Enhanced ISGylation reduces respiratory distress following Francisella novicida infection

The Interferon-Stimulated Gene 15 (ISG15) is a ubiquitin-like protein induced by viral and bacterial infection. ISG15 covalently modifies host and pathogenic proteins in a process called ISGylation. Yet, the consequences of ISGylation on protein fate and function remain to be determined. Here we sought to assess whether ISGylation would be protective following bacterial pneumonia caused by Francisella novicida. We found that infection with F. novicida induces ISGylation both in vitro in macrophages and in vivo in the lung, liver, and spleen of mice infected intranasally. Surprisingly, ISG15 and ISGylation do not affect bacterial burden in the lung in vivo , but in a model of enhanced ISGylation ( usp18C61A/C61A ) mice have decreased respiratory distress relative to Isg15-/- animals. In order to understand the mechanism which underlies this phenotype, we mapped the ISGylome of F. novicida- infected mouse lungs using label-free quantitative mass spectrometry and identified enrichment in ISGylation of proteins involved in the innate immune response and cytosolic nucleotide signaling. We validated ISGylation of the sterile alpha motif and HD-containing protein 1 (SAMHD1) via immunoprecipitation. SAMHD1 depletes cytosolic dinucleotide stores critical for retroviral replication but it is unknown how its activity could affect bacterial infection. Structure-function analysis indicates that ISG15 modification sites in usp18C61A/C61A mice could prevent SAMHD1 dimerization and therefore abrogate function. Accordingly, deletion of SAMHD1 in fibroblasts with enhanced ISGylation reduces bacterial load. Taken together, unchecked ISGylation plays a protective role in F. novicida infection in vivo through improved respiratory function. Thus, inhibiting USP18 may be a promising therapeutic strategy for both viral and bacterial pneumonia. Author summary Francisella tularensis is a bacterial pathogen responsible for the disease tularemia, which can result in severe respiratory infection if as few as ten bacteria are inhaled. Our cells have many ways of managing infections, including the production of proteins designed to fight off foreign pathogens. One protein produced following infection is the interferon-stimulated gene 15 (ISG15). ISG15 is a ubiquitin-like molecule, meaning that it can be chemically attached to other proteins. When bound ISG15 changes the stability, interacting partners, or function of its target in a process termed ISGylation. Here we show that ISG15 is produced following infection with Francisella. We found that enhanced ISGylation led to less severe respiratory symptoms. To better understand the mechanism by which ISGylation protects from infection we identified the ISG15-modified proteins in the lung using mass-spectrometry-based proteomics. We found protein targets that are involved in the control of immune signaling pathways including sterile alpha motif and HD-containing protein 1 (SAMHD1) which, when deleted in cells with enhanced ISGylation, leads to better bacterial clearance. Together, we show that enhanced ISGylation plays a protective role following bacterial pneumonia, indicating that targeting this pathway could prove a beneficial therapeutic in both bacterial and viral respiratory diseases. ### Competing Interest Statement The authors have declared no competing interest.