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A surprising sweetener from enteropathogenic Escherichia coli a
Jaclyn S. Pearson & Elizabeth L Hartland
a
a
Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia Accepted author version posted online: 23 Dec 2014.
Click for updates To cite this article: Jaclyn S. Pearson & Elizabeth L Hartland (2014): A surprising sweetener from enteropathogenic Escherichia coli, Gut Microbes, DOI: 10.4161/19490976.2014.983762 To link to this article: http://dx.doi.org/10.4161/19490976.2014.983762
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A surprising sweetener from enteropathogenic Escherichia coli
1
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Jaclyn S. Pearson1 and Elizabeth L Hartland1*
Department of Microbiology and Immunology, University of Melbourne at the Peter
an M
Addendum for:
Pearson JS, Giogha C, Ong SY, Kennedy CL, Kelly M, Robinson KS, Wong T, Mansell
ed
A, Riedmaier P, Zaid, A, Mühlen S, Oates C, Crepin VF, Marches O, Ang CS,
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Williamson NA, O’Reilly L, Bankovacki A, Nachbur U, Infusini G, Webb AS, Silke J, Strasser A, Frankel G and Hartland EL. A type III effector antagonizes death receptor
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signalling during bacterial gut infection. Nature. 2013. 501:247-51
and
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correspondence to
[email protected] us
*
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Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
Li S, Zhang L, Yao Q, Li L, Dong N, Rong J, Gao W, Ding X, Sun L, Chen X, Chen S, Shao F. Pathogen blocks host death receptor signalling by arginine GlcNAcylation of death domains. Nature 2013. 501: 242-246.
1
Abstract Infections with enteropathogenic Escherichia coli (EPEC) are remarkably devoid of gut inflammation and necrotic damage compared to infections caused by invasive pathogens such as Salmonella and Shigella. Recently, we observed that EPEC blocks cell death
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using the type III secretion system (T3SS) effector NleB. NleB mediated post-
translational modification of death domain containing adaptor proteins by the covalent
cr
attachment of N-acetylglucosamine (GlcNAc) to a conserved arginine in the death
us
domain. N-linked glycosylation of arginine has not previously been reported in
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defined. Although the addition of a single GlcNAc to arginine is a seemingly slight alteration, the impact of NleB is considerable as arginine in this location is critical for
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death domain interactions and death receptor induced apoptosis. Hence, by blocking cell
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gut epithelium.
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death, NleB promotes enterocyte survival and thereby prolongs EPEC attachment to the
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mammalian cell biology and the precise biochemistry of this modification is not yet
2
Glycosylation as a key biological process Protein glycosylation is the most common post-translational modification characterised to date with more than half of all human proteins estimated to contain at least one glycan
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chain 1, 2. Glycans associated with the cell surface and intracellular proteins and lipids influence a diverse range of biological processes in animal systems 3. For example,
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glycan structures contribute to the correct folding and conformational stability of a range
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of cellular proteins synthesised at the endoplasmic reticulum (ER) 4. Surface glycans
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events 5, 6 and also mediate interactions between the vertebrate host and microbial pathogens 7-9. Other processes influenced by glycan structures include innate immune
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signaling, autoimmune reactions and tumour metastases 2, 8.
ed
Glycan structure is determined by the concerted action of numerous genes that code for glycosyltransferases, glycosidases, and other enzymes that synthesise and remodel glycan
pt
chains, as well as accessory enzymes involved in the synthesis and transport of nucleotide
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sugars. Glycosyltransferases (GTs) constitute a family of enzymes that catalyse the biosynthesis of glycosidic linkages by the transfer of a sugar residue from an activated nucleotide sugar donor (such as UDP) to specific acceptor molecules 10. GTs can be
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serve as ligands for glycan-binding proteins in cellular trafficking, adhesion and signaling
classified into families based on their amino acid sequence similarities and their particular GT fold. There are currently 4 known GT folds. GT-A and GT-B are well characterised and are the predominate folds within known GTs, while GT-C and GT-D are more recently described 11.
3
In almost all glycoproteins, carbohydrate units are coupled to a polypeptide backbone either by N- or O-glycosidic bonds or by both types of linkage 2. Alternatively glycans can be linked to the C2 position of Trp (C-mannosylation) or as a linker structure between glycophosphatidylinositol anchors to protein backbones 12. N-linked
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glycosylation is the most studied type of protein glycosylation in eukaryotes and is
essential for efficient protein folding and assembly in the ER and anterograde trafficking
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through the secretory pathway 12. N-glycans are synthesised via the activity of
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oligosaccharidetransferases (OST) at the luminal side of ER membrane. Here, OST
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polypeptide chain during translation. The glycans are linked covalently to asparagine residues within an acceptor peptide sequon, Asn-X-(Ser/Thr) (alternatively and less
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frequently Asn-X-Cys), where X is any amino acid except Pro 12. This specific sequon
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denotes potential N-glycan sites and does not always predetermine glycosylation events.
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O-linked glycosylation is a common covalent modification of mammalian glycoproteins that typically occurs on the hydroxyl moieties of Ser/Thr, and sometimes hydroxylysine
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(collagen) and Tyr (glycogenin) residues 13. O-linked glycans can be grouped into two categories, mucin and non-mucin glycans. Mucin O-glycans are found in all mucous secretions as transmembrane glycoproteins of cell surfaces and as secreted mucins 13.
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transfers glycans from a lipid-linked oligosaccharide (LLO) precursor to a nascent
They begin with covalently α-linked N-acetylgalactosamine (GalNAc) molecules linked to Ser or Thr residues and can be extended with galactose, N-acetylglucosamine (GlcNAc), fucose or sialic acid. Overall mucin O-glycans are highly heterogeneous, with up to 8 possible core structures and hundreds of side chain variations. Non-mucin Oglycans include α-linked O-fucose, β-linked O-xylose, α-linked O-mannose, β-linked O4
GlcNAc, and α- or β-linked O-glucose glycans. O-GlcNAc is one of the most abundant post-translational modifications (PTM) in cytoplasmic and nuclear compartments of all metazoan cells 14. In this form of O-linked glycosylation, the GlcNAc is not elongated or modified to form more complex structures. It is a highly dynamic modification that
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cycles rapidly on and off due to the concerted action of the cellular enzymes, O-GlcNAc
transferase (OGT) and O-GlcNAcase (OGA) 14. OGT is a GT that catalyses the addition
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of a single GlcNAc from UDP-GlcNAc to Ser or Thr residues of target proteins, whereas
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OGA actively removes GlcNAc moieties. A number of consensus sites for O-
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The rapid cycling of O-GlcNAcylation is related to a complex and dynamic interplay
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between O-GlcNAc and O-phosphate, which together regulate a diverse array of proteins involved in signaling, transcription and cytoskeletal regulation. O-GlcNAc can occur in
ed
competition with Ser/Thr-O-phosphorylation, and is therefore an important regulatory
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mechanism in cellular processes 15. O-GlcNAc modifications are highly abundant in the nucleus, often occurring on transcriptional or translational regulatory factors, and also on
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cytoplasmic proteins involved in stress responses, cell signaling, energy metabolism, and cytoskeletal regulation. Thus it is not surprising that O-GlcNAc cycling is highly responsive to external stimuli, some of which include insulin, high glucose and cellular
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GlcNAcylation have been described 12.
stress. In some cases, altered or dysregulated O-GlcNAcylation of proteins contributes to development of neurodegenerative diseases including Alzheimer’s and Parkinson’s disease, and also contributes to the development of diabetes in mammals 14. OGT alleles are also essential in embryogenesis 16.
5
OGT is regulated by a number of complex mechanisms including transcriptional regulation of its expression, differential mRNA splicing, proteolytic processing, PTM, and self-oligomerisation
14
. The most important factor in its regulation however is the
cellular concentration of the donor substrate, UDP-GlcNAc. In a cell UDP-GlcNAc
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levels are highly sensitive to carbohydrate, fatty acid, energy and nitrogen metabolic fluxes, and can change rapidly 14. Upon transfer of GlcNAc, UDP release potently
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inhibits OGT and thus modulates further O-GlcNAcylation events.
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The effect of EPEC infection on general cellular glycosylation events is unknown but recently, Gao et al. 17 identified the T3SS effector NleB from A/E pathogens as a novel
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GlcNAc transferase. NleB and the SseK homologues from Salmonella displayed significant structural homology to the GT-8 family of GTs 17. These enzymes typically
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adopt a GT-A fold consisting of an α/β/α sandwich that resembles a Rossmann fold and a
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DxD catalytic motif which requires a divalent cation for activity, typically Mn2+ 18. The DxD motif interacts with phosphate groups of a nucleotide sugar donor through the
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coordination of the divalent cation and mediates sugar transfer to target proteins.
NleB was previously implicated in the inhibition of NF-κB activation upon EPEC
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EPEC produces a novel glycosyltransferase T3SS effector, NleB
infection
19, 20 21
. Gao et al. 17 reasoned that if O-GlcNAc is involved in the regulation of
signaling networks, including NF-κB 22, NleB may suppress NF-κB activation through its GlcNAc transferase activity. While the study from Gao et al. 17 identified GAPDH as a potential target of NleB activity, exhaustive site-directed mutagenesis of serine or
6
threonine residues within the GAPDH sequence did not reveal a specific amino acid that was modified by NleB.
Subsequently, two independent studies identified host death domain (DD)-containing
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proteins as alternative targets for NleB 23, 24. Both groups utilised the yeast two-hybrid system with NleB as bait to identify FADD, RIPK1 24 and TRADD 23 as potential
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mammalian binding partners for NleB. Both studies showed that NleB functioned as a
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GlcNAc transferase to add a single GlcNAc residue to a conserved arginine residue
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RIPK1 and TRADD to form homotypic and heterotypic DD interactions, thereby shutting down death receptor signaling pathways including inflammation, apoptosis and
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necroptosis 23, 24.
While Li et al 23 found that R235 within the DD of TRADD was specifically modified by
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NleB, our work identified the equivalent residue, R117, within the DD of FADD as the
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target of NleB activity 24. In both studies, mutation of the NleB DxD catalytic motif or the conserved Arg residue within FADD or TRADD, abolished transfer of GlcNAc to the
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target proteins 23, 24. Further to this Li et al. 23 demonstrated that the DDs of TRADD, FADD and RIPK1 were efficiently modified by NleB during in EPEC infection, whereas the DDs of FAS and TNFR1 were only modified by NleB expressed ectopically and not
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within the DD of these proteins. The GlcNAc addition blocked the ability of FADD,
during EPEC infection. Other DD proteins including MyD88 and IRAK1 were not modified by NleB, which is likely due to the fact that they do not contain the equivalent conserved Arg residue 23. Importantly, the study by Li et al. 23 determined that GAPDH was not GlcNAcylated by either OGT or NleB.
7
NleB and inhibition of cell death signaling Consistent with previous work 17, 19, 20, Li et al. 23 also showed that ectopic expression of NleB1 inhibited NF-κB activation in cultured epithelial cells. Our study however, showed that NleB1 had no impact on production of the NF-κB-dependent inflammatory cytokine
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IL-8 during EPEC infection, despite the ability of NleB to modify TRADD and RIPK1,
two key mediators of signaling via TNFR1 23, 24. Given the additional involvement of DD
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proteins in cell death signaling and the strong affinity of NleB for FADD 23, 24, we
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investigated the consequences of NleB-mediated inhibition of Fas-induced extrinsic
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nleB mutants of the murine A/E pathogen, Citrobacter rodentium (CR) are attenuated for
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colonization 25 and more recent experiments have shown that the GlcNAc transferase activity of NleB is important for this phenotype 17, 23. Given the strong inhibition of Fas-
ed
signaling by NleB in vitro through GlcNAcylation of FADD 23, 24, we postulated that
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attenuation of the nleB CR mutant in vivo was due to the elimination of infected enterocytes by Fas ligand (FasL) positive immune cells. Hence in the absence of NleB,
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infected cells would undergo apoptosis and slough off into the lumen of the gut, thereby reducing pathogen load. Consistent with this, the nleB CR mutant induced greater activation of caspase-8, a marker of FasL-induced apoptosis than wild type CR 24.
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apoptosis rather than TNFR1-induced inflammation on the outcome of infection.
Moreover Fas- or FasL-deficient mice experienced greater colitis and took longer to clear CR than wild type mice. Interestingly, the reduced colonization exhibited by the nleB mutant was reversed in Fas-deficient mice, suggesting that nleB mutant attenuation was indeed due to functional Fas signaling 24.
8
The GlcNAcylation of Arg117 in the DD of FADD is a disabling modification. Arg117 is essential for death domain interactions between FADD and the DD of Fas as well as between TRADD and FADD 26. Mutation of Arg117 abrogates cell death signalling through Fas or TNFR1 26. The essential role of this amino acid in Fas signaling is also
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evident in the requirement of Arg117 in FADD for embryogenesis 27, and this has been
explained by structural elucidation of the Fas-FADD signalosome that puts Arg117 at the
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interface of adjacent FADD molecules and the DD of Fas and FADD 28. Upon
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engagement of Fas by FasL, Fas DD binding to FADD increases the overall stability of
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Conclusion
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Prior to the discovery that an EPEC effector protein could mediate arginine glycosylation, there has been only one published report of the same phenomenon in a
ed
biological system. In a brief report, Singh et al. 30 described a protein known as
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amylogenin from sweet corn that autocatalytically added a single β-glucose residue to its own arginine. This self-glucosylation of amylogenin occurred in vitro in the presence of
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UDP-[14C]glucose, a modification that could be reversed by the addition of β-glucosidase 30
. The authors suggested the reaction might be important in priming starch synthesis in
corn however no further reports on the activity of amylogenin or the nature of the
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FADD, including in the death effector domain that binds and stabilizes procaspase-8 29.
modification have been produced. Hence, NleB remains the first example of a glycosyltransferase that can modify arginine in a mammalian cell. The unique nature of this N-linked GlcNAcylation implies that the modification is irreversible, as no mammalian enzymes exist, such as OGAs, to remove GlcNAc from arginine. However,
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to date this has not been investigated and more work is needed to understand the biochemical basis of this surprising activity.
Acknowledgements
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Research Council (NHMRC) APP1044061 awarded to ELH.
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This work was supported by funding from the Australian National Health and Medical
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