Exploring the impact of ISG15 and enhanced ISGylation on host responses and immune cell function during infection with Francisella novicida

Ellen Upton

doi:10.25820/etd.007684

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Dissertation

Exploring the impact of ISG15 and enhanced ISGylation on host responses and immune cell function during infection with Francisella novicida

Ellen Upton

University of Iowa

Doctor of Philosophy (PhD), University of Iowa

Summer 2024

DOI: 10.25820/etd.007684

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SpreadsheetS1 Table- List of Di-glyicne modified peptides following F. novicida infection Embargoed Access, Embargo ends: 08/30/2026

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Supplemental Table 233.11 kB

SpreadsheetS2 Table- Gene ontology analysis of di-glycine enriched proteins Embargoed Access, Embargo ends: 08/30/2026

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Supplemental Table 31.79 MB

SpreadsheetS3 Table- List of whole lung input proteins ranked by significance in comparing uningected to infected samples via t-test Embargoed Access, Embargo ends: 08/30/2026

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Supplemental Table 414.88 kB

SpreadsheetS4 Table- List of dy-glycine sites on SAMHD1 identified via LC-MS/MS Embargoed Access, Embargo ends: 08/30/2026

Abstract

The Interferon-Stimulated Gene 15 (ISG15) is a ubiquitin-like protein produced following viral and bacterial infections. ISG15 covalently modifies host and pathogenic proteins in a process termed ISGylation. Yet, the consequences of ISGylation on protein fate and function remain to be determined. My thesis work utilized models of absent (Isg15-/-) and enhanced ISGylation (Usp18C61A/C61A) to assess the function of ISG15 and ISGylation on cellular response and immune cell function following bacterial pneumonia caused by Francisella novicida. I found that infection with F. novicida induces robust ISG15 expression and ISGylation but does not affect bacterial burden in the lung in vivo. Yet, enhanced ISGylation leads to decreased immunopathology and respiratory distress relative to Isg15-/- animals. Lung histology from infected mice with enhanced ISGylation showed fewer necropurulent lesions and granulomas relative to wildtype and ISG15-deficient mice. In order to understand the mechanism underlying this phenotype, I mapped the ISGylome of F. novicida-infected mouse lungs using label-free quantitative mass spectrometry and identified enrichment in ISGylation of proteins involved in innate immune responses and cell-intrinsic anti-bacterial clearance pathways. I then coupled fluorescence-activated cell sorting to quantitative mass-spectrometry to enumerate myeloid cell recruitment and characterize the proteins expressed by alveolar macrophages and neutrophils following infection. While the difference in absolute number of innate immune cells recruited by genotype was not significant, enhanced ISGylation led to increased proteomic plasticity and activation in both neutrophils and alveolar macrophages. I also showed that ISG15 plays a role in the metabolism of the naturally occurring antibacterial fatty acid monoester Glycerol Monolaurate (GML). I found that GML potently impeded F. novicida growth and survival in vitro, which was previously unknown. To appraise the metabolic response to infection, I used gas-chromatography mass-spectrometry to survey the production of metabolites, and surprisingly, observed intracellular GML production following F. novicida infection. Notably, ISG15 was necessary for increased GML levels induced by bacterial infection, and enhanced ISG15 conjugation correlated with GML levels following serum starvation. These studies together indicate unique mechanisms of action for ISG15 in response to bacterial respiratory infection with F. novicida. These observations further highlight the importance of ISG15 regulation in response to cell stress and infection and propose new advantageous effects of targeting the cellular response by ISG15. Altogether this work adds to the growing literature addressing the role of ISG15 in response to bacterial infections, focusing on innate immune responses and metabolic changes.

Biochemistry

Immunology

Francisella

ISG15

Details

Title: Subtitle: Exploring the impact of ISG15 and enhanced ISGylation on host responses and immune cell function during infection with Francisella novicida
Creators: Ellen Upton
Contributors: Lilliana Radoshevich (Advisor)
Kevin Legge (Advisor)
John Harty (Committee Member)
Balaji Manicassamy (Committee Member)
Josalyn Cho (Committee Member)
Resource Type: Dissertation
Degree Awarded: Doctor of Philosophy (PhD), University of Iowa
Degree in: Biomedical Science (Immunology)
Date degree season: Summer 2024
Publisher: University of Iowa
DOI: 10.25820/etd.007684
Number of pages: xiii, 149 pages
Language: English
Date submitted: 07/22/2024
Description illustrations: Illustrations, tables, graphs, charts
Description bibliographic: Includes bibliographical references (pages 129-149).
Public Abstract (ETD): Our cells have many ways of managing infections, including producing proteins designed to fight off foreign pathogens. My thesis work focused on the effects of one such protein, ISG15, following bacterial infection. ISG15 can bind to and modify hundreds of other molecules that are involved in both normal cell functions and immune responses in a process called ISGylation, but little is known about how this modification changes the function of these targets. While most research has been focused on inhibiting ISGylation, my work looked rather at the role of preventing the removal of ISG15, leading to enhanced ISGylation. My project utilized Francisella tularensis, the bacterial pathogen responsible for the disease tularemia, which, while rare, can result in severe lung infections and pneumonia if as few as ten bacteria are inhaled. I found that ISG15 is produced following infection with Francisella and that enhanced ISGylation led to less severe respiratory symptoms. To better understand the mechanism by which ISGylation protects from infection I identified all the ISG15-modified targets in the lung. I found many proteins involved in immune signaling pathways that were highly modified by ISG15 and I showed that immune cells with enhanced ISGylation were more activated and responsive following infection. I also demonstrated a link between ISG15 and the intracellular production of an antibacterial lipid molecule following infection with Francisella. Together, these data suggest that enhanced ISGylation plays a protective role following bacterial pneumonia, indicating that targeting this pathway could prove a beneficial therapeutic in bacterial respiratory diseases.
Academic Unit: Biomedical Science Program
Record Identifier: 9984698249102771

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