Publications by authors named "Steve Matthews"

80 Publications

Why should HCWs receive priority access to vaccines in a pandemic?

BMC Med Ethics 2021 06 27;22(1):79. Epub 2021 Jun 27.

Plunkett Centre for Ethics, Australian Catholic University, 7 Ice Street, Darlinghurst, NSW, 2010, Australia.

Background: Viral pandemics present a range of ethical challenges for policy makers, not the least among which are difficult decisions about how to allocate scarce healthcare resources. One important question is whether healthcare workers (HCWs) should receive priority access to a vaccine in the event that an effective vaccine becomes available. This question is especially relevant in the coronavirus pandemic with governments and health authorities currently facing questions of distribution of COVID-19 vaccines.

Main Text: In this article, we critically evaluate the most common ethical arguments for granting healthcare workers priority access to a vaccine. We review the existing literature on this topic, and analyse both deontological and utilitarian arguments in favour of HCW prioritisation. For illustrative purposes, we focus in particular on the distribution of a COVID-19 vaccine. We also explore some practical complexities attendant on arguments in favour of HCW prioritisation.

Conclusions: We argue that there are deontological and utilitarian cases for prioritising HCWs. Indeed, the widely held view that we should prioritise HCWs represents an example of ethical convergence. Complexities arise, however, when considering who should be included in the category of HCW, and who else should receive priority in addition to HCWs.
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http://dx.doi.org/10.1186/s12910-021-00650-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8236218PMC
June 2021

A Bacteriophage DNA Mimic Protein Employs a Non-specific Strategy to Inhibit the Bacterial RNA Polymerase.

Front Microbiol 2021 2;12:692512. Epub 2021 Jun 2.

BioBank, The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China.

DNA mimicry by proteins is a strategy that employed by some proteins to occupy the binding sites of the DNA-binding proteins and deny further access to these sites by DNA. Such proteins have been found in bacteriophage, eukaryotic virus, prokaryotic, and eukaryotic cells to imitate non-coding functions of DNA. Here, we report another phage protein Gp44 from bacteriophage SPO1 of , employing mimicry as part of unusual strategy to inhibit host RNA polymerase. Consisting of three simple domains, Gp44 contains a DNA binding motif, a flexible DNA mimic domain and a random-coiled domain. Gp44 is able to anchor to host genome and interact bacterial RNA polymerase the β and β' subunit, resulting in bacterial growth inhibition. Our findings represent a non-specific strategy that SPO1 phage uses to target different bacterial transcription machinery regardless of the structural variations of RNA polymerases. This feature may have potential applications like generation of genetic engineered phages with Gp44 gene incorporated used in phage therapy to target a range of bacterial hosts.
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http://dx.doi.org/10.3389/fmicb.2021.692512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8208478PMC
June 2021

Moral Self-Orientation in Alzheimer's Dementia.

Authors:
Steve Matthews

Kennedy Inst Ethics J 2020 ;30(2):141-166

People with Alzheimer's dementia experience significant neuropsychological decline, and this seems to threaten their sense of self. Yet they continue to have regard for their moral standing, especially from the feedback they receive from others in relation to such things as pride in their work, retaining a valued role, or acting out of a sense of purpose. This continuing self-regard is based on a self-image which often persists through memory loss. I will argue that in care settings the self-image ought to be assumed to remain intact. Treating a person with Alzheimer's dementia supportively and respectfully as the person with a certain role or identity-say as scientist, musician, janitor, parent, or friend-fosters an environment in which they are best able to retain what I call moral self-orientation. The latter notion is central to the well-being of social persons, and so it takes on special significance for people with dementia because, although their remembering selves are fragmenting, their self-image persists. Normative aspects of the self-image, I argue, require a social framework of support to sustain the self-image.
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http://dx.doi.org/10.1353/ken.2020.0009DOI Listing
January 2020

RssB-mediated σ Activation is Regulated by a Two-Tier Mechanism via Phosphorylation and Adaptor Protein - IraD.

J Mol Biol 2021 02 18;433(3):166757. Epub 2020 Dec 18.

BioBank, The First Affiliated Hospital of Xi'an Jiaotong University, Shaanxi 710061, China; Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, SW7 2AZ London, United Kingdom; Instrument Analysis Center of Xi'an Jiaotong University, Xi'an, Shaanxi 710049, China. Electronic address:

Regulation of bacterial stress responding σ is a sophisticated process and mediated by multiple interacting partners. Controlled proteolysis of σ is regulated by RssB which maintains minimal level of σ during exponential growth but then elevates σ level while facing stresses. Bacteria developed different strategies to regulate activity of RssB, including phosphorylation of itself and production of anti-adaptors. However, the function of phosphorylation is controversial and the mechanism of anti-adaptors preventing RssB-σ interaction remains elusive. Here, we demonstrated the impact of phosphorylation on the activity of RssB and built the RssB-σ complex model. Importantly, we showed that the phosphorylation site - D58 is at the interface of RssB-σ complex. Hence, mutation or phosphorylation of D58 would weaken the interaction of RssB with σ. We found that the anti-adaptor protein IraD has higher affinity than σ to RssB and its binding interface on RssB overlaps with that for σ. And IraD-RssB complex is preferred over RssB-σ in solution, regardless of the phosphorylation state of RssB. Our study suggests that RssB possesses a two-tier mechanism for regulating σ. First, phosphorylation of RssB provides a moderate and reversible tempering of its activity, followed by a specific and robust inhibition via the anti-adaptor interaction.
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http://dx.doi.org/10.1016/j.jmb.2020.166757DOI Listing
February 2021

Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer.

Nat Commun 2020 07 30;11(1):3807. Epub 2020 Jul 30.

Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, DD1 5EH, Dundee, UK.

The human genome contains an estimated 600 ubiquitin E3 ligases, many of which are single-subunit E3s (ssE3s) that can bind to both substrate and ubiquitin-loaded E2 (E2~Ub). Within ssE3s structural disorder tends to be located in substrate binding and domain linking regions. RNF4 is a ssE3 ligase with a C-terminal RING domain and disordered N-terminal region containing SUMO Interactions Motifs (SIMs) required to bind SUMO modified substrates. Here we show that, although the N-terminal region of RNF4 bears no secondary structure, it maintains a compact global architecture primed for SUMO interaction. Segregated charged regions within the RNF4 N-terminus promote compaction, juxtaposing RING domain and SIMs to facilitate substrate ubiquitination. Mutations that induce a more extended shape reduce ubiquitination activity. Our result offer insight into a key step in substrate ubiquitination by a member of the largest ubiquitin ligase subtype and reveal how a defined architecture within a disordered region contributes to E3 ligase function.
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http://dx.doi.org/10.1038/s41467-020-17647-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7393505PMC
July 2020

NMR insights into the pre-amyloid ensemble and secretion targeting of the curli subunit CsgA.

Sci Rep 2020 05 12;10(1):7896. Epub 2020 May 12.

Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.

The biofilms of Enterobacteriaceae are fortified by assembly of curli amyloid fibres on the cell surface. Curli not only provides structural reinforcement, but also facilitates surface adhesion. To prevent toxic intracellular accumulation of amyloid precipitate, secretion of the major curli subunit, CsgA, is tightly regulated. In this work, we have employed solution state NMR spectroscopy to characterise the structural ensemble of the pre-fibrillar state of CsgA within the bacterial periplasm, and upon recruitment to the curli pore, CsgG, and the secretion chaperone, CsgE. We show that the N-terminal targeting sequence (N) of CsgA binds specifically to CsgG and that its subsequent sequestration induces a marked transition in the conformational ensemble, which is coupled to a preference for CsgE binding. These observations lead us to suggest a sequential model for binding and structural rearrangement of CsgA at the periplasmic face of the secretion machinery.
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http://dx.doi.org/10.1038/s41598-020-64135-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217966PMC
May 2020

The major subunit of widespread competence pili exhibits a novel and conserved type IV pilin fold.

J Biol Chem 2020 05 9;295(19):6594-6604. Epub 2020 Apr 9.

Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, (ComGC) and (ComGC), revealing that this pilin displays extensive structural conservation. Strikingly, ComGC and ComGC exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.
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http://dx.doi.org/10.1074/jbc.RA120.013316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212644PMC
May 2020

Resonance assignments of N-terminal receiver domain of sigma factor S regulator RssB from Escherichia coli.

Biomol NMR Assign 2019 10 21;13(2):333-337. Epub 2019 Jun 21.

BioBank, The First Affiliated Hospital of Xi'an Jiaotong University, Shaanxi, 710061, China.

Sigma factor S (σ) are master regulator responsible for the survival of bacteria under extreme conditions. Bacteria start specific gene expression via σ promoter recognition, activating various responses to cope with external conditions. Although this self-protection mechanism is vital for bacteria to propagate and evolve, there are many puzzling research questions to be answered. For example, while interactions between σ, transcription regulator RssB, and anti-adaptor Ira proteins are believed to be responsible for controlling the cellular level of σ, their competition mechanism among them remains elusive. Furthermore, there are still debates on the location of the interface of Ira proteins and RssB and whether phosphorylation on the receiver domain is essential for σ activation remains elusive. While there is one crystal structure for the Escherichia coli receiver domain deposited in the database, the missing regions in the structure become an obstacle for functional and interactive studies. Despite attempts, there is no structure for any protein complex in this important biological process, making it one overlooked area in bacterial transcription. Here, using solution-state NMR, our near-complete resonance assignment for the receiver domain of E. coli RssB provides a basis for future structure determination and interaction studies with its many known and putative ligands.
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http://dx.doi.org/10.1007/s12104-019-09901-1DOI Listing
October 2019

Global biochemical and structural analysis of the type IV pilus from the Gram-positive bacterium .

J Biol Chem 2019 04 5;294(17):6796-6808. Epub 2019 Mar 5.

From the Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London SW7 2AZ, United Kingdom,

Type IV pili (Tfp) are functionally versatile filaments, widespread in prokaryotes, that belong to a large class of filamentous nanomachines known as type IV filaments (Tff). Although Tfp have been extensively studied in several Gram-negative pathogens where they function as key virulence factors, many aspects of their biology remain poorly understood. Here, we performed a global biochemical and structural analysis of Tfp in a recently emerged Gram-positive model, In particular, we focused on the five pilins and pilin-like proteins involved in Tfp biology in We found that the two major pilins, PilE1 and PilE2, (i) follow widely conserved principles for processing by the prepilin peptidase PilD and for assembly into filaments; (ii) display only one of the post-translational modifications frequently found in pilins, a methylated N terminus; (iii) are found in the same heteropolymeric filaments; and (iv) are not functionally equivalent. The 3D structure of PilE1, solved by NMR, revealed a classical pilin-fold with a highly unusual flexible C terminus. Intriguingly, PilE1 more closely resembles pseudopilins forming shorter Tff than Tfp-forming major pilins, underlining the evolutionary relatedness among different Tff. Finally, we show that Tfp contain a low abundance of three additional proteins processed by PilD, the minor pilins PilA, PilB, and PilC. These findings provide the first global biochemical and structural picture of a Gram-positive Tfp and have fundamental implications for our understanding of a widespread class of filamentous nanomachines.
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http://dx.doi.org/10.1074/jbc.RA118.006917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6497953PMC
April 2019

H, C and N NMR assignments of Bacillus subtilis bacteriophage SPO1 protein Gp46.

Biomol NMR Assign 2019 04 4;13(1):245-247. Epub 2019 Mar 4.

BioBank, The First Affiliated Hospital of Xi'an Jiaotong University, Shaanxi, 710061, China.

Bacterial antibiotic resistance is a serious threat to public health and bacteriophage therapy is an alternative for antibiotics in the era of multidrug resistance. While phage draws attention in fighting bacterial infection and is used in protein display to study macromolecular interactions, the molecular machinery of the host invasion mechanism remains largely unclear for many bacteriophages. Despite recent studies on T4 and T7 phages of Gram-negative model organism Escherichia coli revealing many interesting features of their invasive strategies, the studies on Gram-positive bacterial phages still lag far behind their counterparts. SPO1 is a lytic phage of model organism Bacillus subtilis and one of the best studied Gram-positive bacterial phages. SPO1 features a unique Host Takeover Module coding for 24 proteins which show little similarity to any previously known proteins. Gp46, located in this module, is an acidic protein that is produced by SPO1 presumably during the host takeover event. Here we describe the complete resonance assignment of Gp46 as the basis for the first structure determination of SPO1 phage protein and further mechanism study.
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http://dx.doi.org/10.1007/s12104-019-09885-yDOI Listing
April 2019

Xenogeneic Regulation of the Bacterial Transcription Machinery.

J Mol Biol 2019 09 15;431(20):4078-4092. Epub 2019 Feb 15.

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

The parasitic life cycle of viruses involves the obligatory subversion of the host's macromolecular processes for efficient viral progeny production. Viruses that infect bacteria, bacteriophages (phages), are no exception and have evolved sophisticated ways to control essential biosynthetic machineries of their bacterial prey to benefit phage development. The xenogeneic regulation of bacterial cell function is a poorly understood area of bacteriology. The activity of the bacterial transcription machinery, the RNA polymerase (RNAP), is often regulated by a variety of mechanisms involving small phage-encoded proteins. In this review, we provide a brief overview of known phage proteins that interact with the bacterial RNAP and compare how two prototypical phages of Escherichia coli, T4 and T7, use small proteins to "puppeteer" the bacterial RNAP to ensure a successful infection.
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http://dx.doi.org/10.1016/j.jmb.2019.02.008DOI Listing
September 2019

Efficient Single-Strand Break Repair Requires Binding to Both Poly(ADP-Ribose) and DNA by the Central BRCT Domain of XRCC1.

Cell Rep 2019 01;26(3):573-581.e5

Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9RQ, UK; Division of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW1E 6BT, UK. Electronic address:

XRCC1 accelerates repair of DNA single-strand breaks by acting as a scaffold protein for the recruitment of Polβ, LigIIIα, and end-processing factors, such as PNKP and APTX. XRCC1 itself is recruited to DNA damage through interaction of its central BRCT domain with poly(ADP-ribose) chains generated by PARP1 or PARP2. XRCC1 is believed to interact directly with DNA at sites of damage, but the molecular basis for this interaction within XRCC1 remains unclear. We now show that the central BRCT domain simultaneously mediates interaction of XRCC1 with poly(ADP-ribose) and DNA, through separate and non-overlapping binding sites on opposite faces of the domain. Mutation of residues within the DNA binding site, which includes the site of a common disease-associated human polymorphism, affects DNA binding of this XRCC1 domain in vitro and impairs XRCC1 recruitment and retention at DNA damage and repair of single-strand breaks in vivo.
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http://dx.doi.org/10.1016/j.celrep.2018.12.082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6334254PMC
January 2019

Physical Determinants of Amyloid Assembly in Biofilm Formation.

mBio 2019 01 8;10(1). Epub 2019 Jan 8.

Department of Chemistry, University of Cambridge, Cambridge, United Kingdom

A wide range of bacterial pathogens have been shown to form biofilms, which significantly increase their resistance to environmental stresses, such as antibiotics, and are thus of central importance in the context of bacterial diseases. One of the major structural components of these bacterial biofilms are amyloid fibrils, yet the mechanism of fibril assembly and its importance for biofilm formation are currently not fully understood. By studying fibril formation , in a model system of two common but unrelated biofilm-forming proteins, FapC from and CsgA from , we found that the two proteins have a common aggregation mechanism. In both systems, fibril formation proceeds via nucleated growth of linear fibrils exhibiting similar measured rates of elongation, with negligible fibril self-replication. These similarities between two unrelated systems suggest that convergent evolution plays a key role in tuning the assembly kinetics of functional amyloid fibrils and indicates that only a narrow window of mechanisms and assembly rates allows for successful biofilm formation. Thus, the amyloid assembly reaction is likely to represent a means for controlling biofilm formation, both by the organism and by possible inhibitory drugs. Biofilms are generated by bacteria, embedded in the formed extracellular matrix. The biofilm's function is to improve the survival of a bacterial colony through, for example, increased resistance to antibiotics or other environmental stresses. Proteins secreted by the bacteria act as a major structural component of this extracellular matrix, as they self-assemble into highly stable amyloid fibrils, making the biofilm very difficult to degrade by physical and chemical means once formed. By studying the self-assembly mechanism of the fibrils from their monomeric precursors in two unrelated bacteria, our experimental and theoretical approaches shed light on the mechanism of functional amyloid assembly in the context of biofilm formation. Our results suggest that fibril formation may be a rate-limiting step in biofilm formation, which in turn has implications on the protein self-assembly reaction as a target for potential antibiotic drugs.
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http://dx.doi.org/10.1128/mBio.02279-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325246PMC
January 2019

Archaic and alternative chaperones preserve pilin folding energy by providing incomplete structural information.

J Biol Chem 2018 11 18;293(44):17070-17080. Epub 2018 Sep 18.

From the Department of Chemistry, University of Turku, Joint Biotechnology Laboratory (JBL), Arcanum, Vatselankatu 2, Turku FIN-20500, Finland and

Adhesive pili are external component of fibrous adhesive organelles and help bacteria attach to biotic or abiotic surfaces. The biogenesis of adhesive pili via the chaperone-usher pathway (CUP) is independent of external energy sources. In the classical CUP, chaperones transport assembly-competent pilins in a folded but expanded conformation. During donor-strand exchange, pilins subsequently collapse, producing a tightly packed hydrophobic core and releasing the necessary free energy to drive fiber formation. Here, we show that pilus biogenesis in non-classical, archaic, and alternative CUPs uses a different source of conformational energy. High-resolution structures of the archaic Csu-pili system from revealed that non-classical chaperones employ a short donor strand motif that is insufficient to fully complement the pilin fold. This results in chaperone-bound pilins being trapped in a substantially unfolded intermediate. The exchange of this short motif with the longer donor strand from adjacent pilin provides the full steric information essential for folding, and thereby induces a large unfolded-to-folded conformational transition to drive assembly. Our findings may inform the development of anti-adhesion drugs (pilicides) to combat bacterial infections.
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http://dx.doi.org/10.1074/jbc.RA118.004170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222105PMC
November 2018

Structural Basis of Phosphatidic Acid Sensing by APH in Apicomplexan Parasites.

Structure 2018 08 14;26(8):1059-1071.e6. Epub 2018 Jun 14.

Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, London SW7 2AZ, UK; BioBank, First Affiliated Hospital, School of Medicine, Xi'an Jiaotong University, Xi'an, 710049, P. R. China. Electronic address:

Plasmodium falciparum and Toxoplasma gondii are obligate intracellular parasites that belong to the phylum of Apicomplexa and cause major human diseases. Their access to an intracellular lifestyle is reliant on the coordinated release of proteins from the specialized apical organelles called micronemes and rhoptries. A specific phosphatidic acid effector, the acylated pleckstrin homology domain-containing protein (APH) plays a central role in microneme exocytosis and thus is essential for motility, cell entry, and egress. TgAPH is acylated on the surface of the micronemes and recruited to phosphatidic acid (PA)-enriched membranes. Here, we dissect the atomic details of APH PA-sensing hub and its functional interaction with phospholipid membranes. We unravel the key determinant of PA recognition for the first time and show that APH inserts into and clusters multiple phosphate head-groups at the bilayer binding surface.
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http://dx.doi.org/10.1016/j.str.2018.05.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6084407PMC
August 2018

The FapF Amyloid Secretion Transporter Possesses an Atypical Asymmetric Coiled Coil.

J Mol Biol 2018 10 8;430(20):3863-3871. Epub 2018 Jun 8.

Department of Life Sciences, Imperial College London, South Kensington Campus, London SW7 2AZ, UK. Electronic address:

Gram-negative bacteria possess specialized biogenesis machineries that facilitate the export of amyloid subunits, the fibers of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialized outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8-Å resolution. This domain forms a novel asymmetric trimeric coiled coil that possesses a single buried tyrosine residue as well as an extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail.
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http://dx.doi.org/10.1016/j.jmb.2018.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173795PMC
October 2018

T7 phage factor required for managing RpoS in .

Proc Natl Acad Sci U S A 2018 06 22;115(23):E5353-E5362. Epub 2018 May 22.

Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, SW7 2AZ London, United Kingdom;

T7 development in requires the inhibition of the housekeeping form of the bacterial RNA polymerase (RNAP), Eσ, by two T7 proteins: Gp2 and Gp5.7. Although the biological role of Gp2 is well understood, that of Gp5.7 remains to be fully deciphered. Here, we present results from functional and structural analyses to reveal that Gp5.7 primarily serves to inhibit Eσ, the predominant form of the RNAP in the stationary phase of growth, which accumulates in exponentially growing as a consequence of the buildup of guanosine pentaphosphate [(p)ppGpp] during T7 development. We further demonstrate a requirement of Gp5.7 for T7 development in cells in the stationary phase of growth. Our finding represents a paradigm for how some lytic phages have evolved distinct mechanisms to inhibit the bacterial transcription machinery to facilitate phage development in bacteria in the exponential and stationary phases of growth.
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http://dx.doi.org/10.1073/pnas.1800429115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003314PMC
June 2018

A Rapid Colorimetric Method to Visualize Protein Interactions.

Chemistry 2018 May 17;24(26):6727-6731. Epub 2018 Apr 17.

College of Chemical Engineering, North China University of Science and Technology, Tangshan, 063210, P. R. China.

As key molecules in most biological pathways, proteins physically contact one or more biomolecules in a highly specific manner. Several driving forces (i.e., electrostatic and hydrophobic) facilitate such interactions and a variety of methods have been developed to monitor these processes both in vivo and in vitro. In this work, a new method is reported for the detection of protein interactions by visualizing a color change of a cyanine compound, a supramolecule complex of 3,3-di-(3-sulfopropyl)-4,5,4',5'-dibenzo-9-methyl-thiacarbocyanine triethylammonium salt (MTC). Nuclear magnetic resonance (NMR) studies suggest that the hydrophobic nature of the protein surfaces drives MTC into different types of aggregates with distinct colors. When proteins interact with other biomolecules, the hydrophobic surface of the complex differs, resulting in a shift in the form of MTC aggregation, which results in a color change. As a result, this in vitro method has the potential to become a rapid tool for the confirmation of protein-biomolecule interactions, without the requirements for sophisticated instrumentation or approaches.
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http://dx.doi.org/10.1002/chem.201800401DOI Listing
May 2018

Introduction: Testing and Refining Marc Lewis's Critique of the Brain Disease Model of Addiction.

Neuroethics 2017 2;10(1):1-6. Epub 2017 Mar 2.

Centre for Moral Philosophy & Applied Ethics, Plunkett Centre for Ethics, Australian Catholic University (ACU), St Vincent's Hospital (Plunkett, ACU), 390 Victoria Street, Darlinghurst, NSW 2010 Australia.

In this introduction we set out some salient themes that will help structure understanding of a complex set of intersecting issues discussed in this special issue on the work of Marc Lewis: (1) conceptual foundations of the disease model, (2) tolerating the disease model given socio-political environments, and (3) A third wave: refining conceptualization of addiction in the light of Lewis's model.
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http://dx.doi.org/10.1007/s12152-017-9310-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486510PMC
March 2017

Full shut-off of Escherichia coli RNA-polymerase by T7 phage requires a small phage-encoded DNA-binding protein.

Nucleic Acids Res 2017 Jul;45(13):7697-7707

MRC Centre for Molecular Microbiology and Infection, Imperial College London, London SW7 2AZ, UK.

Infection of Escherichia coli by the T7 phage leads to rapid and selective inhibition of the bacterial RNA polymerase (RNAP) by the 7 kDa T7 protein Gp2. We describe the identification and functional and structural characterisation of a novel 7 kDa T7 protein, Gp5.7, which adopts a winged helix-turn-helix-like structure and specifically represses transcription initiation from host RNAP-dependent promoters on the phage genome via a mechanism that involves interaction with DNA and the bacterial RNAP. Whereas Gp2 is indispensable for T7 growth in E. coli, we show that Gp5.7 is required for optimal infection outcome. Our findings provide novel insights into how phages fine-tune the activity of the host transcription machinery to ensure both successful and efficient phage progeny development.
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http://dx.doi.org/10.1093/nar/gkx370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569994PMC
July 2017

Stigma and Self-Stigma in Addiction.

J Bioeth Inq 2017 Jun 3;14(2):275-286. Epub 2017 May 3.

Faculty of Health, Medicine and Life Sciences, Maastricht University, Peter Debyeplein 1, 6229 HA, Maastricht, The Netherlands.

Addictions are commonly accompanied by a sense of shame or self-stigmatization. Self-stigmatization results from public stigmatization in a process leading to the internalization of the social opprobrium attaching to the negative stereotypes associated with addiction. We offer an account of how this process works in terms of a range of looping effects, and this leads to our main claim that for a significant range of cases public stigma figures in the social construction of addiction. This rests on a social constructivist account in which those affected by public stigmatization internalize its norms. Stigma figures as part-constituent of the dynamic process in which addiction is formed. Our thesis is partly theoretical, partly empirical, as we source our claims about the process of internalization from interviews with people in treatment for substance use problems.
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http://dx.doi.org/10.1007/s11673-017-9784-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5527047PMC
June 2017

Facet-Dependent Interactions of Islet Amyloid Polypeptide with Gold Nanoparticles: Implications for Fibril Formation and Peptide-Induced Lipid Membrane Disruption.

Chem Mater 2017 Feb 13;29(4):1550-1560. Epub 2017 Feb 13.

Department of Materials, Imperial College London, London SW7 2AZ, U.K.; Department of Bioengineering and Institute of Biomedical Engineering, Imperial College London, London SW7 2AZ, U.K.

A comprehensive understanding of the mechanisms of interaction between proteins or peptides and nanomaterials is crucial for the development of nanomaterial-based diagnostics and therapeutics. In this work, we systematically explored the interactions between citrate-capped gold nanoparticles (AuNPs) and islet amyloid polypeptide (IAPP), a 37-amino acid peptide hormone co-secreted with insulin from the pancreatic islet. We utilized diffusion-ordered spectroscopy, isothermal titration calorimetry, localized surface plasmon resonance spectroscopy, gel electrophoresis, atomic force microscopy, transmission electron microscopy (TEM), and molecular dynamics (MD) simulations to systematically elucidate the underlying mechanism of the IAPP-AuNP interactions. Because of the presence of a metal-binding sequence motif in the hydrophilic peptide domain, IAPP strongly interacts with the Au surface in both the monomeric and fibrillar states. Circular dichroism showed that AuNPs triggered the IAPP conformational transition from random coil to ordered structures (α-helix and β-sheet), and TEM imaging suggested the acceleration of IAPP fibrillation in the presence of AuNPs. MD simulations revealed that the IAPP-AuNP interactions were initiated by the N-terminal domain (IAPP residues 1-19), which subsequently induced a facet-dependent conformational change in IAPP. On a Au(111) surface, IAPP was unfolded and adsorbed directly onto the Au surface, while for the Au(100) surface, it interacted predominantly with the citrate adlayer and retained some helical conformation. The observed affinity of AuNPs for IAPP was further applied to reduce the level of peptide-induced lipid membrane disruption.
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http://dx.doi.org/10.1021/acs.chemmater.6b04144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333186PMC
February 2017

Structural and functional studies of Escherichia coli aggregative adherence fimbriae (AAF/V) reveal a deficiency in extracellular matrix binding.

Biochim Biophys Acta Proteins Proteom 2017 Mar 9;1865(3):304-311. Epub 2016 Dec 9.

Centre for Structural Biology, Department of Life Sciences, Imperial College London, South Kensington, London, United Kingdom. Electronic address:

Enteroaggregative Escherichia coli (EAEC) is an emerging cause of acute and persistent diarrhea worldwide. The pathogenesis of different EAEC stains is complicated, however, the early essential step begins with attachment of EAEC to intestinal mucosa via aggregative adherence fimbriae (AAFs). Currently, five different variants have been identified, which all share a degree of similarity in the gene organization of their operons and sequences. Here, we report the solution structure of Agg5A from the AAF/V variant. While preserving the major structural features shared by all AAF members, only Agg5A possesses an inserted helix at the beginning of the donor strand, which together with altered surface electrostatics, renders the protein unable to interact with fibronectin. Hence, here we characterize the first AAF variant with a binding mode that varies from previously described AAFs.
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http://dx.doi.org/10.1016/j.bbapap.2016.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289312PMC
March 2017

PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2B(Glu2).

Nat Commun 2016 08 17;7:12404. Epub 2016 Aug 17.

Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Science Park Road, Falmer, Brighton BN1 9RQ, UK.

PARP3 is a member of the ADP-ribosyl transferase superfamily that we show accelerates the repair of chromosomal DNA single-strand breaks in avian DT40 cells. Two-dimensional nuclear magnetic resonance experiments reveal that PARP3 employs a conserved DNA-binding interface to detect and stably bind DNA breaks and to accumulate at sites of chromosome damage. PARP3 preferentially binds to and is activated by mononucleosomes containing nicked DNA and which target PARP3 trans-ribosylation activity to a single-histone substrate. Although nicks in naked DNA stimulate PARP3 autoribosylation, nicks in mononucleosomes promote the trans-ribosylation of histone H2B specifically at Glu2. These data identify PARP3 as a molecular sensor of nicked nucleosomes and demonstrate, for the first time, the ribosylation of chromatin at a site-specific DNA single-strand break.
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http://dx.doi.org/10.1038/ncomms12404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992063PMC
August 2016

A distinct sortase SrtB anchors and processes a streptococcal adhesin AbpA with a novel structural property.

Sci Rep 2016 08 5;6:30966. Epub 2016 Aug 5.

Departments of Pediatric Dentistry and Microbiology, University of Alabama at Birmingham School of Dentistry, Birmingham, AL, USA.

Surface display of proteins by sortases in Gram-positive bacteria is crucial for bacterial fitness and virulence. We found a unique gene locus encoding an amylase-binding adhesin AbpA and a sortase B in oral streptococci. AbpA possesses a new distinct C-terminal cell wall sorting signal. We demonstrated that this C-terminal motif is required for anchoring AbpA to cell wall. In vitro and in vivo studies revealed that SrtB has dual functions, anchoring AbpA to the cell wall and processing AbpA into a ladder profile. Solution structure of AbpA determined by NMR reveals a novel structure comprising a small globular α/β domain and an extended coiled-coil heliacal domain. Structural and biochemical studies identified key residues that are crucial for amylase binding. Taken together, our studies document a unique sortase/adhesion substrate system in streptococci adapted to the oral environment rich in salivary amylase.
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http://dx.doi.org/10.1038/srep30966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974636PMC
August 2016

Crystal structure of the CupB6 adhesive tip from the chaperone-usher family of pili from Pseudomonas aeruginosa.

Biochim Biophys Acta 2016 11 29;1864(11):1500-5. Epub 2016 Jul 29.

Department of Life Sciences, Imperial College London, London SW7 2AZ, United Kingdom. Electronic address:

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that can cause chronic infection of the lungs of cystic fibrosis patients. Chaperone-usher systems in P. aeruginosa are known to translocate and assemble adhesive pili on the bacterial surface and contribute to biofilm formation within the host. Here, we report the crystal structure of the tip adhesion subunit CupB6 from the cupB1-6 gene cluster. The tip domain is connected to the pilus via the N-terminal donor strand from the main pilus subunit CupB1. Although the CupB6 adhesion domain bears structural features similar to other CU adhesins it displays an unusual polyproline helix adjacent to a prominent surface pocket, which are likely the site for receptor recognition.
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http://dx.doi.org/10.1016/j.bbapap.2016.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5022761PMC
November 2016

Crystal structure and analysis of HdaB: The enteroaggregative Escherichia coli AAF/IV pilus tip protein.

Protein Sci 2016 10 1;25(10):1898-905. Epub 2016 Aug 1.

School of Biological and Chemical Sciences, Queen Mary University London, London, E1 4NS, United Kingdom.

Enteroaggregative Escherichia coli is the primary cause of pediatric diarrhea in developing countries. They utilize aggregative adherence fimbriae (AAFs) to promote initial adherence to the host intestinal mucosa, promote the formation of biofilms, and mediate host invasion. Five AAFs have been identified to date and AAF/IV is amongst the most prevalent found in clinical isolates. Here we present the X-ray crystal structure of the AAF/IV tip protein HdaB at 2.0 Å resolution. It shares high structural homology with members of the Afa/Dr superfamily of fimbriae, which are involved in host invasion. We highlight surface exposed residues that share sequence homology and propose that these may function in invasion and also non-conserved regions that could mediate HdaB specific adhesive functions.
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http://dx.doi.org/10.1002/pro.2982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5029526PMC
October 2016

Staphylococcal Bap Proteins Build Amyloid Scaffold Biofilm Matrices in Response to Environmental Signals.

PLoS Pathog 2016 06 21;12(6):e1005711. Epub 2016 Jun 21.

Laboratory of Microbial Biofilms, Idab-CSIC-Universidad Pública de Navarra-Gobierno de Navarra and Dpto Producción Agraria, Pamplona, Spain.

Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria.
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http://dx.doi.org/10.1371/journal.ppat.1005711DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4915627PMC
June 2016

Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones.

Sci Rep 2016 Apr 21;6:24656. Epub 2016 Apr 21.

Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK.

Polypeptide aggregation into amyloid is linked with several debilitating human diseases. Despite the inherent risk of aggregation-induced cytotoxicity, bacteria control the export of amyloid-prone subunits and assemble adhesive amyloid fibres during biofilm formation. An Escherichia protein, CsgC potently inhibits amyloid formation of curli amyloid proteins. Here we unlock its mechanism of action, and show that CsgC strongly inhibits primary nucleation via electrostatically-guided molecular encounters, which expands the conformational distribution of disordered curli subunits. This delays the formation of higher order intermediates and maintains amyloidogenic subunits in a secretion-competent form. New structural insight also reveal that CsgC is part of diverse family of bacterial amyloid inhibitors. Curli assembly is therefore not only arrested in the periplasm, but the preservation of conformational flexibility also enables efficient secretion to the cell surface. Understanding how bacteria safely handle amyloidogenic polypeptides contribute towards efforts to control aggregation in disease-causing amyloids and amyloid-based biotechnological applications.
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http://dx.doi.org/10.1038/srep24656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838910PMC
April 2016

Human vulnerability in medical contexts.

Theor Med Bioeth 2016 Feb;37(1):1-7

Plunkett Centre for Ethics at St Vincent's Hospital, Sydney, and School of Philosophy, Australian Catholic University, Sydney, Australia.

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http://dx.doi.org/10.1007/s11017-016-9357-9DOI Listing
February 2016
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