PROGRAM | Biological Sciences

By: Summer Hackenburg Chair: Ramona Neunuebel

ABSTRACT

Intracellular pathogens have evolved unique ways to survive within their eukaryotic hosts. Understanding the mechanisms behind this survival will be crucial for the generation of new drug targets not only to combat the pathogen itself but for generation of new antibiotics targeting unique pathways to help circumvent antibiotic resistance.  Throughout this dissertation the model organism Legionella pneumophila, hereafter Legionella, is used to investigate the molecular mechanism behind its pathogenesis. The largest area of investigation for Legionella pneumophila is the over 300 effector proteins that are secreted into the host cell during infection.  The effector proteins that have currently been studied target multiple cellular processes by targeting host proteins and often share more similarities with eukaryotic proteins than bacterial. It is through this mimicry that Legionella uses its effector proteins to control important cellular processes such as actin organization and immune response regulation for the promotion of bacterial survival.

Legionella pneumophila is a gram-negative bacterium that is naturally found in freshwater and soil environments where it shares this niche with protozoans. By natural consequence of the amoeba attempting to use Legionella as a nutrient source, Legionella overrides amoebas’ phagocytic nature and established a replicative niche where it survives and replicates. Through this coevolution with amoeba, Legionella quickly became an opportunistic pathogen. Upon inhalation of contaminated water droplets, Legionella targets alveolar human lung macrophages. Unfortunately for humans but beneficial for Legionella, the degradative pathways that amoeba use are evolutionarily very similar to that of the human lung macrophage. To this end, Legionella already possess the necessary tools to subvert the innate immune system and avoid degradation within the professional phagocytes of the lung. Upon phagocytosis of an avirulent bacterium, an initial phagosome is formed. This phagosome will then proceed down a pathway known as phagosomal maturation where the initial phagosome will be a part of multiple fusion events with endocytic compartments. The first fusion event that happens is the phagosome with endosomes, beginning the acidification process of the phagosomes. After that the fusion events continue with late endosomes and eventually the lysosome. The fusion with the lysosomes generates what is known as the phagolysosome and in this compartment the bacterium will be degraded. When Legionella is phagocytosed, the bacterium still starts its journey with the phagosome, but the fate of this phagosome is not the same as previously described. Instead of fusion events occurring between the phagosome and endocytic vesicles, Legionella prevents the fusion with those compartments and recruits vesicles traveling between the ER and the Golgi to expand its own vacuole known as the Legionella-containing vacuole (LCV). To accomplish this, Legionella secretes over 300 effector proteins into the host cell that can manipulate multiple cellular processes by manipulating host proteins.  This dissertation is devoted to the functional characterization of effector protein Lem8.

Chapters 2 and 3 of this dissertation describe in detail the characterization of Lem8’s role during Legionella infection. At first it was hypothesized that Lem8 was a deubiquitinating enzyme due to its localization with ubiquitin in transfected and infected cells. However, this hypothesis was disproved due to Lem8s inability to cleave ubiquitin linkages or interact with a suicide probe generated to interact with and inhibit deubiquitinating proteins. The rest of Chapter 2 and 3 focus on determining the interactions between Lem8 and two groups of host proteins, the 14-3-3 family and Rho GTPases (Rac1, RhoA, and Cdc42). To this end, it was determined that Lem8 can directly interact with six isoforms of 14-3-3 and this interaction persists during infection in a Lem8 dependent manner. Additionally, Lem8 also directly interacts with Rac1, RhoA, and Cdc42 and Rac1 can be seen localizing to the LCV in a Lem8 dependent manner. Although Lem8 is predicted to be a protease, there is currently no data indicating that there is proteolytic functioning occurring to any 14-3-3 isoform or any Rho GTPase. However, it was determined that Lem8, 14-3-3, and Rac1 form a stable complex. The current hypothesis is that Lem8 manipulates these host proteins to prevent the LCV from fusing with endocytic compartments during infection.

Chapter 4 of this dissertation is dedicated to development of a new methodology for tracking effector proteins during live cell microscopy. HaloTag is a new reporter protein that can be fused to a protein of interest, in this case effector proteins and then can be visualized using a ligand instead of antibodies. The potential behind this method is the use of the HaloLigand that forms a covalent bond with the HaloTag reporter protein. This generates a more stable interact between HaloTag and the ligand and increases the specificity of the signal seen. In addition, HaloLigands have the potential to be more than 9x brighter than fluorescence produced by traditional antibody. This means that proteins of low abundance, such as effector proteins, can be detected without increasing artifacts and background signal that would be a concern when using a traditional staining method. In chapter 4, a gateway vector containing the HaloTag reporter protein was successfully generated and verified for full length protein production. Using a well characterized effector protein SidM, and Lem8 that was characterized in the previous two chapters, the localization of Halo-Tagged effector proteins was verified in multiple cell lines.

Through the work in Chapter 2-4 of this dissertation there are two key advancements made to the Legionella field. The first being that Lem8 is the first effector protein ever reported to directly interact with 14-3-3 isoforms and Rho GTPases. This advancement is critical as 14-3-3 proteins and Rho GTPases are devastatingly important to the normal function of cells. The second advancement is an advancement for visualization in the Legionella field. Being able to visualize Legionella effectors as they are being secreted from the bacterium is a methodology that is not possible without the use of new technology like HaloTag. Here I have shown that it is possible to label the protein before it is even secreted and can then follow it during infection.

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