Recognition of phylogenetically diverse pathogens through enzymatically amplified recruitment of RNF213.

Innate immunity senses microbial ligands known as pathogen-associated molecular patterns (PAMPs). Except for nucleic acids, PAMPs are exceedingly taxa-specific, thus enabling pattern recognition receptors to detect cognate pathogens while ignoring others. How the E3 ubiquitin ligase RNF213 can respond to phylogenetically distant pathogens, including Gram-negative Salmonella, Gram-positive Listeria, and eukaryotic Toxoplasma, remains unknown.

Ubiquitylation: Sword and shield in the bacterial arsenal.

In two recent studies in Nature, Hör et al. and Chambers et al. report that ubiquitin-like conjugation in bacteria antagonizes phage replication.

TECPR1 conjugates LC3 to damaged endomembranes upon detection of sphingomyelin exposure.

Invasive bacteria enter the cytosol of host cells through initial uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the BCV membrane, thereby exposing to the cytosol intraluminal, otherwise shielded danger signals such as glycans and sphingomyelin. The detection of glycans by galectin-8 triggers anti-bacterial autophagy, but how cells sense and respond to cytosolically exposed sphingomyelin remains unknown. Here, we identify TECPR1 (tectonin beta-propeller repeat containing 1) as a receptor for cytosolically exposed sphingomyelin, which recruits ATG5 into an E3 ligase complex that mediates lipid conjugation of LC3 independently of ATG16L1.

Comparative study of GBP recruitment on two cytosol-dwelling pathogens, Francisella novicida and Shigella flexneri highlights differences in GBP repertoire and in GBP1 motif requirements.

Guanylate-Binding Proteins are interferon-inducible GTPases that play a key role in cell autonomous responses against intracellular pathogens. Despite sharing high sequence similarity, subtle differences among GBPs translate into functional divergences that are still largely not understood. A key GBP feature is the formation of supramolecular GBP complexes on the bacterial surface.

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 autophagy. 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).

Sensing of mycobacterial arabinogalactan by galectin-9 exacerbates mycobacterial infection.

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio-synthetical target for anti-tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin-9 and exacerbates mycobacterial infection. Administration of AG-specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb-infected mice or Mycobacterium marinum-infected zebrafish.

Targeting the Conserved Stem Loop 2 Motif in the SARS-CoV-2 Genome.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle.

SIK2 orchestrates actin-dependent host response upon infection.

is an intracellular pathogen of a substantial global health concern. In order to identify key players involved in 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 infection.

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.

Transbilayer Movement of Sphingomyelin Precedes Catastrophic Breakage of Enterobacteria-Containing Vacuoles.

Pathogenic bacteria enter the cytosol of host cells through uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the vacuolar membrane [1]. Bacterial invaders are sensed either directly, through cytosolic pattern-recognition receptors specific for bacterial ligands, or indirectly, through danger receptors that bind host molecules displayed in an abnormal context, for example, glycans on damaged BCVs [2-4]. In contrast to damage caused by Listeria monocytogenes, a Gram-positive bacterium, BCV rupture by Gram-negative pathogens such as Shigella flexneri or Salmonella Typhimurium remains incompletely understood [5, 6].

Guanylate-binding proteins convert cytosolic bacteria into caspase-4 signaling platforms.

Bacterial lipopolysaccharide triggers human caspase-4 (murine caspase-11) to cleave gasdermin-D and induce pyroptotic cell death. How lipopolysaccharide sequestered in the membranes of cytosol-invading bacteria activates caspases remains unknown. Here we show that in interferon-γ-stimulated cells guanylate-binding proteins (GBPs) assemble on the surface of Gram-negative bacteria into polyvalent signaling platforms required for activation of caspase-4.

CALCOCO2/NDP52 initiates selective autophagy through recruitment of ULK and TBK1 kinase complexes.

The selective macroautophagy of prospective cargo necessitates activity of the autophagy machinery at cargo-determined locations. Whether phagophore membranes are recruited to, or are generated at, the cargo is unknown. In our recent study we show that damaged -containing vacuoles, marked by LGALS8/galectin-8, engage the cargo receptor CALCOCO2/NDP52 to recruit the autophagy-initiating ULK and TBK1 complexes and cause the formation of WIPI2-positive phagophore membranes.

The Cargo Receptor NDP52 Initiates Selective Autophagy by Recruiting the ULK Complex to Cytosol-Invading Bacteria.

Xenophagy, a selective autophagy pathway that protects the cytosol against bacterial invasion, relies on cargo receptors that juxtapose bacteria and phagophore membranes. Whether phagophores are recruited from a constitutive pool or are generated de novo at prospective cargo remains unknown. Phagophore formation in situ would require recruitment of the upstream autophagy machinery to prospective cargo.

Spatiotemporal Control of ULK1 Activation by NDP52 and TBK1 during Selective Autophagy.

Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis.

Measuring Antibacterial Autophagy.

Bacteria that escape from membrane-enclosed vacuoles to the cytosol of cells are targeted by autophagy, which recognizes and captures bacteria into autophagosomes wherein their proliferation is restricted. Here we discuss two means by which antibacterial autophagy is assessed: (1) the visualization and enumeration of autophagy protein recruitment to the vicinity of cytosolic bacteria by means of immunofluorescence microscopy and (2) the measurement of autophagy-dependent restriction of bacterial proliferation by means of colony-forming unit assay.

Galectin-8-mediated selective autophagy protects against seeded tau aggregation.

Assembled tau can transfer between cells and seed the aggregation of soluble tau. This process is thought to underlie the amplification and propagation of tau inclusions throughout the brain in neurodegenerative diseases, including Alzheimer's disease. An understanding of the mechanisms involved may provide strategies for limiting assembled tau propagation.

GBPs Inhibit Motility of Shigella flexneri but Are Targeted for Degradation by the Bacterial Ubiquitin Ligase IpaH9.8.

Interferon exposure boosts cell-autonomous immunity for more efficient pathogen control. But how interferon-enhanced immunity protects the cytosol against bacteria and how professionally cytosol-dwelling bacteria avoid clearance are insufficiently understood. Here we demonstrate that the interferon-induced GTPase family of guanylate-binding proteins (GBPs) coats Shigella flexneri in a hierarchical manner reliant on GBP1.

LUBAC-synthesized linear ubiquitin chains restrict cytosol-invading bacteria by activating autophagy and NF-κB.

Cell-autonomous immunity relies on the ubiquitin coat surrounding cytosol-invading bacteria functioning as an 'eat-me' signal for xenophagy. The origin, composition and precise mode of action of the ubiquitin coat remain incompletely understood. Here, by studying Salmonella Typhimurium, we show that the E3 ligase LUBAC generates linear (M1-linked) polyubiquitin patches in the ubiquitin coat, which serve as antibacterial and pro-inflammatory signalling platforms.

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.

TBK1 directs WIPI2 against Salmonella.

Defense of the mammalian cell cytosol against Salmonella invasion is reliant upon capture of the infiltrating bacteria by macroautophagy (hereafter autophagy), a process controlled by the kinase TBK1. In our recent study we showed that recruitment of TBK1 activity to Salmonella stabilizes the key autophagy regulator WIPI2 on those bacteria, a novel and essential function for TBK1 in the control of the early steps of antibacterial autophagy. Substantial redundancy exists in the precise recruitment mechanism for TBK1 because engagement with any of several Salmonella-associated 'eat-me' signals, including host-derived glycans, and K48- and K63-linked ubiquitin chains, suffices to recruit TBK1 functionality.

Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2-dependent antibacterial autophagy.

Mammalian cells deploy autophagy to defend their cytosol against bacterial invaders. Anti-bacterial autophagy relies on the core autophagy machinery, cargo receptors, and "eat-me" signals such as galectin-8 and ubiquitin that label bacteria as autophagy cargo. Anti-bacterial autophagy also requires the kinase TBK1, whose role in autophagy has remained enigmatic.

Rubicon swaps autophagy for LAP.

Phagocytic cells engulf their prey into vesicular structures called phagosomes, of which a certain proportion becomes demarcated for enhanced maturation by a process called LC3-associated phagocytosis (LAP). Light has now been shed on the molecular requirements of LAP, establishing a central role for the protein Rubicon in the immune response to Aspergillus fumigatus.