Guanylate-binding proteins as mediators of inflammasome activation in response to bacterial infection
Creators: name: Feng, Shouya
Publisher: name: The Australian National University
Year: 2023

description: Guanylate-binding proteins (GBPs) are a family of interferon-inducible GTPases expressed by mammalian cells to promote immune defences against pathogens. GBPs can be recruited to bacterial membranes, pathogen-containing vacuoles or viral replication complex and mediate the disruption of these structures. Following the rupture of intracellular pathogens, microbial ligands are liberated and sensed by innate immune sensors such as absent in melanoma 2 (AIM2)-like receptors. Certain immune sensors can form cytosolic signalling complexes, known as inflammasomes. Inflammasome activation controls and eradicates intracellular pathogens by driving the secretion of pro-inflammatory cytokines, IL-1b and IL-18, and pyroptosis, a form of inflammatory cell death mediated by the pore-forming protein gasdermin D. GBPs can promote inflammasome activation in response to specific pathogens. This PhD thesis investigates how each member of the GBP family discriminates microbes and cooperates with one another to drive pathogen-selective inflammasome responses. To comprehensively study the individual contributions of GBPs in inflammasome activation, the CRISPR-Cas9 gene editing technology was used to generate mice lacking each of the five GBPs on the chromosome 3 locus and four GBPs on the chromosome 5 locus. Bone marrow-derived macrophages (BMDMs) were generated from wild-type and knockout mice and infected with pathogens such as Moraxella catarrhalis, a major cause of otitis media and chronic obstructive pulmonary disease and Francisella tularensis subsp. novicida, a cause of tularemia. The level of inflammasome activation, bacterial proliferation and the subcellular location of GBPs were assessed following infection. To further investigate how GBPs control bacterial burden in the host, mice were infected with M. catarrhalis or F. novicida and analysed for bacterial load and cytokine levels. Recombinant mouse GBPs were also purified to assess their antimicrobial activity against different pathogens. To understand the mechanisms of microbial killing by GBPs, putative antimicrobial regions were identified using bioinformatic tools and synthesised for testing on clinically important pathogens. Further, multiple imaging techniques, such as correlative light and electron microscopy, were performed to gain insights into the antimicrobial mechanisms of GBPs. I found that GBP1, GBP2, GBP3 and GBP5 contribute to M. catarrhalis-induced Caspase-11-NLRP3 inflammasome activation and F. novicida-induced AIM2 inflammasome activation. GBP1 and GBP3 were not required for inflammasome activation by other bacterial pathogens including Listeria monocytogenes, Salmonella enterica serovar Typhimurium, Escherichia coli and Citrobacter rodentium. The selectivity of GBPs towards specific pathogens suggests that GBPs may have pattern-recognition capabilities. Indeed, using immunofluorescence staining, I found that endogenous GBP1, GBP2, GBP3 and GBP5, the same GBPs required for inflammasome activation, co-localised with M. catarrhalis and F. novicida in BMDMs, whereas GBP7 did not. Moreover, I observed increased bacterial burden in cells and in mouse lacking GBPs, suggesting restriction of bacterial replication by GBPs. Recombinantly expressed full-length GBP1 also caused selective antimicrobial killing of F. novicida. I further investigated the mechanism of how GBP1 kills bacteria. I identified a region within the N-terminal domain of GBP1, which contains charged and hydrophobic amino acids that can bind to and facilitate direct killing of bacteria. This thesis highlights a crucial role for GBPs in the host defence against two bacteria, through a mechanism dependent on inflammasome signalling. These results reveal that GBPs may discriminate between pathogens, confer activation of innate immunity, and provide a host-inspired roadmap for the design of highly selective antimicrobial peptides for use against emerging and re-emerging pathogens.