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.
No MeSH data available.
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Mentions: To determine which signaling pathway was responsible for the Listeria-induced ISG15 transcript and to help identify the cellular compartment in which bacteria are sensed, we made use of Listeria strains that are impaired at different stages of infection. Incubation of cells with L. innocua, a non-pathogenic Listeria species which cannot invade cells, did not induce ISG15 induction, demonstrating that external pathogen recognition receptors were not involved (Figure 2A). We then used a strain of L. innocua that expresses Internalin B (InlB), a L. monocytogenes virulence factor that mediates entry into nonphagocytic cells (Dramsi et al., 1995). These bacteria enter the cell and are entrapped in a membrane-bound phagosome, but lack the required virulence factors to escape from it. This strain was also unable to induce an ISG15 signal, suggesting that the pathogen recognition receptors that survey the phagosome are not sufficient for ISG15 induction (Figure 2A). Listeria's hemolysin, listeriolysin O (LLO) is an extremely potent virulence factor, which triggers vacuolar escape of the bacterium as well as a plethora of changes in the host cell (Hamon et al., 2012). Strikingly, we found that the Δhly mutant was able to potently induce ISG15 (Figure 2A). Thus, LLO is not necessary for ISG15 induction. However, in several human epithelial cell lines the mutant that lacks LLO (Δhly) can still escape into the cytosol (Portnoy et al., 1988; Marquis et al., 1995). Listeria expresses two phospholipases that can compensate for the lack of LLO in the Δhly mutant in order to free the bacterium from the phagosome in human epithelial cells (Marquis et al., 1995; Smith et al., 1995). To assess whether Listeria trapped in the phagosome could induce ISG15, we constructed a triple mutant (lacking LLO, PLCA, and PLCB) of Listeria. This mutant is unable to escape the phagosome of human epithelial cells (Figure 2—figure supplement 1). Single mutants (either in PLCA or LLO), which escape into the cytosol, induce a strong ISG15 signal relative to non-infected cells (Figure 2B). In contrast, the triple mutant that is confined to the phagosome does not induce ISG15 (Figure 2B). We thus conclude that only cytoplasmic bacteria induce ISG15. In fact, the only other mutant to induce less ISG15 production was the ΔactA mutant (Figure 2A). This mutant is unable to spread from cell to cell and cannot escape autophagic recognition, degradation, and lysis (Gouin et al., 2005; Yoshikawa et al., 2009). As a result bacterial load is much lower compared to wild-type bacteria, providing an explanation for the reduced ISG15 signal (Figure 2A). Taken together, our results reveal that ISG15 induction stems from cytosolic bacteria.10.7554/eLife.06848.005Figure 2.Listeria induces ISG15 via the cytosolic surveillance pathway (CSP) which senses bacterial DNA.
No MeSH data available.