You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: ISG15 counteracts Listeria monocytogenes infection}
Article:
Abstract:
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.
Introduction:
Listeria monocytogenes is a food-borne pathogen that can cause enteric infections. In addition, in immunocompromised individuals it can cross the blood–brain barrier and in pregnant women the feto-placental barrier potentially leading to cases of meningitis and septicemia. To be fully virulent, Listeria must evade macrophage killing, enter and replicate in epithelial cells and spread from cell to cell. Towards these aims Listeria subverts a number of normal host cell functions in order to promote its own replication and dissemination through a plethora of well-characterized virulence factors( Cossart and Lebreton, 2014). Conversely, Listeria induces a rapid and sterilizing CD8+ T cell-mediated adaptive immune response that has been extensively characterized( Lara-Tejero and Pamer, 2004; Pamer, 2004). A more recent area of investigation has been the innate immune response to the pathogen( Stavru et al., 2011). Since Listeria is able to survive and replicate in the cytosol, several groups have sought to elucidate how bacteria are sensed within macrophages and more recently within nonphagocytic cells. Once Listeria has escaped from the phagosome, its multidrug efflux pumps secrete small molecules leading to activation of an IRF3-dependent cytosolic surveillance pathway( CSP), resulting in type I interferon activation( Crimmins et al., 2008). One of these small molecules, cyclic-di-AMP, is sufficient to activate interferon β production in macrophages( Woodward et al., 2010). In nonphagocytic cells, type I interferon induction seems to emanate from sensing of triphosphorylated RNA molecules via a RIG-I and MAVS-dependent pathway( Abdullah et al., 2012; Hagmann et al., 2013). Type I interferon production subsequently leads to autocrine or paracrine activation of interferon-stimulated genes( ISGs). We have recently shown that Listeria also activates the type III interferon pathway( Lebreton et al., 2011; Bierne et al., 2012), a pathway which was discovered much later than type I interferon( Kotenko et al., 2003; Sheppard et al., 2003). The type III interferon receptor has a more limited tissue expression pattern than the receptor for type I interferon but activates a signaling pathway similar to that of the type I interferon receptor. Several laboratories including ours have recently contributed to the understanding of the type III interferon-dependent response to intracellular viral and bacterial infections. Strikingly, the type III response occurs via peroxisomal MAVS( Dixit et al., 2010; Odendall et al., 2014). The role of one particular ISG, ISG15, during bacterial infection remains elusive. ISG15 is a linear di-ubiquitin-like molecule( ubl) that is conserved from zebrafish to human; however, it is much less well characterized than other ubls( Bogunovic et al., 2013). It can conjugate to over 300 cellular proteins and can also function as a cytokine to induce interferon-γ production in peripheral blood mononuclear cells( D'Cunha et al., 1996; Giannakopoulos et al., 2005; Zhao et al., 2005). Since Listeria, as other pathogenic bacteria, often targets post-translational modifications during infection( Bonazzi et al., 2008; Ribet and Cossart, 2010; Ribet et al., 2010), we were interested in investigating the interplay between the interferon-stimulated ubl ISG15 and Listeria. ISG15 plays an important role in the innate immune response to viruses. Isg15 expression becomes rapidly upregulated, and the protein is subsequently conjugated to cellular and/or viral targets following type I interferon induction( Zhang and Zhang, 2011). Mice deficient in ISG15 are susceptible to infection with Influenza, Sindbis, and Herpes viruses( D'Cunha et al., 1996; Lenschow et al., 2005, 2007). Furthermore, many viruses encode proteins that specifically impair ISGylation( Frias-Staheli et al., 2007). ISG15 seems to be unique among ubls, as it can both modify specific target proteins and non-specifically modify proteins cotranslationally( Frias-Staheli et al., 2007; Durfee et al., 2010; Zhao et al., 2010). Since ISG15 is strongly induced by type I interferon, which is produced during bacterial infection, we aimed to decipher whether ISG15 is induced during Listeria infection and if so whether ISGylation acts as a means of host defense against invading bacteria. Here, we show that in nonphagocytic cells ISG15 is dramatically induced upon Listeria infection and that, surprisingly, early induction can be type I interferon independent. Listeria-mediated ISG15 induction depends on the CSP, which senses bacterial DNA and signals through STING, TBK1, IRF3, and IRF7. Most importantly, we demonstrate that ISG15 counteracts Listeria infection both in vitro and in vivo. We identified protein targets of ISGylation following overexpression of ISG15 using stable isotope labeling in tissue culture( SILAC) analysis and uncovered a prominent enrichment in integral membrane proteins of the endoplasmic reticulum and Golgi apparatus. This enrichment correlated with an increase in canonical secretion of cytokines known to control infection, highlighting a new mechanism of regulation of the host response to an intracytosolic pathogen.
Discussion:
Listeria has long been used as a model organism to understand the adaptive immune response, however more recently interest in the field has focused on the innate immune pathways required to produce type I and type III interferons and bacterial modulators of type I and type III interferons during Listeria infection of nonphagocytic cells( Lebreton et al., 2011; Abdullah et al., 2012; Bierne et al., 2012; Hagmann et al., 2013; Odendall et al., 2014). In phagocytic cells, the primary type I interferon response to Listeria is mediated by Listeria cyclic-di-AMP( Woodward et al., 2010). In nonphagocytic cells, type I interferon is produced downstream of RIG-I sensing of triphosphorylated RNA molecules( Abdullah et al., 2012; Hagmann et al., 2013). Here, we highlight a complementary pathway, which can lead to rapid and direct interferon-independent induction of ISG15, and potentially other ISGs, emanating from sensing of bacterial DNA. Notably, we implicate the same molecular players required for the CSP in macrophages. However cyclic-di-AMP, the PAMP sensed by the CSP in mouse macrophages, was not sufficient to directly induce ISG15 in an interferon-independent manner in nonphagocytic cells. Interestingly, a similar pathway was implicated in sensing the cytosolic bacterial DNA of Mycobacterium tuberculosis in macrophages( Manzanillo et al., 2012) and sensing viral DNA in nonphagocytic cells( Hasan et al., 2013). Our study complements and supports these findings as it also alludes to the importance of this response in nonphagocytic cells during Listeria infection. It is still an open question whether specific ISGs can contribute to a functional antibacterial state similar to an antiviral state. We hypothesized that the massive induction of ISG15 following Listeria infection must have a phenotypic effect on host cells. Indeed, we have demonstrated that ISG15 counteracts Listeria infection both in vitro and in vivo. The in vitro susceptibility is clearly conjugation-dependent and we have preliminary data suggesting that the conjugation incompetent mutant of ISG15 does not protect cells from infection( data not shown). During viral infections ISG15 can restrict infection by direct modification of viral proteins( Zhao et al., 2010), by modification of host proteins that are important to viral replication( Pincetic et al., 2010), by non-specific modification of newly translated proteins to target viral capsid( Durfee et al., 2010) and finally in the context of infection in vivo it can have non-conjugation dependent roles either within the cell or as a cytokine when secreted from polymorphonuclear leukocytes( D'Cunha et al., 1996; Werneke et al., 2011; Bogunovic et al., 2012). Here, we highlight a novel mode of action of ISG15 on canonical cytokine secretion that correlates with modification of ER and Golgi proteins. Interestingly, ubiquitin itself can have an effect on secretion by playing a role in determining COPII vesicle size and cargo through specific Cullin E3 ligases( Lu and Pfeffer, 2014). To our knowledge, a link between the intracellular function of ISG15 and canonical secretion of other cytokines has not been reported, thus our work identifies a novel function of ISG15, which could also play a role in other bacterial and viral infections. Two recent studies have underscored the importance of ISG15 in the human innate immune response to pathogens by characterizing patients who lack a functional ISG15 protein( Bogunovic et al., 2012; Zhang et al., 2014). ISG15-deficient patients were susceptible to Mycobacterium bovis Bacille Calmette-Guérin( BCG). These individuals were shown to have reduced production of interferon γ, which was linked to ISG15's extracellular role as a cytokine. In light of our results, ISG15's intracellular role as a modulator of cytokine secretion in nonphagocytic cells could also be responsible for reduced production of interferon γ. Increased cytokine secretion mediated by ISG15 is a previously uncharacterized way for the host to engage the immune system to combat both bacterial and viral infection; the first cells that encounter Listeria,( i. e., nonphagocytic cells) which are able to induce ISG15 at early time points could raise the alarm against an invading pathogen and as a result help alert innate immune cells to the site of infection. In particular, IL-6 and IL-8 are key mediators of neutrophil and monocyte recruitment in vivo and IL-6 has been repeatedly characterized as a critical mediator of the innate immune response to Listeria( Baggiolini and Clark-Lewis, 1992; Dalrymple et al., 1995; Personnic et al., 2010). If cytokine secretion in the absence of ISG15 is less efficient, it could help to explain the moderate but statistically significant susceptibility of Isg15−/− mice to systemic Listeria infection. These data coupled with the aforementioned human ISG15-deficient patient susceptibility to BCG infection highlight the in vivo relevance of ISG15 as an antibacterial factor( Bogunovic et al., 2012). Finally, the interferon-independent induction of ISG15 and potentially other ISGs may help to explain the still perplexing phenotype of interferon receptor-deficient mice( Ifnar−/−). IFNAR-deficient mice are highly resistant to Listeria infection when infected intravenously( Auerbuch et al., 2004; Carrero et al., 2004; O'Connell et al., 2004; Stockinger et al., 2004). If certain subsets of cells are in fact able to induce ISGs in the absence of interferon and if these ISGs like ISG15 could contribute to an antibacterial state, this may explain why these mice are resistant to Listeria infection.
Listeria monocytogenes is a bacterium that can cause serious food poisoning in humans . Infections with this bacterium can be particularly dangerous to young children , pregnant women , the elderly , and individuals with weakened immune systems because they are more susceptible to developing serious complications that can sometimes lead to death . The bacteria infect cells in the lining of the human gut . Cells that detect the bacteria respond by producing proteins called interferons and other signaling proteins that activate the body's immune system to fight the infection . One of the genes that the interferons activate encodes a protein called ISG15 , which helps to defend the body against viruses . However , it is not clear what role ISG15 plays in fighting bacterial infections . Here , Radoshevich et al . studied the role of ISG15 in human cells exposed to L . monocytogenes . The experiments show that ISG15 levels increase in the cells , but that the initial increase does not depend on Interferon proteins . Instead , ISG15 production is triggered by an alternative pathway called the cytosolic surveillance pathway , which is activated by the presence of bacterial DNA inside the cell . Further experiments found that ISG15 can counteract the infections of L . monocytogenes both in cells grown in cultures and in living mice . ISG15 modifies other proteins in the cell to promote the release of proteins called cytokines that help the body to eliminate the bacteria . Radoshevich et al . 's findings reveal a new role for ISG15 in fighting bacterial infections . A future challenge will be to understand the molecular details of how ISG15 triggers the release of cytokines .