The influenza replication blocking inhibitor LASAG does not sensitize human epithelial cells for bacterial infections.

Severe influenza virus (IV) infections still represent a major challenge to public health. To combat IV infections, vaccines and antiviral compounds are available. However, vaccine efficacies vary with very limited to no protection against newly emerging zoonotic IV introductions. In addition, the development of resistant virus variants against currently available antivirals can be rapidly detected, in consequence demanding the design of novel antiviral strategies. Virus supportive cellular signaling cascades, such as the NF-κB pathway, have been identified to be promising antiviral targets against IV in in vitro and in vivo studies and clinical trials. While administration of NF-κB pathway inhibiting agents, such as LASAG results in decreased IV replication, it remained unclear whether blocking of NF-κB might sensitize cells to secondary bacterial infections, which often come along with viral infections. Thus, we examined IV and Staphylococcus aureus growth during LASAG treatment. Interestingly, our data reveal that the presence of LASAG during superinfection still leads to reduced IV titers. Furthermore, the inhibition of the NF-κB pathway resulted in decreased intracellular Staphylococcus aureus loads within epithelial cells, indicating a dependency on the pathway for bacterial uptake. Unfortunately, so far it is not entirely clear if this phenomenon might be a drawback in bacterial clearance during infection.

In Vitro Models to Study Influenza Virus and Staphylococcus aureus Super-Infection on a Molecular Level.

Investigation of pathogen-host interactions on a molecular level requires sophisticated in vitro infection procedures, especially in the presence of different pathogens.Super-infections of influenza viruses (IV) and bacteria, with increasing incidence of Staphylococcus aureus (S. aureus) cases, are a long-known phenomenon and represent a major complication in IV-infected patients. Although several in vivo studies have improved our knowledge about pathogenesis and immune responses of super-infections that result in increased morbidity and mortality, the consequences of the direct interplay of viruses and bacteria on a molecular level in affected cells that may contribute to the deadly synergism of these pathogens are so far poorly characterized. Here we describe different infection schemes to study IV and S. aureus coinfections of distinct cell populations in vitro. Depending on the focus of interest, regulation of cell responses such as signalling mechanisms or pro- and anti-inflammatory cytokine expression, or consequences for the viral or bacterial life cycle, can be analyzed. The described infection procedures could be used as guidelines and adapted to super-infection settings of other viral and bacterial pathogens.

Staphylococcus aureus triggers a shift from influenza virus-induced apoptosis to necrotic cell death.

Superinfections with Staphylococcus aureus are a major complication of influenza disease, causing excessive inflammation and tissue damage. This enhanced cell-damaging effect is also observed in superinfected tissue cultures, leading to a strong decrease in overall cell viability. In our analysis of the underlying molecular mechanisms, we observed that, despite enhanced cell damage in superinfection, S. aureus did not increase but rather inhibited influenza virus (IV)-induced apoptosis in cells on the level of procaspase-8 activation. This apparent contradiction was solved when we observed that S. aureus mediated a switch from apoptosis to necrotic cell death of IV-infected cells, a mechanism that was dependent on the bacterial accessory gene regulator ( agr) locus that promotes bacterial survival and spread. This so far unknown action may be a bacterial strategy to enhance dissemination of intracellular S. aureus and may thereby contribute to increased tissue damage and severity of disease.-Van Krüchten, A., Wilden, J. J., Niemann, S., Peters, G., Löffler, B., Ludwig, S., Ehrhardt, C. Staphylococcus aureus triggers a shift from influenza virus-induced apoptosis to necrotic cell death.

Mitogen-activated protein kinases (MAPKs) regulate IL-6 over-production during concomitant influenza virus and Staphylococcus aureus infection.

Bacterial super-infections are a major complication of influenza virus (IV) infections and often lead to severe pneumonia. One hallmark of IV-associated Staphylococcus aureus (S. aureus) infection is rapid progression to a serious disease outcome. Changes in immune and inflammatory host responses increase morbidity and complicate efficient therapy. A key player during inflammation is the multifunctional cytokine IL-6. Although increased IL-6 levels have been observed after severe disease upon IV and/or bacterial super-infection, the underlying molecular mechanisms still remain to be elucidated. In the present study, we focused on cellular signalling pathways regulating IL-6 production upon IV/S. aureus super-infection. Additionally, infection with viable bacteria was mimicked by lipoteichoic acid stimulation in this model. Analyses of cellular signalling mechanisms revealed synergistically increased activation of the MAPK p38 as well as enhanced phosphorylation of the MAPKs ERK1/2 and JNK in the presence of super-infecting bacteria. Interestingly, inhibition of MAPK activity indicated a strong dependence of IL-6 expression on p38 and ERK1/2, while the MAPK JNK seems not to be involved. Thus, our results provide new molecular insights into the regulation of IL-6, a marker of severe disease, which might contribute to the lethal synergism of IV and S. aureus.

Super-infection with Staphylococcus aureus inhibits influenza virus-induced type I IFN signalling through impaired STAT1-STAT2 dimerization.

Bacterial super-infections are a major complication in influenza virus-infected patients. In response to infection with influenza viruses and bacteria, a complex interplay of cellular signalling mechanisms is initiated, regulating the anti-pathogen response but also pathogen-supportive functions. Here, we show that influenza viruses replicate to a higher efficiency in cells co-infected with Staphylococcus aureus (S. aureus). While cells initially respond with increased induction of interferon beta upon super-infection, subsequent interferon signalling and interferon-stimulated gene expression are rather impaired due to a block of STAT1-STAT2 dimerization. Thus, S. aureus interrupts the first line of defence against influenza viruses, resulting in a boost of viral replication, which may lead to enhanced viral pathogenicity.