogenous protein translocated from wildtype L. pneumophila strains. The antibody was determined to be specific to Lpg2411 and highly sensitive to the protein. In chapter 3, the antibody was tested for its ability to detect Lpg2411 ex vivo. The antibody was purified and optimized for use in immunofluorescence. With the newfound antibody, Lpg2411 protein signal was tracked throughout an infection to determine when L. pneumophila expresses the protein. Protein localization was then categorized across a series of infections in order to characterize the subcellular distribution of the protein. ☐ In chapter 4, Lpg2411 co-localization and interaction with host proteins was investigated. Lpg2411 co-localized with a variety of host proteins involved in the autophagy pathway. The protein also co-localized with a few unexpected proteins that are involved in separate protein degradation pathways. Lpg2411 interaction was investigated with two of the proteins it localized with. Lpg2411 was determined to associate with HDAC6, a protein fundamental in the formation of the aggresome, a collection of aggregated proteins. Lpg2411 displayed no association with LC3B, an integral membrane protein of the autophagosome, despite showing extensive localization with the protein and others associated with the autophagosome. Together, this suggests that Lpg2411 could be impacting protein degradation in more ways than just targeting the autophagy pathway. Overall, this research has furthered the characterization of Lpg2411 and expanded our understanding on how L. pneumophila may seek to impact the autophagy pathway to serve the bacteria’s needs throughout the course of infection.
Description
Keywords
Aggrephagy, Autophagy, Effector protein, Legionella pneumophila, Legionnaires' disease, Lpg2411