Publications by authors named "Teresa L M Thurston"

14 Publications

  • Page 1 of 1

Interesting Biochemistries in the Structure and Function of Bacterial Effectors.

Front Cell Infect Microbiol 2021 24;11:608860. Epub 2021 Feb 24.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom.

Bacterial effector proteins, delivered into host cells by specialized multiprotein secretion systems, are a key mediator of bacterial pathogenesis. Following delivery, they modulate a range of host cellular processes and functions. Strong selective pressures have resulted in bacterial effectors evolving unique structures that can mimic host protein biochemical activity or enable novel and distinct biochemistries. Despite the protein structure-function paradigm, effectors from different bacterial species that share biochemical activities, such as the conjugation of ubiquitin to a substrate, do not necessarily share structural or sequence homology to each other or the eukaryotic proteins that carry out the same function. Furthermore, some bacterial effectors have evolved structural variations to known protein folds which enable different or additional biochemical and physiological functions. Despite the overall low occurrence of intrinsically disordered proteins or regions in prokaryotic proteomes compared to eukaryotes proteomes, bacterial effectors appear to have adopted intrinsically disordered regions that mimic the disordered regions of eukaryotic signaling proteins. In this review, we explore examples of the diverse biochemical properties found in bacterial effectors that enable effector-mediated interference of eukaryotic signaling pathways and ultimately support pathogenesis. Despite challenges in the structural and functional characterisation of effectors, recent progress has been made in understanding the often unusual and fascinating ways in which these virulence factors promote pathogenesis. Nevertheless, continued work is essential to reveal the array of remarkable activities displayed by effectors.
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http://dx.doi.org/10.3389/fcimb.2021.608860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7943720PMC
February 2021

Salmonella Effector SteE Converts the Mammalian Serine/Threonine Kinase GSK3 into a Tyrosine Kinase to Direct Macrophage Polarization.

Cell Host Microbe 2020 01 17;27(1):41-53.e6. Epub 2019 Dec 17.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK. Electronic address:

Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. In addition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatory transcriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3, and phosphorylation of SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate signal transducer and activator of transcription 3 (STAT3) on tyrosine-705. This results in STAT3 activation, which along with GSK3 is required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker interleukin-4Rα (IL-4Rα). Overall, the conversion of GSK3 to a tyrosine-directed kinase represents a tightly regulated event that enables a bacterial virulence protein to reprogram innate immune signaling and establish an anti-inflammatory environment.
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http://dx.doi.org/10.1016/j.chom.2019.11.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6953433PMC
January 2020

Structure-function analyses of the bacterial zinc metalloprotease effector protein GtgA uncover key residues required for deactivating NF-κB.

J Biol Chem 2018 09 26;293(39):15316-15329. Epub 2018 Jul 26.

From the Section of Microbiology, MRC Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ and

The closely related type III secretion system zinc metalloprotease effector proteins GtgA, GogA, and PipA are translocated into host cells during infection. They then cleave nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) transcription factor subunits, dampening activation of the NF-κB signaling pathway and thereby suppressing host immune responses. We demonstrate here that GtgA, GogA, and PipA cleave a subset of NF-κB subunits, including p65, RelB, and cRel but not NF-κB1 and NF-κB2, whereas the functionally similar type III secretion system effector NleC of enteropathogenic and enterohemorrhagic cleaved all five NF-κB subunits. Mutational analysis of NF-κB subunits revealed that a single nonconserved residue in NF-κB1 and NF-κB2 that corresponds to the P1' residue Arg-41 in p65 prevents cleavage of these subunits by GtgA, GogA, and PipA, explaining the observed substrate specificity of these enzymes. Crystal structures of GtgA in its apo-form and in complex with the p65 N-terminal domain explained the importance of the P1' residue. Furthermore, the pattern of interactions suggested that GtgA recognizes NF-κB subunits by mimicking the shape and negative charge of the DNA phosphate backbone. Moreover, structure-based mutational analysis of GtgA uncovered amino acids that are required for the interaction of GtgA with p65, as well as those that are required for full activity of GtgA in suppressing NF-κB activation. This study therefore provides detailed and critical insight into the mechanism of substrate recognition by this family of proteins important for bacterial virulence.
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http://dx.doi.org/10.1074/jbc.RA118.004255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166728PMC
September 2018

Methylthioadenosine Suppresses Salmonella Virulence.

Infect Immun 2018 09 22;86(9). Epub 2018 Aug 22.

Department of Molecular Genetics and Microbiology, School of Medicine, Duke University, Durham, North Carolina, USA

In order to deploy virulence factors at appropriate times and locations, microbes must rapidly sense and respond to various metabolite signals. Previously, we showed a transient elevation of the methionine-derived metabolite methylthioadenosine (MTA) concentration in serum during systemic serovar Typhimurium infection. Here we explored the functional consequences of increased MTA concentrations on Typhimurium virulence. We found that MTA, but not other related metabolites involved in polyamine synthesis and methionine salvage, reduced motility, host cell pyroptosis, and cellular invasion. Further, we developed a genetic model of increased bacterial endogenous MTA production by knocking out the master repressor of the methionine regulon, Like MTA-treated Typhimurium, the Δ mutant displayed reduced motility, host cell pyroptosis, and invasion. These phenotypic effects of MTA correlated with suppression of flagellar and pathogenicity island 1 (SPI-1) networks. Typhimurium Δ had reduced virulence in oral and intraperitoneal infection of C57BL/6J mice independently of the effects of MTA on SPI-1. Finally, Δ bacteria induced a less severe inflammatory cytokine response in a mouse sepsis model. Together, these data indicate that exposure of Typhimurium to MTA or disruption of the bacterial methionine metabolism pathway suppresses Typhimurium virulence.
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http://dx.doi.org/10.1128/IAI.00429-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105896PMC
September 2018

Structural basis for the glycosyltransferase activity of the effector SseK3.

J Biol Chem 2018 04 15;293(14):5064-5078. Epub 2018 Feb 15.

From the Molecular Structure of Cell Signalling Laboratory, Francis Crick Institute, 1 Midland Road, London NW1 1AT, United Kingdom,

The -secreted effector SseK3 translocates into host cells, targeting innate immune responses, including NF-κB activation. SseK3 is a glycosyltransferase that transfers an -acetylglucosamine (GlcNAc) moiety onto the guanidino group of a target arginine, modulating host cell function. However, a lack of structural information has precluded elucidation of the molecular mechanisms in arginine and GlcNAc selection. We report here the crystal structure of SseK3 in its apo form and in complex with hydrolyzed UDP-GlcNAc. SseK3 possesses the typical glycosyltransferase type-A (GT-A)-family fold and the metal-coordinating DD motif essential for ligand binding and enzymatic activity. Several conserved residues were essential for arginine GlcNAcylation and SseK3-mediated inhibition of NF-κB activation. Isothermal titration calorimetry revealed SseK3's preference for manganese coordination. The pattern of interactions in the substrate-bound SseK3 structure explained the selection of the primary ligand. Structural rearrangement of the C-terminal residues upon ligand binding was crucial for SseK3's catalytic activity, and NMR analysis indicated that SseK3 has limited UDP-GlcNAc hydrolysis activity. The release of free -acetyl α-d-glucosamine, and the presence of the same molecule in the SseK3 active site, classified it as a retaining glycosyltransferase. A glutamate residue in the active site suggested a double-inversion mechanism for the arginine -glycosylation reaction. Homology models of SseK1, SseK2, and the orthologue NleB1 reveal differences in the surface electrostatic charge distribution, possibly accounting for their diverse activities. This first structure of a retaining GT-A arginine -glycosyltransferase provides an important step toward a better understanding of this enzyme class and their roles as bacterial effectors.
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http://dx.doi.org/10.1074/jbc.RA118.001796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892559PMC
April 2018

Salmonella SPI-2 Type III Secretion System Effectors: Molecular Mechanisms And Physiological Consequences.

Cell Host Microbe 2017 Aug;22(2):217-231

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, Armstrong Road, London SW7 2AZ, UK. Electronic address:

Serovars of Salmonella enterica cause both gastrointestinal and systemic diseases in a broad range of mammalian hosts, including humans. Salmonella virulence depends in part on its pathogenicity island 2 type III secretion system (SPI-2 T3SS), which is required to translocate at least 28 effector proteins from vacuolar-resident bacteria into host cells. Comparative genomic analysis reveals that all serovars encode a subset of "core" effectors, suggesting that they are critical for virulence in different hosts. An additional subset of effectors is found sporadically throughout different serovars, and several inhibit activation of the innate immune system. In this Review, we summarize the biochemical activities, host cell interaction partners, and physiological functions of SPI-2 T3SS effectors in the context of the selective pressures encountered by S. enterica in vivo. We also consider some of the remaining challenges to achieve a unified understanding of how effector activities work together to promote Salmonella virulence.
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http://dx.doi.org/10.1016/j.chom.2017.07.009DOI Listing
August 2017

Correction for Günster et al., "SseK1 and SseK3 Type III Secretion System Effectors Inhibit NF-κB Signaling and Necroptotic Cell Death in Salmonella-Infected Macrophages".

Infect Immun 2017 06 23;85(6). Epub 2017 May 23.

Section of Microbiology, Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom.

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http://dx.doi.org/10.1128/IAI.00242-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5442618PMC
June 2017

SseK1 and SseK3 Type III Secretion System Effectors Inhibit NF-κB Signaling and Necroptotic Cell Death in Salmonella-Infected Macrophages.

Infect Immun 2017 03 23;85(3). Epub 2017 Feb 23.

Section of Microbiology, Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, United Kingdom

Within host cells such as macrophages, translocates virulence (effector) proteins across its vacuolar membrane via the SPI-2 type III secretion system. Previously, it was shown that when expressed ectopically, the effectors SseK1 and SseK3 inhibit tumor necrosis factor alpha (TNF-α)-induced NF-κB activation. In this study, we show that ectopically expressed SseK1, SseK2, and SseK3 suppress TNF-α-induced, but not Toll-like receptor 4- or interleukin-induced, NF-κB activation. Inhibition required a DXD motif in SseK1 and SseK3, which is essential for the transfer of -acetylglucosamine to arginine residues (arginine-GlcNAcylation). During macrophage infection, SseK1 and SseK3 inhibited NF-κB activity in an additive manner. SseK3-mediated inhibition of NF-κB activation did not require the only known host-binding partner of this effector, the E3-ubiquitin ligase TRIM32. SseK proteins also inhibited TNF-α-induced cell death during macrophage infection. Despite SseK1 and SseK3 inhibiting TNF-α-induced apoptosis upon ectopic expression in HeLa cells, the percentage of infected macrophages undergoing apoptosis was SseK independent. Instead, SseK proteins inhibited necroptotic cell death during macrophage infection. SseK1 and SseK3 caused GlcNAcylation of different proteins in infected macrophages, suggesting that these effectors have distinct substrate specificities. Indeed, SseK1 caused the GlcNAcylation of the death domain-containing proteins FADD and TRADD, whereas SseK3 expression resulted in weak GlcNAcylation of TRADD but not FADD. Additional, as-yet-unidentified substrates are likely to explain the additive phenotype of a strain lacking both SseK1 and SseK3.
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http://dx.doi.org/10.1128/IAI.00010-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5328493PMC
March 2017

Growth inhibition of cytosolic Salmonella by caspase-1 and caspase-11 precedes host cell death.

Nat Commun 2016 11 3;7:13292. Epub 2016 Nov 3.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, Flowers Building Exhibition Road, London SW7 2AZ, UK.

Sensing bacterial products in the cytosol of mammalian cells by NOD-like receptors leads to the activation of caspase-1 inflammasomes, and the production of the pro-inflammatory cytokines interleukin (IL)-18 and IL-1β. In addition, mouse caspase-11 (represented in humans by its orthologs, caspase-4 and caspase-5) detects cytosolic bacterial LPS directly. Activation of caspase-1 and caspase-11 initiates pyroptotic host cell death that releases potentially harmful bacteria from the nutrient-rich host cell cytosol into the extracellular environment. Here we use single cell analysis and time-lapse microscopy to identify a subpopulation of host cells, in which growth of cytosolic Salmonella Typhimurium is inhibited independently or prior to the onset of cell death. The enzymatic activities of caspase-1 and caspase-11 are required for growth inhibition in different cell types. Our results reveal that these proteases have important functions beyond the direct induction of pyroptosis and proinflammatory cytokine secretion in the control of growth and elimination of cytosolic bacteria.
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http://dx.doi.org/10.1038/ncomms13292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097160PMC
November 2016

TBK1 directs WIPI2 against Salmonella.

Autophagy 2016 12 18;12(12):2508-2509. Epub 2016 Oct 18.

a MRC Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Francis Crick Avenue , Cambridge , UK.

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. We therefore propose that buffering TBK1 recruitment against potential bacterial interference might be of evolutionary advantage to the host.
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http://dx.doi.org/10.1080/15548627.2016.1235126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5172500PMC
December 2016

Erratum for O'Neill et al., Cytosolic Replication of Group A Streptococcus in Human Macrophages.

mBio 2016 06 28;7(3). Epub 2016 Jun 28.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, United Kingdom

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http://dx.doi.org/10.1128/mBio.00931-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937218PMC
June 2016

Cytosolic Replication of Group A Streptococcus in Human Macrophages.

mBio 2016 Apr 12;7(2):e00020-16. Epub 2016 Apr 12.

MRC Centre for Molecular Bacteriology and Infection, Imperial College London, United Kingdom

Unlabelled: As key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, including group A Streptococcus(GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from within human neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using several quantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealed that despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas the major virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impaired for intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophage cytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted by the autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture, indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicate within viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagy receptors to bacteria.

Importance: Classically regarded as an extracellular pathogen, GAS can persist within human epithelial cells, as well as neutrophils and macrophages. Some studies suggest that GAS can modulate its intracellular vacuole to promote survival and perhaps replicate in macrophages. However, an in-depth single-cell analysis of the dynamics of survival and replication is lacking. We used macrophage-like cell lines and primary macrophages to measure the intracellular growth of GAS at both the population and single-cell levels. While CFU counts revealed no increase in overall bacterial growth, quantitative fluorescence microscopy, flow cytometry, and time-lapse imaging revealed bacterial replication in a proportion of infected macrophages. This study emphasizes the importance of single-cell analysis especially when studying the intracellular fate of a pathogen that is cytotoxic and displays heterogeneity in terms of intracellular killing and growth. To our knowledge, this study provides the first direct visualization of GAS replication inside human cells.
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http://dx.doi.org/10.1128/mBio.00020-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959517PMC
April 2016

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

Nature 2012 Jan 15;482(7385):414-8. Epub 2012 Jan 15.

MRC Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Hills Road, Cambridge CB2 0QH, UK.

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.
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http://dx.doi.org/10.1038/nature10744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3343631PMC
January 2012

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

Nat Immunol 2009 Nov 11;10(11):1215-21. Epub 2009 Oct 11.

Medical Research Council Laboratory of Molecular Biology, Division of Protein and Nucleic Acid Chemistry, Cambridge, UK.

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.
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http://dx.doi.org/10.1038/ni.1800DOI Listing
November 2009