Natural killer cell-intrinsic type I IFN signaling controls Klebsiella pneumoniae growth during lung infection.

Klebsiella pneumoniae is a significant cause of nosocomial pneumonia and an alarming pathogen owing to the recent isolation of multidrug resistant strains. Understanding of immune responses orchestrating K. pneumoniae clearance by the host is of utmost importance. Here we show that type I interferon (IFN) signaling protects against lung infection with K. pneumoniae by launching bacterial growth-controlling interactions between alveolar macrophages and natural killer (NK) cells. Type I IFNs are important but disparate and incompletely understood regulators of defense against bacterial infections. Type I IFN receptor 1 (Ifnar1)-deficient mice infected with K. pneumoniae failed to activate NK cell-derived IFN-γ production. IFN-γ was required for bactericidal action and the production of the NK cell response-amplifying IL-12 and CXCL10 by alveolar macrophages. Bacterial clearance and NK cell IFN-γ were rescued in Ifnar1-deficient hosts by Ifnar1-proficient NK cells. Consistently, type I IFN signaling in myeloid cells including alveolar macrophages, monocytes and neutrophils was dispensable for host defense and IFN-γ activation. The failure of Ifnar1-deficient hosts to initiate a defense-promoting crosstalk between alveolar macrophages and NK cell was circumvented by administration of exogenous IFN-γ which restored endogenous IFN-γ production and restricted bacterial growth. These data identify NK cell-intrinsic type I IFN signaling as essential driver of K. pneumoniae clearance, and reveal specific targets for future therapeutic exploitations.

A trans-kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection.

Bacteria can inhibit the growth of other bacteria by injecting effectors using a type VI secretion system (T6SS). T6SS effectors can also be injected into eukaryotic cells to facilitate bacterial survival, often by targeting the cytoskeleton. Here, we show that the trans-kingdom antimicrobial T6SS effector VgrG4 from Klebsiella pneumoniae triggers the fragmentation of the mitochondrial network. VgrG4 colocalizes with the endoplasmic reticulum (ER) protein mitofusin 2. VgrG4 induces the transfer of Ca2+ from the ER to the mitochondria, activating Drp1 (a regulator of mitochondrial fission) thus leading to mitochondrial network fragmentation. Ca2+ elevation also induces the activation of the innate immunity receptor NLRX1 to produce reactive oxygen species (ROS). NLRX1-induced ROS limits NF-κB activation by modulating the degradation of the NF-κB inhibitor IκBα. The degradation of IκBα is triggered by the ubiquitin ligase SCFß-TrCP, which requires the modification of the cullin-1 subunit by NEDD8. VgrG4 abrogates the NEDDylation of cullin-1 by inactivation of Ubc12, the NEDD8-conjugating enzyme. Our work provides an example of T6SS manipulation of eukaryotic cells via alteration of the mitochondria.

Klebsiella pneumoniae Reduces SUMOylation To Limit Host Defense Responses.

Klebsiella pneumoniae is an important cause of multidrug-resistant infections worldwide. Understanding the virulence mechanisms of K. pneumoniae is a priority and timely to design new therapeutics. Here, we demonstrate that K. pneumoniae limits the SUMOylation of host proteins in epithelial cells and macrophages (mouse and human) to subvert cell innate immunity. Mechanistically, in lung epithelial cells, Klebsiella increases the levels of the deSUMOylase SENP2 in the cytosol by affecting its K48 ubiquitylation and its subsequent degradation by the ubiquitin proteasome. This is dependent on Klebsiella preventing the NEDDylation of the Cullin-1 subunit of the ubiquitin ligase complex E3-SCF-ßTrCP by exploiting the CSN5 deNEDDylase. Klebsiella induces the expression of CSN5 in an epidermal growth factor receptor (EGFR)-phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-extracellular signal-regulated kinase (ERK)-glycogen synthase kinase 3 beta (GSK3ß) signaling pathway-dependent manner. In macrophages, Toll-like receptor 4 (TLR4)-TRAM-TRIF-induced type I interferon (IFN) via IFN receptor 1 (IFNAR1)-controlled signaling mediates Klebsiella-triggered decrease in the levels of SUMOylation via let-7 microRNAs (miRNAs). Our results revealed the crucial role played by Klebsiella polysaccharides, the capsule, and the lipopolysaccharide (LPS) O-polysaccharide, to decrease the levels of SUMO-conjugated proteins in epithelial cells and macrophages. A Klebsiella-induced decrease in SUMOylation promotes infection by limiting the activation of inflammatory responses and increasing intracellular survival in macrophages.IMPORTANCEKlebsiella pneumoniae has been singled out as an urgent threat to human health due to the increasing isolation of strains resistant to "last-line" antimicrobials, narrowing the treatment options against Klebsiella infections. Unfortunately, at present, we cannot identify candidate compounds in late-stage development for treatment of multidrug-resistant Klebsiella infections; this pathogen is exemplary of the mismatch between unmet medical needs and the current antimicrobial research and development pipeline. Furthermore, there is still limited evidence on K. pneumoniae pathogenesis at the molecular and cellular levels in the context of the interactions between bacterial pathogens and their hosts. In this research, we have uncovered a sophisticated strategy employed by Klebsiella to subvert the activation of immune defenses by controlling the modification of proteins. Our research may open opportunities to develop new therapeutics based on counteracting this Klebsiella-controlled immune evasion strategy.

Identification of bacterial biofilm and the Staphylococcus aureus derived protease, staphopain, on the skin surface of patients with atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by an impaired epidermal barrier, dysregulation of innate and adaptive immunity, and a high susceptibility to bacterial colonization and infection. In the present study, bacterial biofilm was visualized by electron microscopy at the surface of AD skin. Correspondingly, Staphylococcus aureus (S. aureus) isolates from lesional skin of patients with AD, produced a substantial amount of biofilm in vitro. S. aureus biofilms showed less susceptibility to killing by the antimicrobial peptide LL-37 when compared with results obtained using planktonic cells. Confocal microscopy analysis showed that LL-37 binds to the S. aureus biofilms. Immuno-gold staining of S. aureus biofilm of AD skin detected the S. aureus derived protease staphopain adjacent to the bacteria. In vitro, staphopain B degraded LL-37 into shorter peptide fragments. Further, LL-37 significantly inhibited S. aureus biofilm formation, but no such effects were observed for the degradation products. The data presented here provide novel information on staphopains present in S. aureus biofilms in vivo, and illustrate the complex interplay between biofilm and LL-37 in skin of AD patients, possibly leading to a disturbed host defense, which facilitates bacterial persistence.

Proteolytic inactivation of LL-37 by karilysin, a novel virulence mechanism of Tannerella forsythia.

Tannerella forsythia is a gram-negative bacterium strongly associated with the development and/or progression of periodontal disease. Here, we have shown that a newly characterized matrix metalloprotease-like enzyme, referred to as karilysin, efficiently cleaved the antimicrobial peptide LL-37, significantly reducing its bactericidal activity. This may contribute to the resistance of T. forsythia to the antibacterial activity of LL-37, since their vitality was found not to be affected by LL-37 at concentrations up to 2.2 muM. Furthermore, proteolysis of LL-37 by karilysin not only abolished its ability to bind lipopolysaccharide (LPS) to quench endotoxin-induced proinflammatory activity, but LL-37 cleavage also caused the release of active endotoxin from the LPS/LL-37 complex. Proteolytic inactivation of LL-37 bactericidal activity by karilysin may protect LL-37-sensitive species in the subgingival plaque and maintain the local inflammatory reaction driven by LPS from gram-negative bacteria. Consequently, the karilysin protease may directly contribute to periodontal tissue damage and the development and/or progression of chronic periodontitis.