NIHRDataInsights/HRCSData huggingface dataset:
Title: Unravelling a Novel Mode of Multiple Antibiotic Resistance: Mechanism and Inhibition of Radical-SAM RNA Methyltransferases
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Members of the radical SAM (S-adenosyl methionine) enzyme family catalyse a wide variety of chemically challenging biotransformations, often at unreactive centres. These include methylation of a range of substrates, of which one is the 23S RNA component of the bacterial ribosome. The radical SAM enzyme Cfr catalyses methylation at the 8-position of a specific nucleotide base (A2503) resulting in resistance to a range of antibiotics that inhibit translation by binding to the large ribosomal subunit. Most significantly, these include linezolid, the first oxazolidinone antibiotic in clinical use and a key agent for treatment of infections by multi-resistant Gram-positive bacterial pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). Transferrable linezolid resistance was previously unknown; however Cfr is now beginning to disseminate amongst clinical MRSA strains. Radical-SAM-catalysed methyl transfer remains incompletely understood. Accordingly, we propose to develop spectroscopic (fluorescence and absorbance-based) and biochemical methods with which to study the Cfr-catalysed rRNA methylation reaction. Using these we will screen directed libraries of small-molecule compounds with the aim of identifying Cfr inhibitors and investigate the relationship between substrate (SAM and rRNA) binding and the oxidation state of the enzyme to obtain new mechanistic information. We will test the ability of the inhibitors we identify to restore the effectiveness of linezolid against Cfr-expressing S.aureus strains, and initiate a programme of crystallisation experiments with the aim of determining crystal structures of Cfr:RNA and Cfr:inhibitor complexes. Our results will identify routes towards inhibiting a new and challenging mechanism of antibiotic resistance in Gram-positive bacteria and provide new information on how radical-SAM enzymes catalyse unfavourable chemical reactions.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Development of epidermicin NI01 for nasal decolonization
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The World Health Organisation have stated that infections caused by antibiotic resistant bacteria (e.g. MRSA) are one of the greatest current threats to public health and there is an urgent need for novel antibiotics to combat drug resistant infections. We have discovered epidermicin, an exciting and important new antibiotic that rapidly kills MRSA and other bacteria. The new antibiotic is more active than current antibiotics, is non-toxic and bacteria don't develop resistance to it. These are significant advantages over current antibiotics so we plan to develop epidermicin for clinical use. People who carry MRSA in the nose have a high risk of developing infection and transmitting MRSA, so we will examine how effective epidermicin is for removing MRSA from the nose. We will also carry out experiments designed to help us to understand how epidermicin kills bacteria and why they do not become resistant and carry out work to ensure that the antibiotic is not toxic when used clinically.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: CamChain - Campylobacter in chicken production: survival, virulence and control
Funder: Biotechnology and Biological Sciences Research Council

Abstract: We comprise groups in Europe, Thailand and Viet Nam. The Thai government will fund work there and that in Viet Nam will be funded by Wellcome Trust and the Dutch Government. Our work will address knowledge gaps on the behaviour of Campylobacter in the poultry chain. We need to better understand interactions between Campylobacter, chickens and the environment and how these affect food safety. We will create a holistic picture of Campylobacter behaviour in chicken production and fundamental data on survival, how environmental exposures affect virulence and on-farm population biology and on pre- and/or probiotic intervention on-farm to reduce Campylobacter levels entering the food chain. We will use modelling and risk assessment tools to identify and test the potential efficacy of different interventions that can be utilised by the international poultry industry. We will determine the role of flies as vectors of Campylobacter to broilers and develop improved surveillance tools in order to reduce the number of Campylobacter in broiler meat We aim to better understand the behaviour of Campylobacter in poultry production to improve control. We will generate understanding of mechanisms for: In-flock transmission and risk in production including bird to bird spread in-house and downstream in the production chain Virulence encompassing both broiler colonisation and extra-intestinal spread Interventions for improving host resistance based on enhancing gut health and, in so doing, finding points for control Environmental survival and the impact of environmental exposures on virulence. During these processes will be deterine pathogen source and persistence mechanisms Population evolution to examine how the production chain selects certain sub-types of Campylobacter and whether the selection processes change virulence potential


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Vector competence of European mosquitoes to Rift Valley fever virus
Funder: Biotechnology and Biological Sciences Research Council

Abstract: This Emida project, RiftVectors, will address topics relating to arthropod vectors of RVFV. Firstly, we will improve methods for identification of RVFV vectors (with special focus on European counterparts of tropical mosquito species) through the use of molecular tools, and the provision of quantitative estimates of species dynamics in European areas with potential RVFV vectors. Secondly, we aim to gain further insights into the competence of potential RVFV vectors, especially that of potential European vectors in comparison to their African counterparts; we will experimentally asses whether RVFV can adapt to European mosquitoes, and determine environmental factors influencing maintenance and vertical transmission. Thirdly, we will study the induction and nature of innate immune responses of RVFV vectors, followed by investigations on how these host responses control RVFV replication. Finally, we will carry out a detailed investigation whether, and if so how, Wolbachia bacteria can be used to inhibit RVFV replication and transmission in vector mosquitoes, as has been shown for other arboviruses. These questions will be addressed by five work packages. Two partners will be based in the UK (University of Oxford, MRC-University of Glasgow Centre for Virus Research).


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: The ubiquitin ligase NleG type III secretion system effectors of E. coli O157
Funder: Biotechnology and Biological Sciences Research Council

Abstract: 'Attaching & effacing' (AE) E. coli exert a substantial burden on human and animal health and use Type III secretion to colonise intestinal epithelia and induce pathology. The locus of enterocyte effacement (LEE)-encoded Type III secretion system of such pathogens mediates injection of tens of bacterial effectors into enterocytes. These orchestrate cellular pathways in a manner requiring exquisite temporal and spatial control. Among the EHEC effectors are the NleG family members that mimic eukaryotic RING finger/U-box ubiquitin E3 ligases. Our data show that the EHEC EDL933 ubiquitin ligase effectors NleG and NleG7, which target the nucleus and cytosol respectively, contain a carboxy terminal PDZ-binding motif. Moreover, one of the NleG effectors in the EHEC-like mouse pathogen Citrobacter rodetnium, which is homologues to the NleG-EDL (we named NleG-CR) also contains a PDZ-binding motif. Following ectopic expression we found co-localisation of NleG and NleG-CR with ubiquitin in the nucleus, which coincided with diminished histone H3 straining. Moreover, both NleG and NleG-CR bind PDZ1 of the mammalian scaffold protein Na(+) /H(+) exchanger regulatory factor 1 (NHERF1). The PDZ binding partner of NleG7 is not yet known. The focus of this study is the NleG effectors with a PDZ-binding motif. We will employ biochemical, protein-protein interaction, cellular and molecular methodologies to determine the intracellular trafficking of the NleGs, the involvement of NHERF1, their host cell partner and substrate proteins, the effect of NleG on the nuclear proteome and the transcriptome, and the effect of NleG7 on the cytosolic proteome and cell signalling. We will use bovine explants ex vivo and the mouse model to study the role NleGs play in infection of mucosal surfaces, colonisation and pathology. Unravelling the mode of action of the NleGs would have major implications on our understudying of EHEC infection and potentially for designing novel control strategies.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Design of high affinity ligands for C. albicans Als adhesins: inhibitors of biofilm formation
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Candida spp. are ubiquitous commensals in the human flora. They are also the main source of opportunistic mycoses worldwide. Among them, C. albicans is the most commonly isolated species. C. albicans is a highly adhesive organism and can colonise almost any host surface, an unusual ability among microbial pathogens. Candida adhesion manifests in different forms, such as attachment to various cell types, recognition of specific proteins in the host cell surface and extracellular matrix, autoaggregation and the development of biofilms The Als surface glycoproteins are fundamental to the adhesive properties of C. albicans. This work builds on progress from a previous funding period that solved the NT-Als domain structure and revealed a binding mechanism that recognizes up to seven C-terminal residues of peptides in an extended conformation. Our goal is to use cellular, biochemical and structural analyses to understand the aggregative properties of Als adhesins, and to place these results into the context of C. albicans ability to form biofilms Specifically, it is suggested that a conserved amyloid-forming region can act as a switch between the ligand-bound form of Als adhesins or a free form that promotes cell aggregation. This proposal also explores the use of Als adhesins as templates for the development of antifungal drugs. Blocking the activity of NT-Als domains is predicted to interfere with fungal adhesion and virulence. The peptide-binding site of NT-Als domains contains conserved features amenable for identification of antifungal compounds, e.g. using fragment-based screening by biophysical techniques including crystallography, NMR and thermofluorometry.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: A novel bacterial defence system against antimicrobial peptides: Implications for host colonisation in the food-borne pathogen Campylobacter jejuni
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Research on the molecular basis of colonisation by Campylobacter jejuni of the chicken host is required to inform the rational design of strategies to eradicate this pathogen from poultry flocks. It is not clear, however, how C. jejuni evades avian innate immunity, particularly that mediated by cationic antimicrobial peptides (CAMPs). Many strains of C. jejuni are known to be naturally resistant to these molecules but the genetic and structural basis for this has not, up to now, been adequately explained. We have discovered an entirely novel, previously unrecognized, multi-component defence system in C. jejuni that can explain high-level CAMP resistance, which we believe is likely to be necessary for successful host colonisation. This system consists of a membrane bound sensor protein and a small DNA binding protein which control the expression of three genes encoding an inner membrane protein, an outer membrane anchored lipoprotein and a periplasmic protein, as well as certain dsb genes and genes involved in peptide transport and metabolism. We have solved the structure of the lipoprotein, shown that it binds the model CAMP polymyxin B and that a cognate null mutant is highly sensitive to polymyxin B killing. Our aim is to determine the function of each of the components of this complex system and their contribution to chicken colonisation. This will be achieved by investigating the mechanism of action of the lipoprotein and determining if it provides resistance to structurally diverse CAMPs; determining the roles of the uncharacterised genes by mutant studies; determining what the role of the Dsb system is in CAMP resistance; investigating the connection between peptide transport and CAMP transport and degradation; and determining the importance of this system in vivo by chicken colonisation studies with selected mutants. We believe this work could provide new avenues for intervention and will impact on future eradication programmes for C. jejuni in chickens.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Cute-Egg, improvement of eggshell cuticle quality to reduce vertical transmission of zoonotic and pathogenic organisms
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The cuticle is a glycosylated protein layer which covers the external surface of the egg and fills the gas exchange pores through which the embryo 'breathes'. The cuticle is critical to minimising the risk of vertical and horizontal transmission of pathogenic or zoonotic organisms to the egg contents which is un-desirable for food safety, animal and human health. This project will use physiochemical, physiological and genetic methods to provide the information and tools necessary to make precision genetic selection for improved cuticle quality in both meat and egg laying hens. The objective of the work is; 1) to use excitation-emission-matrix spectroscopy to determine the parameters for discrimination of the cuticle from background fluorescence and to design and build an instrument that can rapidly and accurately measure cuticle coverage, 2) to apply this direct measurement to estimate reliable heritabilities for cuticle coverage in meat and egg laying strains of poultry and to determine genetic correlations with important production parameters to ensure there are no major negative consequences, 3) to test the hypothesis that (i) moderate levels of stress, (ii) events determining the timing of oviposition and (iii) bird age influences the level of cuticle coverage and to estimate the contribution of these environmental factors to cuticle variation, 4) to develop a method to measure the degree of glycosylation of the cuticle using time resolved fluorescence, 5) to test the hypothesis that the cuticle and its degree of glycosylation acts both as a physical and a chemical barrier to bacteria using penetration and recovery experiments with a range of GFP bacteria, 6) Using animals killed during the transition from shell to cuticle formation and oviposition we will profile the expression of genes to increase our understanding of the control of cuticle formation. We will also specifically measure genes we know from proteomic studies that encode cuticle proteins.


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS
Health categories: INFECTION
Title: Effects of Nod-like receptor activity on protective immunity against Salmonella infection
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Infectious diseases remain major problems with significant economic and social impact despite the availability of antimicrobials and vaccines. The development of resistance to antimicrobials by microorganisms means that alternative control measures are required. Control of food-borne zoonotic bacterial infections is particularly challenging because some bacteria can behave like commensals in livestock under the right circumstances (for example Salmonella species) yet are pathogens in humans. The molecular basis for this species-specific bacterial "tolerance" is not understood which makes design of control strategies difficult. Vaccination strategies have reduced disease incidence for some bacteria, but many current vaccines have been generated empirically which often results in a product with limited efficacy. Alternative approaches to combat infectious disease in livestock are to use selective breeding for disease resistance traits or to generate genetically modified animals to alter their susceptibility to infection. To design suitable control strategies it is essential to determine how pathogens interface with host immunity Pathogen recognition by host pattern recognition receptors (PRRs) activates innate and adaptive immunity. There are major differences in the repertoires of PRRs and in how they respond to ligands between different species of mammals and birds. The Nucleotide Oligomerisation Domain (Nod)-like receptors (NLRs) show wide species differences in repertoire with mice having 30, humans 22, and chickens 7. Our data show that the NLRs NLRC4 and Nod2 that are missing from chickens are important regulators of TH1 immunity against Salmonella infection. Using comparative studies between mouse and chicken cells this research will identify the NLR-signaling pathways driving the host response to infection. By expressing human NLRC4 or Nod2 in chickens we will determine whether these PRRs contribute to species-specific host responses to salmonellae.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Kinase-dependent control of DNA replication and repair as a drug target in Trypanosoma brucei
Funder: Biotechnology and Biological Sciences Research Council

Abstract: DNA replication and repair are central processes in the transmission of all genomes. The activity of each process is regulated to occur at specific cell cycle stages, allowing the cell to monitor their success. In kinetoplastid organisms, including Trypanosoma species, no work to date has analysed the protein kinase-dependent checkpoints that link replication and repair to cell cycle progression. Unconventional aspects of cell division, the cell cycle, replication and repair have been detailed in kinetoplastid parasites, potentially making the protein kinases (PKs) that co-ordinate these central cell reactions viable drug targets. Identifying these PKs is a key step in understanding and validating this. To do this comprehensively, we propose to use a recently developed RNAi library that targets the complete repertoire of 183 PKs in T. brucei. PKs involved in DNA replication and repair will be identified by comparing, before and after RNAi induction, nucleotide incorporation rates, subcellular localisation of components of the replication machinery and the level of chromatin-association of replication components. PKs that act in repair will be identified by using cell imaging to evaluate when RNAi alters a conserved response to induced DNA breaks, the formation of subnuclear foci, and using next generation sequencing to identify those PKs whose RNAi targeting results in cell killing specifically in the presence of DNA damage. This work will reveal the network of PKs that regulate genome transmission in these diverged eukaryotes. To complement the above RNAi approach, and to move rapidly to the identification of compounds that target PKs that act in replication and repair, we will employ high-content cell imaging and assays developed above to screen available libraries of chemicals that have been designed to target PKs. Any compounds identified will be tested thoroughly for their mode and strength of anti-parasite activity and for the PKs that they target.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Mechanism of binding of mycobacterial glycolipids to mincle, a stimulatory glycan-binding receptor on macrophages
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Mincle is a macrophage receptor that binds several structurally distinct oligosaccharide ligands and which is able to activate cytokine secretion. The goal of this project is to develop a molecular understanding of the basis for sugar recognition and how sugar binding might lead to intracellular signalling events. Ligands for mincle that are of particular interest are glycolipids found in the outer membrane of mycobacteria. Analysis of the binding of these ligands will provide insight into the role of mincle in mycobacterial infection leading to tuberculosis in humans and in cows and will also inform attempts to exploit the stimulatory properties of mincle in development of novel adjuvants. The objectives of the project are: (1) To define the ligand-binding specificity of mincle by developing a panel of synthetic soluble analogues of trehalose dimycolate and testing these compounds in binding competition assays along with screening of arrays of synthetic and pathogen-derived glycans. (2) To investigate the mechanism of ligand binding by creating suitable targets for crystallisation of mincle-ligand complexes. (3) To characterise mincle from key animal species: mouse because it is used as a model for study of mincle function in vitro and cow because of the potential importance of mincle in mycobacterial infection leading to bovine tuberculosis. (4) To analyse the oligomeric structure of mincle and examine the molecular basis for the interaction between mincle polypeptides and the ITAM-containing FcRgamma subunits, which leads to downstream signalling.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION