PROGRAM | Biological Sciences

By: Rebecca Noll Chair: Ramona Neunuebel

ABSTRACT

Intracellular bacterial pathogens are a major concern for public health worldwide. Under normal circumstances, professional phagocytes (macrophages, neutrophils, and dendritic cells) clear infection by ingesting microbes in a plasma membrane-derived vacuole (or phagosome) that gradually acquires microbicidal properties through coordinated fusion events with specific compartments of the endocytic pathway. This process, known as phagosome maturation, is a major target of intracellular bacterial pathogens that replicate within a vacuole. Shielded by multiple layers of membranes, intracellular pathogens become inaccessible to antibiotics and are vulnerable only when venturing out of their protective environment to find a new host. The vacuole plays a critical role for bacterial survival, providing both protection and nutrition 1. However, despite the crucial role of the replication vacuole, a clear understanding of the mechanisms involved is currently lacking.

Here, we investigate the intracellular pathogen Legionella pneumophila, a Gram-negative bacterium that infects macrophages in the human lung where it arrests phagosome maturation and establishes a replication-permissive compartment. Though in nature, this bacterium infects amoebae2-4, human infection can develop following inhalation of contaminated aerosolized water droplets5,6. L. pneumophila causes hospital- and community-acquired pneumonia known as Legionnaires’ disease5,7,8 and when contracted in a hospital setting this disease has a fatality rate as high as 25% despite antibiotic treatment.

In the lung, alveolar macrophages engulf L. pneumophila by phagocytosis, but are unable to degrade it through phagosome maturation. Instead, L. pneumophila reroutes the newly formed phagosome, remodeling the cytosolic façade of the membrane to resemble the endoplasmic reticulum. Remarkably, as soon as 5 minutes post-infection, vesicles derived from the endoplasmic reticulum surround the Legionella-containing vacuole (LCV) indicating that the cytosolic surface of the vacuolar membrane is undergoing remodeling9. This process is dependent on a specialized secretion system that serves as a conduit for translocation of L. pneumophila proteins directly into the host cell. These proteins, termed effectors, mimic functionalities of host proteins in order to disrupt cellular processes and facilitate survival. For example, the phosphoinositide labeling system utilized by the host to label organelles has been hijacked by effectors to alter phosphoinositide composition on various compartments, serve as anchors for membrane localization, and/or disrupt normal trafficking events regulated by these lipids 10. In this dissertation, we address known phosphoinositide-targeting effectors and explore effectors newly identified to harbor this function.

In chapter 2, we analyzed a phosphoinositide-binding effector, AnkX, to understand how it is contributing to survival. We investigated the potential host interaction partners of AnkX through a novel protein array called a Nucleic Acid Programmable Protein Array (NAPPA). Of the 10,000 human proteins tested, we identified eight potential interaction partners of AnkX and aimed our focus on the top candidate, PLEKHN1. We confirmed AnkX and PLEKHN1 as interaction partners via multiple independent in vitro pull-down, co-immunoprecipitation, and cell-based assays. Additionally, we found that the central region of AnkX, harboring ankyrin repeats, co-localized with PLEKHN1 and may be responsible for the interaction with this host protein. We identified that an additional host protein, HuR, a published interaction partner of PLEKHN1, also co-localizes with AnkX. HuR is an RNA-binding protein involved in upregulation of cytochrome c translation and could potentially reveal additional functions of AnkX.

In chapters 3 and 4, we investigated the function of two effectors newly identified to have phosphoinositide-binding capabilities. In chapter 3, we characterized the effector Lpg2411. We found that this effector binds specifically to PI(3)P on autophagosomes. It associates with an autophagic receptor as well as ubiquitinated proteins at sites where PI(3)P is present. We identified that during infection, Lpg2411 accumulates in the infected cell at late time points where it stimulates the host autophagic process. This discovery is intriguing since L. pneumophila effectors have mainly been found to inhibit this process. Then, in chapter 4, we sought to characterize Lpg0405. We found that this effector also has specificity for PI(3)P, using the lipid as an anchor for membrane localization. Unlike Lpg2411, Lpg0405 lacks specificity for a particular membrane compartment, and rather localizes to many different PI(3)P-harboring organelles. We found that Lpg0405 and Lpg2411 associate with each other and Lpg0405 also possesses the ability to bind ubiquitinated proteins. How these proteins functionally relate to each other has yet to be uncovered, but will add a fascinating layer of regulation between effectors. Overall, our data provide advancements to the Legionella field that increase knowledge of how phosphoinositide-binding effectors manipulate the infected host cell.

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