Subversion of host endocytic trafficking pathways by the Legionella pneumophila effector protein AnkX
Date
2020
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Publisher
University of Delaware
Abstract
Bacterial pathogens have developed effective strategies to promote their survival during infection of human cells. Many of these pathogens manage to evade innate immune responses, facilitating their growth and replication within the host. Pathogenic bacteria survive in the host by interacting with the host organelles and disrupting signaling pathway to obtain nutrients while evading degradation. ☐ Legionella pneumophila is an opportunistic Gram-negative bacterium that causes Legionnaires’ disease, a type of pneumonia with increased severity for individuals with a weakened immune system. L. pneumophila is phagocytosed by alveolar macrophages and remains enclosed in a membrane-derived vacuole throughout infection. Its ability to survive and replicate in this compartment is due to the type IV secretion system that facilitates the translocation of over 300 effector proteins into the host cytosol. Many of these proteins function to subvert vesicular trafficking to hijack vesicles from the endoplasmic reticulum-to-Golgi trafficking pathway to camouflage the Legionella-containing vacuole (LCV). ☐ The L. pneumophila effector protein AnkX catalyzes a novel post-translational modification of Rab1 and Rab35 through covalent addition of a phosphocholine moiety to a serine residue on the target protein. Although this biochemical function of AnkX had been previously characterized, the biological consequence of AnkX-catalyzed phosphocholination was not known prior to this work. Here, we broaden the understanding of AnkX function by presenting evidence that it disrupts recycling endocytosis and endocytic trafficking. ☐ In chapter 2, we examine intracellular localization of the effector protein AnkX and determined its impact on the recruitment of Rab GTPases to the LCV. We observed that AnkX disrupts the integrity of the endocytic recycling complex and quantified the extent to which AnkX disrupts endocytic trafficking by using the DQ Red BSA assay. ☐ In chapter 3, we determined AnkX’s subcellular localization to the plasma membrane and tubular membrane compartments by using superresolution microscopy and immunogold transmission electron microscopy. We observed that mCherry-AnkX colocalizes with Rab35, a Rab GTPase in charge to regulating the recycling of cargo back to the plasma membrane via the fast recycling pathway. Furthermore, the C-terminal region of AnkX was responsible for association with the plasma membrane. We observed that mCherry-AnkX co-localized with the major histocompatibility class I protein (MHC-I), a key immunoregulatory protein whose recycling from and back to the plasma membrane is Rab35-dependent. We report that during infection of macrophages, AnkX is responsible for the disruption of endocytic recycling of transferrin, and AnkX’s phosphocholination activity is critical for this function. ☐ In chapter 4, we examine interaction of AnkX with two potential host partners identified by a proteomic screen, PLEKHN1 and GPBP1. We report that the AnkX-mediated phosphocholination of small GTPases is important for interacting with host innate immune responses. ☐ In chapter 5, we optimize the HaloTag technology to be used along with Janelia Fluor Halo ligands to investigate the intracellular localization of L. pneumophila effector proteins in live infected cells. We focused on characterization of AnkX and other known effectors during infection. We report the ability to pre-label the protein within the bacteria in live and fixed cells before infection. We generated HT-AnkX constructs and were able to observe localization in bacteria and following translocation into host cells. ☐ Overall, our data support the hypothesis that AnkX is responsible for disrupting recycling endocytosis and helps L. pneumophila avoid degradation by interfering with phagosome maturation. Additionally, optimization of the HaloTag technology to be used in the context of macrophage infection by L. pneumophila open new avenues for studying the localization of secreted bacterial effector proteins as well as their identification of host protein targets through a number of applications including super resolution microscopy, protein-protein binding assay, and protein purification.
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Keywords
AnkX, Effector, HaloTag, Fluor, Janelia, Legionella