Felix Randow

The TBK1 adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin-coated bacteria.

Cell-autonomous innate immune responses against bacteria attempting to colonize the cytosol of mammalian cells are incompletely understood. Polyubiquitylated proteins can accumulate on the surface of such bacteria, and bacterial growth is restricted by Tank-binding kinase (TBK1). Here we show that NDP52, not previously known to contribute to innate immunity, recognizes ubiquitin-coated Salmonella enterica in human cells and, by binding the adaptor proteins Nap1 and Sintbad, recruits TBK1. Knockdown of NDP52 and TBK1 facilitated bacterial proliferation and increased the number of cells containing ubiquitin-coated salmonella. NDP52 also recruited LC3, an autophagosomal marker, and knockdown of NDP52 impaired autophagy of salmonella. We conclude that human cells utilize the ubiquitin system and NDP52 to activate autophagy against bacteria attempting to colonize their cytosol.

Specific Recognition of Linear Ubiquitin Chains by NEMO Is Important for NF-κB 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.

Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion

Autophagy defends the mammalian cytosol against bacterial infection. Efficient pathogen engulfment is mediated by cargo-selecting autophagy adaptors that rely on unidentified pattern-recognition or danger receptors to label invading pathogens as autophagy cargo, typically by polyubiquitin coating. Here we show in human cells that galectin 8 (also known as LGALS8), a cytosolic lectin, is a danger receptor that restricts Salmonella proliferation. Galectin 8 monitors endosomal and lysosomal integrity and detects bacterial invasion by binding host glycans exposed on damaged Salmonella-containing vacuoles. By recruiting NDP52 (also known as CALCOCO2), galectin 8 activates antibacterial autophagy. Galectin-8-dependent recruitment of NDP52 to Salmonella-containing vesicles is transient and followed by ubiquitin-dependent NDP52 recruitment. Because galectin 8 also detects sterile damage to endosomes or lysosomes, as well as invasion by Listeria or Shigella, we suggest that galectin 8 serves as a versatile receptor for vesicle-damaging pathogens. Our results illustrate how cells deploy the danger receptor galectin 8 to combat infection by monitoring endosomal and lysosomal integrity on the basis of the specific lack of complex carbohydrates in the cytosol.

Neuropilin-1 Expression on Regulatory T Cells Enhances Their Interactions with Dendritic Cells during Antigen Recognition

Summary The interaction of T cells with dendritic cells (DCs) determines whether an immune response is launched or not. Recognition of antigen leads to formation of immunological synapses at the interface between the cells. The length of interaction is likely to determine the functional outcome, because it limits the number of MHC class II-peptide complexes that can be recruited into the synapse. Here, we show that regulatory T (Treg) cells and naive helper T (Th) cells interact differently with DCs in the absence of proinflammatory stimuli. Although differences in T cell receptor repertoire might contribute, Foxp3-induced phenotypic differences play a major role. We found that Neuropilin-1 (Nrp-1), which is expressed by most Treg cells but not naive Th cells, promoted prolonged interactions with immature DCs (iDCs), resulting in higher sensitivity to limiting amounts of antigen. This is likely to give Treg cells an advantage over naive Th cells, with the same specificity leading to a “default” suppression of immune responses in the absence of “danger signals.”

Viral avoidance and exploitation of the ubiquitin system.

The versatility of ubiquitin in regulating protein function and cell behaviour through post-translational protein modification makes it a particularly attractive target for viruses. Here we review how viruses manipulate the ubiquitin system to favour their propagation by redirecting cellular ubiquitin enzymes or encoding their own ubiquitin components to enable replication, egress and immune evasion. These studies not only reveal the many cellular processes requiring ubiquitin but also illustrate how viruses usurp their host cells.

LC3C, Bound Selectively by a Noncanonical LIR Motif in NDP52, Is Required for Antibacterial Autophagy

Summary Autophagy protects cellular homeostasis by capturing cytosolic components and invading pathogens for lysosomal degradation. Autophagy receptors target cargo to autophagy by binding ATG8 on autophagosomal membranes. The expansion of the ATG8 family in higher eukaryotes suggests that specific interactions with autophagy receptors facilitate differential cargo handling. However, selective interactors of ATG8 orthologs are unknown. Here we show that the selectivity of the autophagy receptor NDP52 for LC3C is crucial for innate immunity since cells lacking either protein cannot protect their cytoplasm against Salmonella. LC3C is required for antibacterial autophagy because in its absence the remaining ATG8 orthologs do not support efficient antibacterial autophagy. Structural analysis revealed that the selectivity of NDP52 for LC3C is conferred by a noncanonical LIR, in which lack of an aromatic residue is balanced by LC3C-specific interactions. Our report illustrates that specificity in the interaction between autophagy receptors and autophagy machinery is of functional importance to execute selective autophagy.

SINTBAD, a novel component of innate antiviral immunity, shares a TBK1‐binding domain with NAP1 and TANK

The expression of antiviral genes during infection is controlled by inducible transcription factors such as IRF3 (interferon regulatory factor). Activation of IRF3 requires its phosphorylation by TBK1 (TANK‐binding kinase) or IKKi (inhibitor of nuclear factor κB kinase, inducible). We have identified a new and essential component of this pathway, the adaptor protein SINTBAD (similar to NAP1 TBK1 adaptor). SINTBAD constitutively binds TBK1 and IKKi but not related kinases. Upon infection with Sendai virus, SINTBAD is essential for the efficient induction of IRF‐dependent transcription, as are two further TBK1 adaptors, TANK and NAP1. We identified a conserved TBK1/IKKi‐binding domain (TBD) in the three adaptors, predicted to form an α‐helix with residues essential for kinase binding clustering on one side. Isolated TBDs compete with adaptor binding to TBK1 and prevent poly(I:C)‐induced IRF‐dependent transcription. Our results suggest that efficient signal transduction upon viral infection requires SINTBAD, TANK and NAP1 because they link TBK1 and IKKi to virus‐activated signalling cascades.

Inhibition of IκB Kinase by Vaccinia Virus Virulence Factor B14

The IκB kinase (IKK) complex is a key regulator of signal transduction pathways leading to the induction of NF-κB-dependent gene expression and production of pro-inflammatory cytokines. It therefore represents a major target for the development of anti-inflammatory therapeutic drugs and may be targeted by pathogens seeking to diminish the host response to infection. Previously, the vaccinia virus (VACV) strain Western Reserve B14 protein was characterised as an intracellular virulence factor that alters the inflammatory response to infection by an unknown mechanism. Here we demonstrate that ectopic expression of B14 inhibited NF-κB activation in response to TNFα, IL-1β, poly(I:C), and PMA. In cells infected with VACV lacking gene B14R (vΔB14) there was a higher level of phosphorylated IκBα but a similar level of IκBα compared to cells infected with control viruses expressing B14, suggesting B14 affects IKK activity. Direct evidence for this was obtained by showing that B14 co-purified and co-precipitated with the endogenous IKK complex from human and mouse cells and inhibited IKK complex enzymatic activity. Notably, the interaction between B14 and the IKK complex required IKKβ but not IKKα, suggesting the interaction occurs via IKKβ. B14 inhibited NF-κB activation induced by overexpression of IKKα, IKKβ, and a constitutively active mutant of IKKα, S176/180E, but did not inhibit a comparable mutant of IKKβ, S177/181E. This suggested that phosphorylation of these serine residues in the activation loop of IKKβ is targeted by B14, and this was confirmed using Ab specific for phospho-IKKβ.

Cellular Self-Defense: How Cell-Autonomous Immunity Protects Against Pathogens

Defense and Counter-Defense Provided a pathogen can enter the body and survive coughing and spluttering, peristalsis, and mucus, the first active responses the host evokes to an invading organism will be at the level of the first cell encountered, well before classical cellular immunity and antibody responses are initiated. Randow et al. (p. 701) review the range of intracellular defenses against incoming pathogens and describe how compartmental boundaries within the cell provide multiple levels at which pathogens can be thwarted in their attempts to subjugate the cell to do their bidding. Baxt et al. (p. 697) review the range of evasion tactics that bacterial pathogens can summon to counter host repulsion and establish a niche in which to replicate and ensure onward transmission. Our prevailing view of vertebrate host defense is strongly shaped by the notion of a specialized set of immune cells as sole guardians of antimicrobial resistance. Yet this view greatly underestimates a capacity for most cell lineages—the majority of which fall outside the traditional province of the immune system—to defend themselves against infection. This ancient and ubiquitous form of host protection is termed cell-autonomous immunity and operates across all three domains of life. Here, we discuss the organizing principles that govern cellular self-defense and how intracellular compartmentalization has shaped its activities to provide effective protection against a wide variety of microbial pathogens.

A LC3-Interacting Motif in the Influenza A Virus M2 Protein Is Required to Subvert Autophagy and Maintain Virion Stability

Summary Autophagy recycles cellular components and defends cells against intracellular pathogens. While viruses must evade autophagocytic destruction, some viruses can also subvert autophagy for their own benefit. The ability of influenza A virus (IAV) to evade autophagy depends on the Matrix 2 (M2) ion-channel protein. We show that the cytoplasmic tail of IAV M2 interacts directly with the essential autophagy protein LC3 and promotes LC3 relocalization to the unexpected destination of the plasma membrane. LC3 binding is mediated by a highly conserved LC3-interacting region (LIR) in M2. The M2 LIR is required for LC3 redistribution to the plasma membrane in virus-infected cells. Mutations in M2 that abolish LC3 binding interfere with filamentous budding and reduce virion stability. IAV therefore subverts autophagy by mimicking a host short linear protein-protein interaction motif. This strategy may facilitate transmission of infection between organisms by enhancing the stability of viral progeny.