Investigating the interaction of Legionella pneumophila with host endocytic recycling pathways
Date
2022
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Legionella pneumophila is an opportunistic intracellular pathogen which can be found to infected human alveolar macrophages to cause a severe form of pneumonia known as Legionnaires’ disease. Under normal conditions bacterial pathogens are cleared from the body through engulfment by professional phagocytic cells such as macrophages, neutrophils and dendritic cells, however Legionella has evolved to manipulate these cells to form a plasma membrane derived intracellular replicative compartment. This is accomplished through secretion of 300+ bacterial effector proteins into the host cytosol through a specialized dot/icm type 4 secretion system. These effector proteins target a wide range of host process to prevent fusion with lysosome and allow efficient intracellular replication through nutrient acquisition and dampening of host immune processes. One strategy Legionella employs to avoid fusion with lysosomes is to target regulators of vesicular trafficking. Understanding how Legionella modulates these host pathways is of critical importance for identifying the mechanisms intracellular pathogens employ for survival. ☐ Rab GTPases are small proteins responsible for the coordination of vesicular trafficking and by targeting these proteins through post-translational modification Legionella recruits vesicle trafficked between the endoplasmic reticulum and the Golgi apparatus. However, the interaction with Rab GTPases regulating endocytic recycling has not been thoroughly explored. Endocytic recycling is a critical cellular process responsible for the recycling of the plasma membrane along with any receptors or molecules embedded in the membrane. ☐ In chapter 2, I pursue identification the role of endocytic recycling associated Rab GTPases Rab4, Rab11 and Rab35 during infection. I determined that distinct endocytic recycling Rab GTPases such Rab4 and Rab11are localized to LCV during early infection while Rab35 is prevented from localizing to the LCV indicating that within the recycling pathways specific Rabs are recruited based on their effect on intracellular replication. Using purified Rab4 and Rab11 incubated with Legionella cell lysate, I was able to identify several candidate effectors that may interact (directly or indirectly) with these endocytic recycling Rab GTPases. ☐ In chapter 3, I examined the impact of the specific effector AnkX on vesicular trafficking and particularly the recycling of transferrin. AnkX was previously shown to post-translationally modify host substrates through phosphocholination enzymatic activity. Fluorescence and electron microscopy showed that AnkX localized at the plasma membrane as well endosome and tubules. To identify which endocytic recycling pathway was targeted by AnkX I performed a co-localization experiment that identified AnkX as localizing to Rab35 positive endosomes within the host cell. Additionally, a defect in avoidance of phagosome maturation through acquisition of the late endosomal marker LAMP2 was observed in host cells infected with mutant strains lacking AnkX. In addition to effects on phagosomal maturation I observed a defect in endocytic recycling of transferrin during early infection that requires the phosphocholination activity of AnkX. ☐ In chapter 4 of this dissertation, I examined the interaction of another effector protein, SdeC, with Rab11 dependent endocytic recycling. SdeC belongs to a family of effectors known as the SidE family that catalyze the ubiquitination of host targets independent of the normal host ubiquitination machinery. I identified SdeC in a pulldown of Rab11 with Legionella effectors and then pursued a better understanding of the impact if SdeC on the localization of Rab11 during infection. Intriguingly I not only saw a disruption of Rab11 localization but also observed the localization of Rab11 endogenous effector Rab11-FIP1a (FIP1) appears to be clustered toward the microtubule organizing center indicating a possible defect in vesicular trafficking. To elucidate the mechanisms of this effect we performed pull-down of Rab11 that indicates SdeC is preventing the interaction between Rab11 and Rab11-FIP1a.
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Keywords
AnkX, Legionella pneumophila, Recycling, Human alveolar macrophages, Plasma membrane