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Journal articleByers P, Matthews S, Kennett J, 2021,
Truthfulness in dementia care
, BIOETHICS, Vol: 35, Pages: 839-841, ISSN: 0269-9702 -
Journal articlePathania M, Tosi T, Millership C, et al., 2021,
Structural basis for the inhibition of the Bacillus subtilis c-di-AMP cyclase CdaA by the phosphoglucomutase GlmM
, Journal of Biological Chemistry, Vol: 297, Pages: 1-15, ISSN: 0021-9258Cyclic-di-adenosine monophosphate (c-di-AMP) is an important nucleotide signaling molecule that plays a key role in osmotic regulation in bacteria. c-di-AMP is produced from two molecules of ATP by proteins containing a diadenylate cyclase (DAC) domain. In Bacillus subtilis, the main c-di-AMP cyclase, CdaA, is a membrane-linked cyclase with an N-terminal transmembrane domain followed by the cytoplasmic DAC domain. As both high and low levels of c-di-AMP have a negative impact on bacterial growth, the cellular levels of this signaling nucleotide are tightly regulated. Here we investigated how the activity of the B. subtilis CdaA is regulated by the phosphoglucomutase GlmM, which has been shown to interact with the c-di-AMP cyclase. Using the soluble B. subtilis CdaACD catalytic domain and purified full-length GlmM or the GlmMF369 variant lacking the C-terminal flexible domain 4, we show that the cyclase and phosphoglucomutase form a stable complex in vitro and that GlmM is a potent cyclase inhibitor. We determined the crystal structure of the individual B. subtilis CdaACD and GlmM homodimers and of the CdaACD:GlmMF369 complex. In the complex structure, a CdaACD dimer is bound to a GlmMF369 dimer in such a manner that GlmM blocks the oligomerization of CdaACD and formation of active head-to-head cyclase oligomers, thus suggesting a mechanism by which GlmM acts as a cyclase inhibitor. As the amino acids at the CdaACD:GlmM interphase are conserved, we propose that the observed mechanism of inhibition of CdaA by GlmM may also be conserved among Firmicutes.
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Journal articleMcKenna S, Giblin SP, Bunn RA, et al., 2021,
A highly efficient method for the production and purification of recombinant human CXCL8
, PLoS One, Vol: 16, Pages: 1-12, ISSN: 1932-6203Chemokines play diverse and fundamental roles in the immune system and human disease, which has prompted their structural and functional characterisation. Production of recombinant chemokines that are folded and bioactive is vital to their study but is limited by the stringent requirements of a native N-terminus for receptor activation and correct disulphide bonding required to stabilise the chemokine fold. Even when expressed as fusion proteins, overexpression of chemokines in E. coli tends to result in the formation of inclusion bodies, generating the additional steps of solubilisation and refolding. Here we present a novel method for producing soluble chemokines in relatively large amounts via a simple two-step purification procedure with no requirements for refolding. CXCL8 produced by this method has the correct chemokine fold as determined by NMR spectroscopy and in chemotaxis assays was indistinguishable from commercially available chemokines. We believe that this protocol significantly streamlines the generation of recombinant chemokines.
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Journal articleCrone M, Randell P, Herm Z, et al., 2021,
Rapid design and implementation of an adaptive pooling workflow for SARS-CoV-2 testing in an NHS diagnostic laboratory: a proof-of-concept study
, Wellcome Open Research, Vol: 6, Pages: 1-13, ISSN: 2398-502XBackground: Diagnostic laboratories are currently required to provide routine testing of asymptomatic staff and patients as a part of their clinical screening for SARS-CoV-2 infection. However, these cohorts display very different disease prevalence from symptomatic individuals and testing capacity for asymptomatic screening is often limited. Group testing is frequently proposed as a possible solution to address this; however, proposals neglect the technical and operational feasibility of implementation in a front-line diagnostic laboratory.Methods: Between October and December 2020, as a seven-week proof of concept, we took into account scientific, technical and operational feasibility to design and implement an adaptive pooling strategy in an NHS diagnostic laboratory in London (UK). We assessed the impact of pooling on analytical sensitivity and modelled the impact of prevalence on pooling strategy. We then considered the operational constraints to model the potential gains in capacity and the requirements for additional staff and infrastructure. Finally, we developed a LIMS-agnostic laboratory automation workflow and software solution and tested the technical feasibility of our adaptive pooling workflow.Results: First, we determined the analytical sensitivity of the implemented SARS-CoV-2 assay to be 250 copies/mL. We then determined that, in a setting with limited analyser capacity, the testing capacity could be increased by two-fold with pooling, however, in a setting with limited reagents, this could rise to a five-fold increase. These capacity increases could be realized with modest additional resource and staffing requirements whilst utilizing up to 76% fewer plastic consumables and 90% fewer reagents. Finally, we successfully implemented a plate-based pooling workflow and tested 920 patient samples using the reagents that would usually be required to process just 222 samples.Conclusions: Adaptive pooled testing is a scientifically, technically and operatio
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Journal articleFang Y, Liu Z, Qiu Y, et al., 2021,
Inhibition of viral suppressor of RNAi proteins by designer peptides protects from enteroviral infection in vivo
, Immunity, Vol: 54, Pages: 2231-2244.e6, ISSN: 1074-7613RNA interference (RNAi) is the major antiviral mechanism in plants and invertebrates, but the absence of detectable viral (v)siRNAs in mammalian cells upon viral infection has questioned the functional relevance of this pathway in mammalian immunity. We designed a series of peptides specifically targeting enterovirus A71 (EV-A71)-encoded protein 3A, a viral suppressor of RNAi (VSR). These peptides abrogated the VSR function of EV-A71 in infected cells, and resulted in the accumulation of vsiRNAs and reduced viral replication. These vsiRNAs were functional, as evidenced by RISC-loading and silencing of target RNAs. The effects of VSR-targeting peptides (VTPs) 32 on infection with EV-A71 as well as another enterovirus, Coxsackievirus-A16, were ablated upon deletion of Dicer1 or AGO2, core components of the RNAi pathway. In vivo,VTP treatment protected mice against lethal EV-A71 challenge, with detectable vsiRNAs. Our findings provide evidence for the functional relevance of RNAi in mammalian immunity and present a therapeutic strategy for infectious disease.
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Journal articleFerreira JL, Coleman I, Addison ML, et al., 2021,
The "Jack-of-all-Trades" Flagellum From Salmonella and E. coli Was Horizontally Acquired From an Ancestral B-Proteobacterium (vol 12, 643180, 2021)
, FRONTIERS IN MICROBIOLOGY, Vol: 12 -
Journal articleHumphrey S, San Millan A, Toll-Riera M, et al., 2021,
Staphylococcal phages and pathogenicity islands drive plasmid evolution
, Nature Communications, Vol: 12, Pages: 1-15, ISSN: 2041-1723Conjugation has classically been considered the main mechanism driving plasmid transfer in nature. Yet bacteria frequently carry so-called non-transmissible plasmids, raising questions about how these plasmids spread. Interestingly, the size of many mobilizable and non transmissible plasmids coincides with the average size of phages (~40kb) or that of a family of pathogenicity islands, the phage-inducible chromosomal islands (PICIs, ~11 kb). Here, we show that phages and PICIs from Staphylococcus aureus can mediate intra- and inter-species plasmid transfer via generalised transduction, potentially contributing to non-transmissible plasmid spread in nature. Further, staphylococcal PICIs enhance plasmid packaging efficiency, and phages and PICIs exert selective pressures on plasmids via the physical capacity of their capsids, explaining the bimodal size distribution observed for non-conjugative plasmids. Our results highlight that transducing agents (phages, PICIs) have important roles in bacterial plasmid evolution and, potentially, in antimicrobial resistance transmission.
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Journal articleMullineaux Sanders C, Carson D, Hopkins E, et al., 2021,
Citrobacter amalonaticus inhibits the growth of Citrobacter rodentium in the gut lumen
, mBio, Vol: 5, Pages: 1-19, ISSN: 2150-7511The gut microbiota plays a crucial role in susceptibility to enteric pathogens, including Citrobacter rodentium, a model extracellular mouse pathogen that colonizes the colonic mucosa. C. rodentium infection outcomes vary between mouse strains, with C57BL/6 and C3H/HeN mice clearing or succumbing to the infection respectively. Kanamycin (Kan) treatment at the peak of C57BL/6 mouse infection with Kan-resistant C. rodentium resulted in re-localisation of the pathogen from the colonic mucosa and cecum to solely the cecal luminal contents; cessation of the Kan treatment resulted in rapid clearance of the pathogen. We now show that in C3H/HeN mice, following Kan-induced displacement of C. rodentium to the cecum, the pathogen stably colonizes the cecal lumen of 65% of the mice in the absence of continued antibiotic treatment, a phenomenon we term antibiotic-induced bacterial commensalisation (AIBC). AIBC C. rodentium was well-tolerated by the host, which showed little signs of inflammation; passaged AIBC C. rodentium robustly infected naïve C3H/HeN mice suggesting that the AIBC state is transient and did not select for genetically avirulent C. rodentium mutants. Following withdrawal of antibiotic treatment, 35% of C3H/HeN mice were able to prevent C. rodentium commensalisation in the gut lumen. These mice presented a bloom of a commensal species, Citrobacter amalonaticus, which inhibited the growth of C. rodentium in vitro in a contact-dependant manner, and luminal growth of AIBC C. rodentium in vivo. Overall our data suggest that commensal species can confer colonization resistance against closely-related pathogenic species.
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Journal articleBarski MS, Vanzo T, Zhao XZ, et al., 2021,
Publisher Correction: Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures
, Nature Communications, Vol: 12, ISSN: 2041-1723 -
Journal articleHaag AF, Podkowik M, Ibarra-Chavez R, et al., 2021,
A regulatory cascade controls Staphylococcus aureus pathogenicity island activation
, Nature Microbiology, Vol: 6, Pages: 1300-1308, ISSN: 2058-5276Staphylococcal pathogenicity islands (SaPIs) are a family of closely related mobile chromosomal islands that encode and disseminate the superantigen toxins, toxic shock syndrome toxin 1 and superantigen enterotoxin B (SEB). They are regulated by master repressors, which are counteracted by helper phage–encoded proteins, thereby inducing their excision, replication, packaging and intercell transfer. SaPIs are major components of the staphylococcal mobilome, occupying five chromosomal att sites, with many strains harbouring two or more. As regulatory interactions between co-resident SaPIs could have profound effects on the spread of superantigen pathobiology, we initiated the current study to search for such interactions. Using classical genetics, we found that, with one exception, their regulatory systems do not cross-react. The exception was SaPI3, which was originally considered defective because it could not be mobilized by any known helper phage. We show here that SaPI3 has an atypical regulatory module and is induced not by a phage but by many other SaPIs, including SaPI2, SaPIbov1 and SaPIn1, each encoding a conserved protein, Sis, which counteracts the SaPI3 repressor, generating an intracellular regulatory cascade: the co-resident SaPI, when conventionally induced by a helper phage, expresses its sis gene which, in turn, induces SaPI3, enabling it to spread. Using bioinformatics analysis, we have identified more than 30 closely related coancestral SEB-encoding SaPI3 relatives occupying the same att site and controlled by a conserved regulatory module, immA–immR–str′. This module is functionally analogous but unrelated to the typical SaPI regulatory module, stl–str. As SaPIs are phage satellites, SaPI3 and its relatives are SaPI satellites.
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Journal articleEllis KM, Lucantoni L, Chavchich M, et al., 2021,
The Novel bis-1,2,4-Triazine MIPS-0004373 Demonstrates Rapid and Potent Activity against All Blood Stages of the Malaria Parasite
, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Vol: 65, ISSN: 0066-4804- Cite
- Citations: 8
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Journal articleXie SC, Metcalfe RD, Mizutani H, et al., 2021,
Design of proteasome inhibitors with oral efficacy in vivo against <i>Plasmodium falciparum</i> and selectivity over the human proteasome
, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 118, ISSN: 0027-8424- Cite
- Citations: 31
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Journal articleSchmidt R, Curry S, Hatch A, 2021,
Creating SPACE to evolve academic assessment
, eLife, Vol: 10, ISSN: 2050-084XUniversities and research institutions have to assess individuals when making decisions about hiring, promotion and tenure, but there are concerns that such assessments are overly reliant on metrics and proxy measures of research quality that overlook important factors such as academic rigor, data sharing and mentoring. These concerns have led to calls for universities and institutions to reform the methods they use to assess research and researchers. Here we present a new tool called SPACE that has been designed to help universities and institutions implement such reforms. The tool focuses on five core capabilities and can be used by universities and institutions at all stages of reform process.
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Journal articleGumbart JC, Ferreira JL, Hwang H, et al., 2021,
Lpp positions peptidoglycan at the AcrA-ToIC interface in the AcrAB-ToIC multidrug efflux pump
, BIOPHYSICAL JOURNAL, Vol: 120, Pages: 3973-3982, ISSN: 0006-3495- Cite
- Citations: 16
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Journal articleChatterjee S, Choi A, Frankel G, 2021,
A systematic review of Sec24 cargo interactome
, Traffic, Vol: 22, Pages: 412-424, ISSN: 1398-9219Endoplasmic reticulum (ER)-to-Golgi trafficking is an essential and highly conserved cellular process. The coat protein complex-II (COPII) arm of the trafficking machinery incorporates a wide array of cargo proteins into vesicles through direct or indirect interactions with Sec24, the principal subunit of the COPII coat. Approximately one-third of all mammalian proteins rely on the COPII mediated secretory pathway for membrane insertion or secretion. There are four mammalian Sec24 paralogs and three yeast Sec24 paralogs with emerging evidence of paralog-specific cargo interaction motifs. Furthermore, individual paralogs also differ in their affinity for a subset of sorting motifs present on cargo proteins. As with many aspects of protein trafficking, we lack a systematic and thorough understanding of the interaction of Sec24 with cargoes. This systematic review focuses on the current knowledge of cargo binding to both yeast and mammalian Sec24 paralogs and their ER export motifs. The analyses show that Sec24 paralog specificity of cargo (and cargo receptors) range from exclusive paralog dependence or preference to partial redundancy. Wealso discuss how the Sec24 secretion system is hijacked by viral (e.g., VSV-G, Hepatitis B envelope protein) and bacterial (e.g., the enteropathogenic E. coli type III secretion system effector NleA/EspI) pathogens.
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Journal articleEhsan M, Wang H, Cecchetti C, et al., 2021,
Maltose-bis(hydroxymethyl)phenol (MBPs) and Maltosetris(hydroxymethyl)phenol (MTPs) Amphiphiles for Membrane Protein Stability
, ACS Chemical Biology, Vol: 16, Pages: 1779-1790, ISSN: 1554-8929Membrane protein structures provide a fundamental understanding of their molecular actions and are of importance for drug development. Detergents are widely used to solubilize, stabilize, and crystallize membrane proteins, but membrane proteins solubilized in conventional detergents are prone to denaturation and aggregation. Thus, developing novel detergents with enhanced efficacy for protein stabilization remains important. We report herein the design and synthesis of a class of phenol-derived maltoside detergents. Using two different linkers, we prepared two sets of new detergents, designated maltose-bis(hydroxymethyl)phenol (MBPs) and maltose-tris(hydroxymethyl)phenol (MTPs). The evaluation of these detergents with three transporters and two G-protein coupled receptors allowed us to identify a couple of new detergents (MBP-C9 and MTP-C12) that consistently conferred enhanced stability to all tested proteins compared to a gold standard detergent (DDM). Furthermore, the data analysis based on the detergent structures provides key detergent features responsible for membrane protein stabilization that together will facilitate the future design of novel detergents.
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Journal articleKondor A, Santmarti A, Mautner A, et al., 2021,
On the BET surface area of nanocellulose determined using volumetric, gravimetric and chromatographic adsorption methods
, Frontiers in Chemical Engineering, Vol: 3, Pages: 1-12, ISSN: 2673-2718Volumetric N2 adsorption at –196 °C is generally accepted as “gold standard” for estimating the Brunauer-Emmet-Teller (BET) surface area of nanocellulose. It is unclear however, whether the BET surface area of nanocellulose obtained at such low temperatures and pressures is meaningful at an absolute sense, as nanocellulose is used at ambient temperature and pressure. In this work, a systematic evaluation of the BET surface area of nanocellulose using a highly crystalline bacterial cellulose (BC) as model nanocellulose was undertaken to achieve a comprehensive understanding of the limitations of BET method for nanocellulose. BET surface area obtained using volumetric N2 adsorption at –196 °C was compared with the BET surface area acquired from gravimetric experiments using n-octane adsorption measured using dynamic vapour sorption (DVS) and n-octane adsorption determined by inverse gas chromatography (iGC), both at 25 °C. It was found that the BET surface area calculated from volumetric N2 adsorption data was 25% lower than that of n-octane adsorption at 25 °C obtained using DVS and iGC adsorption methods. These results supported the hypothesis that the BET surface area of nanocellulose is both a molecular scale (N2 vs n-octane, molecular cross section of 0.162 nm2 vs 0.646 nm2) and temperature (–196 °C vs 25 °C) dependent property. This study also demonstrates the importance of selecting appropriate BET pressure range based on established criteria and would suggest that the room temperature gravimetric measurement is more relevant for many nanocellulose applications.
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Journal articleKelsall IR, McCrory EH, Xu Y, et al., 2021,
HOIL-1-catalysed ubiquitylation of unbranched glucosaccharides and its activation by ubiquitin oligomers
<jats:title>Abstract</jats:title> <jats:p> HOIL-1, a component of the Linear Ubiquitin Assembly Complex (LUBAC), ubiquitylates serine and threonine residues in proteins, forming ester bonds (Kelsall et al, 2019, PNAS 116, 13293-13298). Here we report that mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant accumulate polyglucosan in brain, cardiac muscle and other organs, indicating that HOIL-1’s E3 ligase activity is essential to prevent these toxic polysaccharide deposits from accumulating. We found that HOIL-1 monoubiquitylates glycogen and α1:4-linked maltoheptaose <jats:italic>in vitro</jats:italic> and identify the C6 hydroxyl moiety of glucose as the site of ester-linked ubiquitylation. The HOIL-1-catalysed monoubiquitylation of maltoheptaose was accelerated >100-fold by Met1-linked or Lys63-linked ubiquitin oligomers, which interact with the catalytic RBR domain of HOIL-1. HOIL-1 also transferred preformed ubiquitin oligomers to maltoheptaose <jats:italic>en bloc</jats:italic> , producing polyubiquitylated maltoheptaose in one catalytic step. The Sharpin and HOIP components of LUBAC, but not HOIL-1, bound to amylose resin <jats:italic>in vitro</jats:italic> , suggesting a potential function in targeting HOIL-1 to unbranched glucosaccharides in cells. We suggest that monoubiquitylation of unbranched glucosaccharides may initiate their removal by glycophagy to prevent precipitation as polyglucosan. </jats:p>
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Journal articleGhilarov D, Inaba-Inoue S, Stepien P, et al., 2021,
Molecular mechanism of SbmA, a promiscuous transporter exploited by antimicrobial peptides
, Science Advances, Vol: 7, Pages: 1-10, ISSN: 2375-2548Antibiotic metabolites and antimicrobial peptides mediate competition between bacterial species. Many of them hijack inner and outer membrane proteins to enter cells. Sensitivity of enteric bacteria to multiple peptide antibiotics is controlled by the single inner membrane protein SbmA. To establish the molecular mechanism of peptide transport by SbmA and related BacA, we determined their cryo–electron microscopy structures at 3.2 and 6 Å local resolution, respectively. The structures show a previously unknown fold, defining a new class of secondary transporters named SbmA-like peptide transporters. The core domain includes conserved glutamates, which provide a pathway for proton translocation, powering transport. The structures show an outward-open conformation with a large cavity that can accommodate diverse substrates. We propose a molecular mechanism for antibacterial peptide uptake paving the way for creation of narrow-targeted therapeutics.
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Journal articleToh M, Chengan K, Hanson T, et al., 2021,
A high-yield <i>Streptomyces</i> Transcription-translation toolkit for synthetic biology and natural product applications
, Journal of Visualized Experiments, Vol: 2021, ISSN: 1940-087XStreptomyces spp. are a major source of clinical antibiotics and industrial chemicals. Streptomyces venezuelae ATCC 10712 is a fast-growing strain and a natural producer of chloramphenicol, jadomycin, and pikromycin, which makes it an attractive candidate as a next-generation synthetic biology chassis. Therefore, genetic tools that accelerate the development of S. venezuelae ATCC 10712, as well as other Streptomyces spp. models, are highly desirable for natural product engineering and discovery. To this end, a dedicated S. venezuelae ATCC 10712 cell-free system is provided in this protocol to enable high-yield heterologous expression of high G+C (%) genes. This protocol is suitable for small-scale (10-100 μL) batch reactions in either 96-well or 384-well plate format, while reactions are potentially scalable. The cell-free system is robust and can achieve high yields (~5-10 μM) for a range of recombinant proteins in a minimal setup. This work also incorporates a broad plasmid toolset for real-time measurement of mRNA and protein synthesis, as well as in-gel fluorescence staining of tagged proteins. This protocol can also be integrated with high-throughput gene expression characterization workflows or the study of enzyme pathways from high G+C (%) genes present in Actinomycetes genomes.
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Working paperMiguel-Romero L, Alqasmi M, Bacarizo J, et al., 2021,
Non-canonical <i>Staphylococcus aureus</i> pathogenicity island repression
<jats:title>ABSTRACT</jats:title> <jats:p> Mobile genetic elements (MGEs) control their life cycles by the expression of a master repressor, whose function must be disabled to allow the spread of these elements in nature. Here we describe an unprecedented repression-derepression mechanism involved in the transfer of the <jats:italic>Staphylococcus aureus</jats:italic> pathogenicity islands (SaPIs). Contrary to the classical phage and SaPI repressors, which are dimers, the SaPI1 repressor Stl <jats:sup>SaPI1</jats:sup> presents a unique tetrameric conformation, never seen before. Importantly, not just one but two tetramers are required for SaPI1 repression, which increases the novelty of the system. To derepress SaPI1, the phage-encoded protein Sri binds to and induces a conformational change in the DNA binding domains of Stl <jats:sup>SaPI1</jats:sup> , preventing the binding of the repressor to its cognate Stl <jats:sup>SaPI1</jats:sup> sites. Finally, our findings demonstrate that this system is not exclusive to SaPI1 but widespread in nature. Overall, our results characterise a novel repression-induction system involved in the transfer of MGE-encoded virulence factors in nature. </jats:p> <jats:sec> <jats:title>Significance</jats:title> <jats:p>While most repressors controlling the transfer of mobile genetic elements are dimers, we demonstrate here that the Staphylococcal pathogenicity island 1 (SaPI1) is repressed by two tetramers, which have a novel structural fold in their body that has never been seen before in other proteins. Moreover, by solving the structure of the SaPI1 repressor in complex with its inducing protein Sri, we have demo
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Journal articleKaplan M, Oikonomou CM, Wood CR, et al., 2021,
A novel widespread bacterial structure related to the flagellar type III secretion system
<jats:title>Abstract</jats:title> <jats:p>The flagellar type III secretion system (fT3SS) is a suite of membrane-embedded and cytoplasmic proteins responsible for building the bacterial flagellar motility machinery. Homologous proteins form the injectisome machinery bacteria use to deliver effector proteins into eukaryotic cells, and other family members have recently been reported to be involved in the formation of membrane nanotubes. Here we describe a novel, ubiquitous and evolutionarily widespread hat-shaped structure embedded in the inner membrane of bacteria, of yet-unidentified function, that is related to the fT3SS, adding to the already rich repertoire of this family of nanomachines.</jats:p>
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Journal articleGuo M, Rosbottom I, Zhou L, et al., 2021,
Triglycine (GGG) adopts a polyproline II (pPII) conformation in its hydrated crystal form: revealing the role of water in peptide crystallization
, Journal of Physical Chemistry Letters, Vol: 12, Pages: 8416-8422, ISSN: 1948-7185Polyproline II (pPII) is a left-handed 31-helix conformation, which has been observed to be the most abundant secondary structure in unfolded peptides and proteins compared to α-helix and β-sheet. Although pPII has been reported as the most stable conformation for several unfolded short chain peptides in aqueous solution, it is rarely observed in their solid state. Here, we show for the first time a glycine homopeptide (gly-gly-gly) adopting the pPII conformation in its crystalline dihydrate structure. The single crystal X-ray structure with molecular dynamic simulation suggests that a network of water and the charged carboxylate group is critical in stabilizing the pPII conformation in solid state, offering an insight into the structures of unfolded regions of proteins and the role of water in peptide crystallization.
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Journal articleKaplan M, Chreifi G, Metskas LA, et al., 2021,
In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryotomograpy
, ELIFE, Vol: 10, ISSN: 2050-084X- Author Web Link
- Cite
- Citations: 18
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Journal articledel Gaudio I, Hunter-Sellars E, Parkin IP, et al., 2021,
Water sorption and diffusion in cellulose acetate: The effect of plasticisers
, CARBOHYDRATE POLYMERS, Vol: 267, ISSN: 0144-8617- Cite
- Citations: 11
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Journal articleChatterjee S, Lekmeechai S, Constantinou N, et al., 2021,
The type III secretion system effector EspO of enterohaemorrhagic Escherichia coli inhibits apoptosis through an interaction with HAX-1
, Cellular Microbiology, Vol: 23, ISSN: 1462-5814Many enteric pathogens employ a type III secretion system (T3SS) to translocate effector proteins directly into the host cell cytoplasm, where they subvert signalling pathways of the intestinal epithelium. Here, we report that the anti-apoptotic regulator HS1-associated protein X1 (HAX-1) is an interaction partner of the T3SS effectors EspO of enterohaemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium, OspE of Shigella flexneri and Osp1STYM of Salmonella enterica serovar Typhimurium. EspO, OspE and Osp1STYM have previously been reported to interact with the focal adhesions protein integrin linked kinase (ILK). We found that EspO localizes both to the focal adhesions (ILK localisation) and mitochondria (HAX-1 localisation), and that increased expression of HAX-1 leads to enhanced mitochondrial localisation of EspO. Ectopic expression of EspO, OspE and Osp1STYM protects cells from apoptosis induced by staurosporine and tunicamycin. Depleting cells of HAX-1 indicates that the anti-apoptotic activity of EspO is HAX-1 dependent. Both HAX-1 and ILK were further confirmed as EspO1-interacting proteins during infection using T3SS-delivered EspO1. Using cell detachment as a proxy for cell death we confirmed that T3SS-delivered EspO1 could inhibit cell death induced during EPEC infection, to a similar extent as the anti-apoptotic effector NleH, or treatment with the pan caspase inhibitor z-VAD. In contrast, in cells lacking HAX-1, EspO1 was no longer able to protect against cell detachment, while NleH1 and z-VAD maintained their protective activity. Therefore, during both infection and ectopic expression EspO protects cells from cell death by interacting with HAX-1. These results suggest that despite the differences between EHEC, C. rodentium, Shigella and S. typhimurium infections, hijacking HAX-1 anti-apoptotic signalling is a common strategy to maintain the viability of infected cells.Take AwayEspO homologues are found in EHEC, Shigella, S. typhimurium and some E
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Journal articleLi X, Heng JYY, 2021,
The critical role of agitation in moving from preliminary screening results to reproducible batch protein crystallisation
, Chemical Engineering Research and Design, Vol: 173, Pages: 81-88, ISSN: 0263-8762This study investigated the important role of agitation in obtaining consistent and reproducible results when moving from preliminary qualitative screenings for protein crystallisation to quantitative batch crystallisation experiments. Lysozyme-thaumatin binary protein mixture was used as the model protein system in this study. Poor reproducibility between batches were observed for non-agitated crystallisation conditions even if the same sampling timing and frequency applied. With agitation, from 0 to 200 rpm investigated in this study, improved reproducibility of protein crystallisation was observed with increased agitation. Additionally, agitation also had impacts on supersaturation exhaustion rate, yield and crystal size. Moreover, in agitated batch crystallisation, it was found that target protein crystallisation process was decelerated in the presence of protein impurity. In conclusion, we emphasised the essential role of agitation in protein crystallisation experiments else misleading conclusions with inconsistency might be drawn from non-agitated systems.
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Journal articleCheung PCW, Williams DR, Barrett J, et al., 2021,
On the Origins of Some Spectroscopic Properties of "Purple Iron" (the Tetraoxoferrate(VI) Ion) and Its Pourbaix Safe-Space
, MOLECULES, Vol: 26- Cite
- Citations: 6
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Journal articleLarrouy-Maumus G, Katy J, katheryn H, et al., 2021,
Detection of colistin resistance in Pseudomonas aeruginosa using the MALDIxin test on the routine MALDI Biotyper Sirius mass spectrometer
, Frontiers in Microbiology, Vol: 12, ISSN: 1664-302XColistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in Pseudomonas aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-L-arabinose (L-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of L-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant Pseudomonas aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240-247) and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1 hour, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant Pseudomonas aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.
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Journal articleFillol-Salom A, Bacigalupe R, Humphrey S, et al., 2021,
The secret life (cycle) of temperate bacteriophages
<jats:title>Abstract</jats:title> <jats:p> Lysogenic induction ends the stable association between a bacteriophage and its host, and the transition to the lytic cycle begins with prophage <jats:underline>e</jats:underline> xcision followed by DNA <jats:underline>r</jats:underline> eplication and <jats:underline>p</jats:underline> ackaging (ERP) – a temporal program that is considered universal for most temperate phages. Here we report that the long-standing ERP program is an artefact of the experimentally favoured <jats:italic>Salmonella</jats:italic> phage P22 ts <jats:italic> c <jats:sub>2</jats:sub> 29 </jats:italic> heat-inducible mutant, and that wildtype P22 actually follows a replication-packaging-excision (RPE) program. We found that unlike P22 ts <jats:italic> c <jats:sub>2</jats:sub> 29 </jats:italic> , P22 delayed excision to just before it was detrimental to phage production. Thus, at minimal expense to itself, P22 has tuned the timing of excision to balance propagation with lateral transduction, powering the evolution of its host through gene transfer in the interest of self-preservation. </jats:p> <jats:sec> <jats:title>One Sentence Summary</jats:title> <jats:p>Genetic analyses propose a new life cycle for temperate bacteriophages.</jats:p> </jats:sec>
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