Dissertation
The conjugation-dependent antibacterial mechanisms of the interferon stimulated gene 15
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Summer 2023
DOI: 10.25820/etd.007079
Abstract
ISG15 is an interferon-induced ubiquitin-like protein that has been implicated as a central player in antimicrobial immunity. Upon induction, ISG15 can covalently modify host and pathogen-derived proteins (ISGylation), noncovalently interact with intracellular proteins, or act as an immunomodulatory cytokine outside the cell. Historically, the role of ISG15 has been mainly studied in the context of viral infections. It was shown recently that ISG15 also protects against bacterial infections in a conjugation-dependent manner. However, the underlying mechanisms and molecular consequences of ISG15 and ISGylation are poorly defined. This is largely due to the limited knowledge of ISG15 substrates and the lack of identification of the precise sites of modifications.
To investigate the functional consequences of ISGylation during bacterial infection, we used Listeria monocytogenes infection as the model, which robustly induces ISG15 and its conjugation machinery. We devised a genetic strategy and mapped the endogenous in vivo ISGylation following L. monocytogenes infection with label-free quantitative proteomics. We identified ISGylated targets enriched in cellular metabolic processes, including upstream regulators and critical enzymes of autophagy. Additionally, our proteomic screen revealed proteins of actin polymerization machinery, hijacked by L. monocytogenes for actin-based motility, as ISG15 conjugates. Computational analysis of substrate structures suggested that ISGylation preferentially occurs at the catalytic sites of enzymes and/or protein-protein interfaces.
While overexpression of ISG15 alone has been demonstrated to restrict bacterial replication effectively, enhanced ISGylation, achieved by selective inactivation of the ISG15-specific isopeptidase activity, failed to confer host resistance against L. monocytogenes infection. The further study validated that ISGylation of the mammalian target of rapamycin (mTOR), a key negative regulator of autophagy, led to the de-repression of autophagy. Animals and cells with enhanced ISGylation have increased basal and infection-induced autophagy. Furthermore, ISGylation of the Arp2/3 complex, the major actin nucleator, slowed actin filament formation and stiffened Arp2/3-dependent structures including cortical actin and lamellipodia. In wildtype conditions, conjugation is properly controlled, and ISGylation-induced increases in autophagy and restrictions of Arp2/3-mediated actin polymerization serve as host defense strategies to inhibit bacterial growth as well as pathogen-mediated actin-based dissemination. However, in conditions of enhanced ISGylation, irreversible modification of the Arp2/3 complex is exploited by L. monocytogenes, resulting in increased host mortality due to boosted bacterial cell-to-cell spread.
Taken together, these findings established a novel role of ISGylation to modulate autophagy and actin dynamics following infection through direct modification of a subset of metabolic enzymes and actin binding proteins. In addition, our observations necessitate the proper regulation of ISGylation for efficacious disease control and tolerance in antibacterial immunity. Ultimately, our discovery links the host innate immune response to cellular metabolic homeostasis and cytoskeletal remodeling with therapeutic implications for microbial infections.
Details
- Title: Subtitle
- The conjugation-dependent antibacterial mechanisms of the interferon stimulated gene 15
- Creators
- Yifeng Zhang
- Contributors
- Lilliana C. Radoshevich (Advisor)Craig D. Ellermeier (Committee Member)Dominique H. Limoli (Committee Member)Christopher S. Stipp (Committee Member)Mary M. Weber (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Microbiology
- Date degree season
- Summer 2023
- DOI
- 10.25820/etd.007079
- Publisher
- University of Iowa
- Number of pages
- xxiii, 196 pages
- Copyright
- Copyright 2023 Yifeng Zhang
- Language
- English
- Date submitted
- 06/12/2023
- Description illustrations
- Illustrations, tables, graphs, charts
- Description bibliographic
- Includes bibliographical references (pages 162-196).
- Public Abstract (ETD)
Listeria monocytogenes is a ubiquitous bacterium with a highly adaptable lifestyle that can be commonly found in the soil and decaying vegetation. It is an opportunistic foodborne pathogen causing listeriosis, a rare but remarkably fatal infection, particularly in high-risk populations. The high lethality of L. monocytogenes infection is due to the pathogen’s ability to survive various harsh defense strategies from the mammalian host. Interferon stimulated gene 15 (ISG15) is one of those effectors massively produced by the host when L. monocytogenes is detected in the cytosol. Following induction, ISG15 can covalently modify intracellular proteins through a regulated enzymatic cascade. ISG15 confers host protection against L. monocytogenes in a conjugation-dependent manner. However, the consequence of ISGylation during L. monocytogenes infection is poorly defined.
My thesis focuses on understanding the conjugation-dependent mechanisms by which ISG15 protects the host from L. monocytogenes. We have identified endogenous ISG15 substrates following L. monocytogenes infection. My work has built on those findings and validated that ISG15 modifies key regulators of autophagy as well as critical proteins of the actin polymerization machinery. When properly regulated, this serves as a host defense strategy to restrict bacterial growth and dissemination. However, dysregulated ISGylation of actin polymerization machinery is exploited by L. monocytogenes, resulting in increased host mortality. Taken together, my work contributes to our mechanistic understanding of how ISGylation facilitates bacterial clearance and inhibits pathogen dissemination. Additionally, our findings emphasize the importance of proper regulation of ISGylation in host control of bacterial infections.
- Academic Unit
- Microbiology and Immunology
- Record Identifier
- 9984454435802771
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