ISG15 counteracts Listeria monocytogenes infection.
Bottom Line: ISG15 is an interferon-stimulated, linear di-ubiquitin-like protein, with anti-viral activity.We made use of stable isotope labeling in tissue culture (SILAC) to identify ISGylated proteins that could be responsible for the protective effect.Strikingly, infection or overexpression of ISG15 leads to ISGylation of ER and Golgi proteins, which correlates with increased secretion of cytokines known to counteract infection.
Affiliation: Unité, Institut Pasteur, Paris, France.
ISG15 is an interferon-stimulated, linear di-ubiquitin-like protein, with anti-viral activity. The role of ISG15 during bacterial infection remains elusive. We show that ISG15 expression in nonphagocytic cells is dramatically induced upon Listeria infection. Surprisingly this induction can be type I interferon independent and depends on the cytosolic surveillance pathway, which senses bacterial DNA and signals through STING, TBK1, IRF3 and IRF7. Most importantly, we observed that ISG15 expression restricts Listeria infection in vitro and in vivo. We made use of stable isotope labeling in tissue culture (SILAC) to identify ISGylated proteins that could be responsible for the protective effect. Strikingly, infection or overexpression of ISG15 leads to ISGylation of ER and Golgi proteins, which correlates with increased secretion of cytokines known to counteract infection. Together, our data reveal a previously uncharacterized ISG15-dependent restriction of Listeria infection, reinforcing the view that ISG15 is a key component of the innate immune response.
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Mentions: We next assessed whether ISG15 has a functional effect on infection. We created a retroviral construct that expresses an epitope-tagged version of ISG15 (3XFlag-6His-ISG15). We then infected cells that stably express 3XFlag-6His-ISG15 with Listeria. As a control, cells were retrovirally transduced with pBabe puro empty vector. For the same multiplicity of infection (MOI) after 3 hr of infection, stable overexpression of ISG15 resulted in 50% fewer cytosolic bacteria as compared to control cells (Figure 3A). We then assessed bacterial uptake by differentiating between the bacteria that are inside the cell or those that remain on the surface (inside-out staining) and did not observe a difference between control and ISG15-overexpressing cells for invasion (Figure 3—figure supplement 1A). During a time course of infection in these cells at 7 and 12 hr, there were still 50% fewer bacteria, and by 24 hr, the levels of bacteria had equalized between the two cell lines (Figure 3—figure supplement 1B). This time course suggests that ISG15 does not impact bacterial replication as an active bacterial clearance mechanism would (Figure 3—figure supplement 1B). We then knocked down ISG15 during infection. This increased bacterial load by nearly twofold after 15 hr (Figure 3B, Figure 3—figure supplement 2). These data strongly suggest that ISG15 plays a role in protection against Listeria infection following uptake. To further explore this phenotype in primary cells, we isolated mouse embryonic fibroblasts (MEFs) from wild-type and Isg15−/− embryos. We infected these cells with Listeria and we observed a fivefold increase in bacterial load in Isg15−/− MEFs compared to wild-type MEFs for the same MOI (Figure 3C,D). Interestingly, Isg15−/− MEFs are not susceptible to other intracellular pathogens such as Shigella flexneri and Salmonella typhimurium, and in HeLa cells only Staphylococcus aureus is able to induce as much ISG15 as Listeria (Figure 3—figure supplement 1C,D). Indeed, S. flexneri and S. typhimurium induce very little ISG15. We next examined whether ISG15 could also play a role during Listeria infection in vivo by assessing the susceptibility of Isg15−/− animals to the pathogen during systemic infection (Osiak et al., 2005). ISG15-deficient mice exhibited a significant increase in bacterial load compared to wild-type animals in both the spleen and liver after 72 hr of systemic sub-lethal Listeria infection (Figure 3E,F). Taken together, these data demonstrate that ISG15 restricts Listeria infection both in vitro and in vivo.10.7554/eLife.06848.008Figure 3.ISG15 protects against Listeria infection in vitro and in vivo.
No MeSH data available.