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LSSS 2015-2016

2015LSSS2016

Life Sciences Seminar Series

 

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Felix Randow

MRC Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Cambridge, UK; Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK

How cells defend their cytosol against bacteria

Selected Publications

Self and nonself: how autophagy targets mitochondria and bacteria.Randow F, Youle RJ
Cell Host Microbe 2014 Apr 9; 15(4):403-11

Abstract

Autophagy is an evolutionarily conserved pathway that transports cytoplasmic components for degradation into lysosomes. Selective autophagy can capture physically large objects, including cell-invading pathogens and damaged or superfluous organelles. Selectivity is achieved by cargo receptors that detect substrate-associated "eat-me" signals. In this Review, we discuss basic principles of selective autophagy and compare the "eat-me" signals and cargo receptors that mediate autophagy of bacteria and bacteria-derived endosymbionts-i.e., mitochondria.

A LC3-interacting motif in the influenza A virus M2 protein is required to subvert autophagy and maintain virion stability.Beale R, Wise H, Stuart A, Ravenhill BJ, Digard P, Randow F
Cell Host Microbe 2014 Feb 12; 15(2):239-47

Abstract

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.

Cellular self-defense: how cell-autonomous immunity protects against pathogens.Randow F, MacMicking JD, James LC
Science 2013 May 10; 340(6133):701-6

Abstract

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.

An essential role for the ATG8 ortholog LC3C in antibacterial autophagy.von Muhlinen N, Akutsu M, Ravenhill BJ, Foeglein Á, Bloor S, Rutherford TJ, Freund SM, Komander D, Randow F
Autophagy 2013 May; 9(5):784-6

Abstract

Autophagy defends the mammalian cytosol against bacterial invasion. Efficient bacterial engulfment by autophagy requires cargo receptors that bind (a) homolog(s) of the ubiquitin-like protein Atg8 on the phagophore membrane. The existence of multiple ATG8 orthologs in higher eukaryotes suggests that they may perform distinct functions. However, no specific role has been assigned to any mammalian ATG8 ortholog. We recently discovered that the autophagy receptor CALCOCO2/NDP52, which detects cytosol-invading Salmonella enterica serovar Typhimurium (S. Typhimurium), preferentially binds LC3C. The CALCOCO2/NDP52-LC3C interaction is essential for cell-autonomous immunity against cytosol-exposed S. Typhimurium, because cells lacking either protein fail to target bacteria into the autophagy pathway. The selectivity of CALCOCO2/NDP52 for LC3C is determined by a novel LC3C interacting region (CLIR), in which the lack of the key aromatic residue of canonical LIRs is compensated by LC3C-specific interactions. Our findings provide a new layer of regulation to selective autophagy, suggesting that specific interactions between autophagy receptors and the ATG8 orthologs are of biological importance.

LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy.von Muhlinen N, Akutsu M, Ravenhill BJ, Foeglein Á, Bloor S, Rutherford TJ, Freund SM, Komander D, Randow F
Mol Cell 2012 Nov 9; 48(3):329-42

Abstract

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.

Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion.Thurston TL, Wandel MP, von Muhlinen N, Foeglein A, Randow F
Nature 2012 Jan 15; 482(7385):414-8

Abstract

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.

Vaccinia virus protein C6 is a virulence factor that binds TBK-1 adaptor proteins and inhibits activation of IRF3 and IRF7.Unterholzner L, Sumner RP, Baran M, Ren H, Mansur DS, Bourke NM, Randow F, Smith GL, Bowie AG
PLoS Pathog 2011 Sep; 7(9):e1002247

Abstract

Recognition of viruses by pattern recognition receptors (PRRs) causes interferon-β (IFN-β) induction, a key event in the anti-viral innate immune response, and also a target of viral immune evasion. Here the vaccinia virus (VACV) protein C6 is identified as an inhibitor of PRR-induced IFN-β expression by a functional screen of select VACV open reading frames expressed individually in mammalian cells. C6 is a member of a family of Bcl-2-like poxvirus proteins, many of which have been shown to inhibit innate immune signalling pathways. PRRs activate both NF-κB and IFN regulatory factors (IRFs) to activate the IFN-β promoter induction. Data presented here show that C6 inhibits IRF3 activation and translocation into the nucleus, but does not inhibit NF-κB activation. C6 inhibits IRF3 and IRF7 activation downstream of the kinases TANK binding kinase 1 (TBK1) and IκB kinase-ε (IKKε), which phosphorylate and activate these IRFs. However, C6 does not inhibit TBK1- and IKKε-independent IRF7 activation or the induction of promoters by constitutively active forms of IRF3 or IRF7, indicating that C6 acts at the level of the TBK1/IKKε complex. Consistent with this notion, C6 immunoprecipitated with the TBK1 complex scaffold proteins TANK, SINTBAD and NAP1. C6 is expressed early during infection and is present in both nucleus and cytoplasm. Mutant viruses in which the C6L gene is deleted, or mutated so that the C6 protein is not expressed, replicated normally in cell culture but were attenuated in two in vivo models of infection compared to wild type and revertant controls. Thus C6 contributes to VACV virulence and might do so via the inhibition of PRR-induced activation of IRF3 and IRF7.

Endoplasmic reticulum chaperone gp96 is essential for infection with vesicular stomatitis virus.Bloor S, Maelfait J, Krumbach R, Beyaert R, Randow F
Proc Natl Acad Sci U S A 2010 Apr 13; 107(15):6970-5

Abstract

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.