Date of Award

2020

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Sunny Shin

Abstract

Host recognition of intracellular bacterial pathogens results in the formation of a multiprotein complex termed the inflammasome, which leads to the recruitment and activation of inflammatory caspases. These caspases promote IL-1 family cytokine release and pyroptosis, a lytic form of cell death, which are critical for anti-bacterial defense. In mice, interferon-gamma (IFN-) is a potent inducer of the both the noncanonical and canonical inflammasomes. Specifically, a family of IFN-inducible GTPases known as guanylate binding proteins (GBPs) promote inflammasome responses to a variety of bacteria in mice. The functions of the mouse GBPs include stimulating the rupture of pathogen-containing vacuoles and bacteriolysis of cytosolic bacteria in order to release pathogen-derived products, such lipopolysaccharide (LPS), into the cytosol for downstream host sensing and inflammasome activation. In contrast to mice, which possess 11 GBPs, humans have 7 GBPs and their role in inflammasome activation in human macrophages is poorly understood. Here, we use Legionella pneumophila, a vacuolar intracellular gram-negative bacterium, to elucidate the functions of human GBPs on inflammasome responses in macrophages. We determined that human GBP1 is essential for maximal inflammasome responses to L. pneumophila as well as is important for disrupting the L. pneumophila-containing vacuole (LCV) in order to make this vacuolar bacterium more accessible for cytosolic sensing. In addition, LPS can possess different acylation states within the same or different species of gram-negative bacteria. The noncanonical inflammasome mediates inflammatory responses to intracellular LPS and is comprised of caspase-11 in mice, and the two putative orthologs in humans, caspase-4 and caspase-5. While tetra-acylated LPS variants evade caspase-11 detection, caspase-4 was found to be activated by a tetra-acylated LPS variant. However, it is still unclear whether caspase-4 and caspase-5 recognize different LPS variants and whether their activation is promoted by IFN-. Here, we use primary or THP-1-derived macrophages and CRISPR/Cas9 technology to test human noncanonical inflammasome responses to LPS variants from different bacteria and whether IFN- and human GBPs promote these responses in macrophages. Our findings elucidate aspects of human innate immune response to gram-negative bacterial pathogens and may provide insight into developing therapeutics to prevent gram-negative sepsis.

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