SIK2 orchestrates actin-dependent host response upon Salmonella infection.

Salmonella is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in Salmonella infection, we performed a global host phosphoproteome analysis subsequent to bacterial infection. Thereby, we identified the kinase SIK2 as a central component of the host defense machinery upon Salmonella infection. SIK2 depletion favors the escape of bacteria from the Salmonella-containing vacuole (SCV) and impairs Xenophagy, resulting in a hyperproliferative phenotype. Mechanistically, SIK2 associates with actin filaments under basal conditions; however, during bacterial infection, SIK2 is recruited to the SCV together with the elements of the actin polymerization machinery (Arp2/3 complex and Formins). Notably, SIK2 depletion results in a severe pathological cellular actin nucleation and polymerization defect upon Salmonella infection. We propose that SIK2 controls the formation of a protective SCV actin shield shortly after invasion and orchestrates the actin cytoskeleton architecture in its entirety to control an acute Salmonella infection after bacterial invasion.

Ubiquitylation of lipopolysaccharide by RNF213 during bacterial infection.

Ubiquitylation is a widespread post-translational protein modification in eukaryotes and marks bacteria that invade the cytosol as cargo for antibacterial autophagy1-3. The identity of the ubiquitylated substrate on bacteria is unknown. Here we show that the ubiquitin coat on Salmonella that invade the cytosol is formed through the ubiquitylation of a non-proteinaceous substrate, the lipid A moiety of bacterial lipopolysaccharide (LPS), by the E3 ubiquitin ligase ring finger protein 213 (RNF213). RNF213 is a risk factor for moyamoya disease4,5, which is a progressive stenosis of the supraclinoid internal carotid artery that causes stroke (especially in children)6,7. RNF213 restricts the proliferation of cytosolic Salmonella and is essential for the generation of the bacterial ubiquitin coat, both directly (through the ubiquitylation of LPS) and indirectly (through the recruitment of LUBAC, which is a downstream E3 ligase that adds M1-linked ubiquitin chains onto pre-existing ubiquitin coats8). In cells that lack RNF213, bacteria do not attract ubiquitin-dependent autophagy receptors or induce antibacterial autophagy. The ubiquitylation of LPS on Salmonella that invade the cytosol requires the dynein-like core of RNF213, but not its RING domain. Instead, ubiquitylation of LPS relies on an RZ finger in the E3 shell. We conclude that ubiquitylation extends beyond protein substrates and that ubiquitylation of LPS triggers cell-autonomous immunity, and we postulate that non-proteinaceous substances other than LPS may also become ubiquitylated.

The receptor DNGR-1 signals for phagosomal rupture to promote cross-presentation of dead-cell-associated antigens.

Type 1 conventional dendritic (cDC1) cells are necessary for cross-presentation of many viral and tumor antigens to CD8+ T cells. cDC1 cells can be identified in mice and humans by high expression of DNGR-1 (also known as CLEC9A), a receptor that binds dead-cell debris and facilitates XP of corpse-associated antigens. Here, we show that DNGR-1 is a dedicated XP receptor that signals upon ligand engagement to promote phagosomal rupture. This allows escape of phagosomal contents into the cytosol, where they access the endogenous major histocompatibility complex class I antigen processing pathway. The activity of DNGR-1 maps to its signaling domain, which activates SYK and NADPH oxidase to cause phagosomal damage even when spliced into a heterologous receptor and expressed in heterologous cells. Our data reveal the existence of innate immune receptors that couple ligand binding to endocytic vesicle damage to permit MHC class I antigen presentation of exogenous antigens and to regulate adaptive immunity.

Strange New World: Bacteria Catalyze Ubiquitylation via ADP Ribosylation.

Three recent papers, including one by Kotewicz et al. (2016) in this issue of Cell Host & Microbe, show that Legionella deploys a novel form of ubiquitylation to generate its replicative vacuole. Without E1 and E2 enzymes, SidE effectors ubiquitylate serine residues in substrates via an ADP-ribosylated ubiquitin intermediate.

Endoplasmic reticulum chaperone gp96 is essential for infection with vesicular stomatitis virus.

The envelope glycoprotein of vesicular stomatitis virus (VSV-G) enables viral entry into hosts as distant as insects and vertebrates. Because of its ability to support infection of most, if not all, human cell types VSV-G is used in viral vectors for gene therapy. However, neither the receptor nor any specific host factor for VSV-G has been identified. Here we demonstrate that infection with VSV and innate immunity via Toll-like receptors (TLRs) require a shared component, the endoplasmic reticulum chaperone gp96. Cells without gp96 or with catalytically inactive gp96 do not bind VSV-G. The ubiquitous expression of gp96 is therefore essential for the remarkably broad tropism of VSV-G. Cells deficient in gp96 also lack functional TLRs, which suggests that pathogen-driven pressure for TLR-mediated immunity maintains the broad host range of VSV-G by positively selecting for the ubiquitous expression of gp96.

Specific recognition of linear ubiquitin chains by NEMO is important for NF-kappaB activation.

Activation of nuclear factor-kappaB (NF-kappaB), a key mediator of inducible transcription in immunity, requires binding of NF-kappaB essential modulator (NEMO) to ubiquitinated substrates. Here, we report that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO selectively binds linear (head-to-tail) ubiquitin chains. Crystal structures of the UBAN motif revealed a parallel coiled-coil dimer that formed a heterotetrameric complex with two linear diubiquitin molecules. The UBAN dimer contacted all four ubiquitin moieties, and the integrity of each binding site was required for efficient NF-kappaB activation. Binding occurred via a surface on the proximal ubiquitin moiety and the canonical Ile44 surface on the distal one, thereby providing specificity for linear chain recognition. Residues of NEMO involved in binding linear ubiquitin chains are required for NF-kappaB activation by TNF-alpha and other agonists, providing an explanation for the detrimental effect of NEMO mutations in patients suffering from X-linked ectodermal dysplasia and immunodeficiency.

Somatic cell genetics for the study of NF-kappaB signaling in innate immunity.

Vertebrates have evolved acquired immunity, but to detect an infection in its early stages they, nonetheless, rely on Toll-like receptors (TLRs) and other innate immune receptors. We have performed genomewide mutagenesis screens in an immortalized murine cell line to study nuclear factor kappaBeta (NF-kappaB) signaling in the context of innate immunity. To enable metabolic and physical selection for alterations in NF-kappaB signaling, we equipped cells with multiple reporter genes. Despite the diploid nature of the cells, multiple mutants unresponsive to lipopolysaccharide and CpG DNA were isolated from as few as 10 million mutagenized cells. Mutant clones may lead to the discovery of novel genes, and in combination with syngeneic wild-type reporter cells, they may allow a detailed functional analysis of NF-kappaB signaling. Compared with the use of whole animals in genetic screens, somatic cell genetics allows the isolation of genes required for innate immunity, even if these genes also have an essential function in development. Our discovery of an essential role for the endoplasmic reticulum chaperone gp96 (Grp94) in the maturation of TLRs and our work on the regulation of the inhibitor of nuclear factor kappaB kinase (IKK) complex by Nemo will be discussed in this context.

Signal processing by its coil zipper domain activates IKK gamma.

NF-kappaB activation occurs upon degradation of its inhibitor I-kappaB and requires prior phosphorylation of the inhibitor by I-kappaB kinase (IKK). Activity of IKK is governed by its noncatalytic subunit IKKgamma. Signaling defects due to missense mutations in IKKgamma have been correlated to its inability to either become ubiquitylated or bind ubiquitin noncovalently. Because the relative contribution of these events to signaling had remained unknown, we have studied mutations in the coil-zipper (CoZi) domain of IKKgamma that either impair signaling or cause constitutive NF-kappaB activity. Certain signaling-deficient alleles neither bound ubiquitin nor were they ubiquitylated by TRAF6. Introducing an activating mutation into those signaling-impaired alleles restored their ubiquitylation and created mutants constitutively activating NF-kappaB without repairing the ubiquitin-binding defect. Constitutive activity therefore arises downstream of ubiquitin binding but upstream of ubiquitylation. Such constitutive activity reveals a signal-processing function for IKKgamma beyond that of a mere ubiquitin-binding adaptor. We propose that this signal processing may involve homophilic CoZi interactions as suggested by the enhanced affinity of CoZi domains from constitutively active IKKgamma.

Retroviral transduction of DT40.

Retroviral transduction of DT40 provides an easy way to obtain population of cells stably expressing a transgene without causing cellular stress or the levels of cell death seen in transfection protocols. By employing Moloney Murine Leukemia Virus based constructs and pseudotyping the viral particles with VSV envelope glycoprotein, it is a highly efficient procedure, routinely resulting in transgene expression in the majority of cells. It is also rapid. From production of the transgene-containing virus to expression takes only four days.