Date on Master's Thesis/Doctoral Dissertation
12-2024
Document Type
Doctoral Dissertation
Degree Name
Ph. D.
Department
Microbiology and Immunology
Degree Program
Microbiology and Immunology, PhD
Committee Chair
Abu Kwaik, Yousef
Committee Member
Uriarte, Silvia
Committee Member
Lawrenz, Matthew
Committee Member
Lamont, Richard
Committee Member
Mitchell, Thomas
Author's Keywords
Legionella pneumophila; legionella longbeachae; effector protein; LamA; neutrophils; inflammation
Abstract
Legionella species are Gram-negative facultative intracellular bacterial pathogens commonly found in freshwater or soil environments where they survive and replicate within their natural protozoan hosts. Humans serve as an accidental host for Legionella species after inhalation of contaminated aerosolized water droplets or soil particles enabling the bacteria to enter the lungs, infect alveolar macrophages, and result in severe pneumonia. Evasion of degradation and proliferation within alveolar macrophages by Legionella species and manifestation of “Legionnaires’ disease” is dependent on the Dot/Icm type IV secretion system (T4SS). The T4SS apparatus is a needle-like channel for translocation of over ~350 effector proteins, depending on the Legionella species. Many currently identified effector proteins harbor eukaryotic-like domains and motifs acquired through inter-kingdom horizontal gene transfer with diverse eukaryotic protozoan hosts. Thus, the co-evolution of Legionella species among diverse protozoan and amoeba hosts has led to these pathogens acquiring a repertoire of tools promoting survival and replication within eukaryotic cells that can modulate the immune response of accidental mammalian hosts. Interestingly, majority of effectors identified in Legionella are not required for survival or proliferation within mammalian macrophages due to functional redundancy of these proteins. In addition to alveolar macrophages, circulating neutrophils are the first line of innate immune defense that acts to restrict most bacterial pathogens. However, a dysregulation of the neutrophil pro-inflammatory response can have devastating consequences on the host and contribute to disease progression. In response to infection, neutrophils kill invading pathogens through a variety of microbicidal activities such as: NETosis, degranulation, mediating fusion of microbicidal granules to pathogen-containing phagosomes, and generation of extracellular or intracellular reactive oxygen species (ROS) by the phagocyte NADPH oxidase complex. Human neutrophils rapidly kill L. pneumophila in response to the T4SS. Specifically, the T4SS-translocated Legionella amylase (LamA) effector of L. pneumophila catalyzes degradation of neutrophil glycogen (glycogenolysis) and promotes a state of cytosolic hyper-glucose within infected neutrophils. LamA is shown to be an amoeba host-adapted effector necessary for subverting amoeba encystation to maintain an intracellular environment permissive to L. pneumophila replication. However, LamA demonstrates a paradoxical upregulation of pro-inflammatory activities within human macrophages and neutrophils in response to glycogenolysis and subsequent cytosolic hyper-glucose, resulting in partial restriction of L. pneumophila within the human host. In response to LamA-mediated glycogenolysis and cytosolic hyper-glucose, human neutrophils undergo immuno-metabolic reprogramming and activation of microbicidal activities for rapid restriction L. pneumophila by generation of ROS via formation of the catalytically active NADPH oxidase complex and fusion of granules to the bacterial phagosome. Additionally, evaluation of pro-inflammatory cytokine response by neutrophils to viable and killed L. pneumophila suggests bacterial effectors involved in modulating immune responses in host cells are incapable of counter-acting rapid degradation of the bacteria promoted by LamA-mediated glycogenolysis. Thus, L. pneumophila exhibits a paradoxical and counter-evolutionary effect of the amoeba-adapted LamA effector in human neutrophils, resulting in rapid restriction and clearance of the pathogen. In contrast to L. pneumophila, human neutrophils fail to upregulate key microbicidal processes or restrict L. longbeachae, the second most prevalent cause of Legionnaires’ disease. Additionally, neutrophils infected with L. longbeachae fail to undergo a robust proinflammatory response, such as degranulation and IL-8 production. Here, I identify three strategies employed by L. longbeachae for evading restriction by neutrophils and inhibiting neutrophil microbicidal responses to other bacteria co-inhabiting the same cell. First, L. longbeachae excludes the cytosolic and membrane-bound subunits of the phagocyte NADPH oxidase complex from its phagosomal membrane. Consequently, infected neutrophils fail to generate robust ROS in response to L. longbeachae. Second, L. longbeachae impedes fusion of azurophilic granules to its phagosome and the phagosomes of bacteria co-inhabiting the same cell. Third, L. longbeachae inhibits recruitment of cytosolic NADPH oxidase components to co-infecting bacterial phagosomes and protects co-inhabiting bacteria from degradation by ROS. Collectively, L. longbeachae utilizes multiple strategies for evading restriction by human neutrophils. Altogether, L. longbeachae and L. pneumophila demonstrate how divergent evolution between species affects host-pathogen interactions and results in differential responses of human neutrophils to these environmental pathogens.
Recommended Citation
Hanford, Hannah E., "Differential responses of human neutrophils to legionella species." (2024). Electronic Theses and Dissertations. Paper 4491.
Retrieved from https://ir.library.louisville.edu/etd/4491
