NIHRDataInsights/HRCSData huggingface dataset:
Title: Combatting the threat of carbapenem resistance in Gram-negative bacterial pathogens
Funder: Medical Research Council

Abstract: Hospital acquired (nosocomial) infections are a growing threat to global public health. Opportunist Gram-negative bacterial pathogens, both the Enterobacteriaceae and non-fermenters such as Pseudomonas aeruginosa or Acinetobacter baumannii, cause a wide variety of such conditions. Carbapenems are increasingly becoming first choice treatments for infections by these organisms, but resistant strains, in particular those carrying various metallo-beta-lactamases (MBLs) are now proliferating. Notably, the NDM-1 MBL is rapidly disseminating in the Enterobacteriaceae on a global scale. No new antibiotics with anti-Gram-negative activity are at or close to the clinic, and MBLs resist the activity of all available beta-lactamase inhibitors. We propose to develop new inhibitors of the most clinically important MBLs that are suitable for progress towards clinical trials as combinations with existing carbapenems We will identify inhibitors by a combination of in structure-based discovery, use of focussed libraries and optimisation of existing molecules; and develop and use new assays with which to test inhibition of MBLs in vitro. We will test their ability to restore carbapenem activity against clinically relevant MBL-producing strains identified by a programme of resistance monitoring, and optimise identified hits by repeated rounds of synthesis and screening that are guided by structural and biophysical studies. We will demonstrate the suitability of MBL inhibitors for further development using in vitro pharmacokinetic/pharmacodynamic models and toxicity tests. An MBL inhibitor of clinical use will transform the outlook for patients with Gram-negative infections, and the tools that we develop will also aid the efforts of the wider community towards this goal.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: The regulation of influenza A virus replication and transcription
Funder: Medical Research Council

Abstract: The influenza A virus RNP, comprising the RNA polymerase and nucleoprotein (NP) assembled on a viral RNA genome segment, has emerged as a major determinant of host range and pathogenicity. However, the underlying molecular mechanisms are still poorly understood due, at least in part, to a lack of knowledge about the regulation of the RNP complex. We have previously proposed a model suggesting that the assembly of polymerase and NP prevents degradation of viral RNA. The key questions addressed in this proposal are to determine the role of NP, and whether host cell nucleases play a role, in RNP activity.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Interplay of RNA structure, protein and miRNA binding in early replication events of hepatitis C virus
Funder: Medical Research Council

Abstract: Hepatitis C virus (HCV) is a globally important human pathogen. With little or no prospect of a vaccine to prevent infection and only partially effective antivirals, we urgently need to identify therapeutic targets to which new drugs can be directed. This means we need an improved understanding of how the virus replicates. HCV is a small single-stranded RNA virus; initial translation of the infecting genome must be terminated to enable negative-strand synthesis, a process achieved in similar viruses by a feedback loop responsive to viral and/or cellular proteins. These often mediate control by interacting with RNA structures in the virus genome. We have defined a specific protein-RNA interaction with a stem-loop (SL87) known to be implicated in viral translation. We hypothesise that binding of this protein is involved in the control of viral translation. We propose this process is also influenced by changes in the template RNA structure and interaction with miR-122, a hepatocyte-specific micro-RNA implicated in HCV replication and possibly involved in ribosome recruitment. Interplay of these three-components (RNA, protein, micro-RNA) determines the critical translation to replication switch, inhibition of which would be a valid and tempting target for development of HCV therapies. Our study has three objectives. 1) To use our extensive experience in RNA structure analysis to accurately quantify the structure ? and structural changes ? of the HCV genome in cytoplasmic extracts of cells that support virus replication, and to investigate the structural influence of a) mutations that inhibit translational control, b) the interaction with miR-122 and c) protein binding to SL87. 2) To identify and characterize the protein that binds to SL87. Using affinity purification and mass spectroscopy we will identify SL87, define the precise SL87 binding site my mutagenesis and footprinting, generate tagged (e.g. GFP) derivatives for colocalisation studies and reagents (e.g. antisera and siRNA) for studies investigating the distribution and depletion of the protein. 3) To conduct functional studies of virus replication and translation with genome templates of known structure ? to enable the role of structure in translational control to be determined ? and in systems that allow depletion/supplementation with micro-RNA and proteins to determine how control is mediated. These studies will improve understanding of a key stage in the early replication of HCV. Cellular proteins usurped by viruses may provide valid invariant targets for development of novel antiviral therapies, that help avoid the problems caused by virus variation and rapid evolution.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: The Assembly of Tetrathionate Reductase in Pathogenic Bacteria
Funder: Medical Research Council

Abstract: According to the Food Standards Agency, there are around 81,000 cases of foodborne illness annually in the UK. Salmonella species are some of the most common bacterial pathogens associated with foodborne illness in adults living in developed countries and are responsible for about 20-30% of food-related deaths. Salmonella is a Gram-negative bacterium and a member of the gamma-Proteobacteria that includes other human pathogens. The bacterium is a ?facultative anaerobe? and can thus use various compounds as replacements for oxygen during respiration. This has recently proven to be very important in the infection process. Experiments in a mouse model identified that the gut inflammation response, induced initially by Salmonella itself, causes the generation of tetrathionate in the gut mucosa through reaction of thiosulphate with reactive oxygen species. As Salmonella expresses a complex tetrathionate reductase enzyme, this tetrathionate can be used by Salmonella as a respiratory electron acceptor in order to out-compete the many other bacteria that lack this enzyme. The overall aim of this proposal is to provide fundamental information on the mechanism of targeting and assembly of tetrathionate reductase. Tetrathionate reductase is a substrate of the twin-arginine translocation (Tat) pathway, which is a remarkable protein targeting system dedicated to the transport of fully folded proteins across the plasma membranes of bacteria. Indeed, animal pathogens can be rendered avirulent if their Tat systems are inactivated. Thus all aspects of this protein transport system are therefore of interest as potential future targets for novel anti-infectives. All proteins destined for export via the Tat translocase are synthesised with N-terminal twin-arginine signal peptides bearing conserved SRRxFLK amino acid motifs. The majority of Salmonella Tat substrates are cofactor-containing complex enzymes with key roles in respiratory electron transport chains. Pre-export assembly of such enzymes involves an important process that regulates access of the signal peptide to the translocase until cofactor loading and protein folding is complete. We call this process Tat proofreading and it involves the direct recognition and physical binding of the signal peptides by specific chaperones. This project involves a detailed study of the structure and function of paradigm members of the TorD family of Tat proofreading proteins, and their role in tetrathionate reductase assembly. By taking a multi-discipinary approach that combines structural biology, molecular biology, biophysics, microbiology, genetics, and the latest synthetic biology technology, we aim to shed light on the mechanism of signal peptide recognition by TorD-like proteins.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: The role of Hepatitis C virus glycoprotein-receptor polymorphism in viral pathogenesis
Funder: Medical Research Council

Abstract: Hepatitis C Virus (HCV) poses a global health problem, with over 170 million infected individuals worldwide at risk of developing cirrhosis and hepatocellular carcinoma. A number of anti-viral replicase molecules are in clinical development, however, drug resistance and viral genotype-specific differences will limit their efficacy, demonstrating the need for combination therapies targeting different steps of the viral replication cycle. Recent progress in the discovery of a strain of HCV that replicates in cell culture has enabled studies on virus assembly, release and entry, offering new targets for intervention. Studies from our laboratory have contributed to the growing understanding that HCV can disseminate via direct contacts between infected and na?ve cells. Cell-to-cell transfer of HCV is resistant to the neutralising effects of antibodies targeting the viral glycoproteins, providing a strategy for the virus to escape host immune responses. Whilst the majority of studies have focused on identifying host and viral pathways required for the genesis and release of infectious cell-free particles and inhibition of their entry into na?ve cells, cell-to-cell infection is poorly understood. We hypothesise that cell-to-cell infection is the dominant route for HCV to disseminate within the infected liver. The central aim of the current proposal is to define the mechanism(s) of cell-to-cell HCV infection and its role in virus dissemination within an infected host and transmission to new hosts in two clinical settings: recurrent HCV infection of the transplanted liver and mother to child vertical infection. Identification of the selective factors that define HCV spread and transmission will have major implications for the future treatment of HCV infection. At present it is not clear how HCV quasispecies are maintained during chronic infection. A dominant mode of cell-to-cell HCV spread will limit virus trafficking between sites and the resulting viruses will follow independent evolutionary histories, i.e. become compartmentalised. Classic genetic models predict that evolution will occur more rapidly in a series of small isolated populations than in a single homogeneous pool. We propose to study the quasispecies structure of the HCV infected liver. This information will have a major impact on our understanding of HCV pathogenesis and the mechanism(s) underlying virus escape from host immune responses and anti-viral therapies.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Genome-wide association studies to map genetic variation underlying mosquito susceptibility to human malaria
Funder: Medical Research Council

Abstract: Understanding the molecular basis of mosquito susceptibility to Plasmodium infection and thus malaria transmission is fundamental to implementing vector-based malaria control strategies. To date, genotypic interactions between Anopheles gambiae and Plasmodium falciparum, the deadliest of the human malarias, have only been examined in one study. This work detected that genotype-by-genotype (g*g) interactions were the major determinants of the outcome of infection such that no parasite genotype was able to infect all mosquito families, and likewise, no mosquito family was able to resist all parasite genotypes. This finding highlights the complexity of the interaction and motivates research particularly aimed at dissecting the molecular basis of g*g interactions: which molecules lead to different infection outcomes due to their genetic variation? I will use genome-wide association studies (GWAS) in the two major sub-Saharan African malaria vectors, A. gambiae and A. arabiensis, to map genetic variation associated with P. falciparum susceptibility. Lab mosquitoes will be infected with different combinations of recently collected Senegalese parasites that will constitutively express green or red fluorescent protein, allowing the infection success of individual parasite genotypes to be assessed when mosquitoes are fed mixtures of two parasite genotypes. Field mosquitoes will be fed parasites from local gametocyte carriers. GWAS will be carried out using a genotyping chip that interrogates 400,000 SNPs in the mosquito genome. By comparing susceptible to refractory mosquitoes, small regions of the mosquito genome associated with large impacts on the outcome of the infection will be identified. Candidate genes falling in these regions and potentially involved in mosquito susceptibility will be phenotypically characterised using RNA-interference gene knockdown techniques. The molecular nature of g*g interactions and the role of mosquito species in P. falciparum susceptibility is crucial to examine because success of any vector control strategy will depend on application to all of the malaria vectors. Molecules found to be consistently involved in infection outcomes become prime targets for transmission blocking interventions, such as drugs and vaccines. Targeting mosquitoes is an effective malaria transmission reduction strategy as evidenced by the historical and current successes of insecticides and bednets. However, insecticides and bednets will fail as mosquitoes evolve insecticide resistance and bednet avoidance. New vector-based malaria control strategies will depend on a deep understanding of the molecular interactions between vectors and parasites. The efforts described here will further our understanding of the connection between genotype and phenotype in a system that is of great human interest.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Characterisation of the bacterial proteins YjeE, YeaZ and YgjD and evaluation as a potential novel antimicrobial target
Funder: Medical Research Council

Abstract: The conserved bacterial proteins YjeE, YeaZ and YgjD form an interacting network, and have been shown to be generally essential for bacterial viability. YjeE and YeaZ are found only in eubacteria, whilst the YgjD protein has orthologues in all other branches of life. The essential nature of these proteins, coupled with the fact that two of them are found only in bacteria, makes the pathway a potential target for antimicrobial chemotherapy. The aims of this project are to explore the interactions between the proteins and to investigate possible agents to disrupt the functions of one or more components of this pathway. Objectives A two-stranded research project is proposed. Firstly to explore the interactions between the proteins YjeE, YeaZ and YgjD to identify preferential partnerships under different conditions; to investigate potential protease and DNA binding activities; and to determine the structure of one or more of the different partner complexes. The second stage of the project will involve the screening of a compound library for agents that impair the function of one or more proteins in the network. Methodology Plasmids will be constructed carrying the genes for the YjeE, YeaZ and YgjD proteins labelled with split yellow fluorescent protein, which will fluoresce when two labelled proteins interact. The individual components will be labelled in pairs to establish preferentially pairing under a variety of culture conditions (including pH, osmolarity, oxygen tension, and different chemical sources). Electrophoretic mobility shift assays will be used to investigate the DNA binding of YeaZ and YgjD to DNA, either in isolation or in partnership. The stability YjeE/YeaZ and YgjD/YeaZ complexes will be probed, for example to determine the effects of nucleotides, metal ions or pH, and of adding the missing third protein from the network. Stability will be assessed by gel filtration and proteins analysed by SDS PAGE. Crystallisation trials will be undertaken to elucidating a high resolution structure for one or more of these complexes. An assay will be developed to permit screening of a compound library of drug-like molecules. Potential assays for development include a reporter gene format, split-YFP labelling, or the measurement of specific protein activity. Scientific and Medical Opportunities Exploration of the YgjD/YjeE/YeaZ network will increase our understanding of the functions of these proteins in Escherichia coli. This will hopefully lead to the discovery of potential new antimicrobial agents acting at a target site not affected by current classes of antibiotic.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Sugar coated armour: exploring UDP galactopyranose mutase (UGM) as a potential antifungal target
Funder: Medical Research Council

Abstract: The fungal cell wall is a dynamic and multi-layered structure almost entirely built from polysaccharides, consisting of a core of cross linked chitin and glucan. Less well characterised is the outermost galactomannan layer of linear mannan branched by short galactofuranose (Galf) chains that are displayed to the host immune system. Crucially, Galf is produced by a single enzyme, UDP-galactopyranose mutase (UGM), that catalyses the reaction UDP-galactopyranose to UDP-Galf. Understanding the biosynthetic pathways in cell wall synthesis is imperative in the drive for structure-based drug design of the next generation of antifungals. This is particularly pressing given the rise in fungal infections in the immunocompromised with cases of invasive aspergillosis due to Aspergillus fumigatus invariably fatal having limited diagnostic and therapeutic options. There is renewed interest in agents targeting the fungal cell wall following the introduction of the echinocandins and this proposal explores a potential new target. Using a multidisciplinary approach covering genetics, structural biology and high throughput screening I aim to provide concerted genetic and chemical validation of A. fumigatus UGM (AfUGM). Specific objectives and methodologies include: AfUGM genetic validation: based on two reported knockout strains, the role of AfUGM catalytic activity will be studied by complementing these strains with catalytically inactive mutant forms followed by phenotypic analyses. AfUGM molecular mechanism: the AfUGM crystal structure will be determined based on initial crystals/recombinant protein already available in the laboratory, an existing enzyme assay will be optimised and the mechanism/active site probed by site-directed mutagenesis. AfUGM inhibitors: the enzyme assay will be developed into a high-throughput screening (HTS) format and fragment/drug-derived libraries available in the drug Discovery Unit screened to evaluate the druggability of AfUGM as a target. The output will be increased knowledge of AfUGM and the functional implications of impaired Galf synthesis alongside rigorous research training.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Genetic Susceptibility to Rheumatic Heart Disease
Funder: Medical Research Council

Abstract: Background: Rheumatic heart disease (RHD) and its precursor acute rheumatic fever (ARF) result from an autoimmune response to infection with Streptococcus pyogenes (Group A Streptococcus, GAS). A significant cause of morbidity and mortality in the developing world, accounting for 500 000 deaths annually, half as many as malaria. Control of the disease is limited to antibiotic prophylaxis. Investment in research and development for ARF is only 0.4% of that for malaria and there is currently little prospect of developing a GAS vaccine effective in the developing world. Objectives: To identify genetic variants associated with susceptibility to RHD allowing for potential confounders. Study sample: Two thousand patients of Melanesian ethnicity from New Caledonia, Oceania, diagnosed with RHD by echocardiography (recruited to a cardiac imaging study planned by our collaborators) and an equivalent number of peer-nominated controls to be recruited from the same population will be consented for enrolment in genetic studies. Complete demographic and epidemiological data will be collected from each individual case and control as well as detailed clinical data including at a minimum a detailed echocardiographic study of all the cases. Association study: Using a case-control association study approach, microarray technology will be used to genotype at least 200 000 single nucleotide polymorphisms in 1000 cases and 1000 controls. Putative associated alleles will be replicated in a further 1000 cases and 1000 controls using genotyping technology such as the Sequenom‘s MassArray primer extension assay. This approach will give 80% power to detect significant association at allelic odds ratio of 1.5 or greater for alleles with a minor allele frequency of 10% or more at genome-wide significance (p-value = 5 x 10-7). Implications: Rheumatic heart disease is unique example of an autoimmune process associated with a known specific pathogen. Therefore, with the potential to reveal genetic variation associated with susceptibility RHD, this study might further our understanding of the complex interaction between human genetics, susceptibility to infection and autoimmunity. In addition the results might generate new approaches to GAS vaccine development.


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