The conjugation-dependent antibacterial mechanisms of the interferon stimulated gene 15
Creators: name: Zhang, Yifeng
Publisher: name: University of Iowa
Year: 2025
Subjects: Actin-based motility
Autophagy
Cellular microbiology
Host pathogen interactions
Listeria monocytogenes
Post-translational modifications
public abstract

description: 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.
description: 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.
description: 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.
description: 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.