NIHRDataInsights/HRCSData huggingface dataset:
Title: Interactions underlying assembly and function of TolC-dependent drug efflux and toxin export machineries
Funder: Medical Research Council

Abstract: Studying mechanisms of virulence and antibiotic resistance in pathogenic bacteria is needed to understand and counteract bacterial diseases. We propose to continue our studies on TolC-dependent membrane machineries that are ubiquitous among Gram-negative pathogens like E.coli, Salmonella, Pseudomonas, Neisseria, Actinobacillus, Serratia and Bordatella. These machineries either export large virulence proteins like cytolytic toxins, or eject antibiotics and other antibacterial inhibitors. They are important to bacterial survival, especially in host infections where they contribute to virulence and multidrug resistance. These machines share a tripartite structure, comprising an outer membrane-anchored TolC protein that projects across the inter-membrane periplasmic space to present a cell exit duct to molecules bound by inner membrane transporters - commonly a traffic ATPase for toxin export, or proton antiporter for drug efflux. These apposed membrane components are structurally and functionally linked by a periplasmic adaptor that is key to recruitment and opening of the TolC exit channel. The assembled pumps span the entire bacterial cell envelope of two membranes and periplasmic space. Over the last 20 years we have defined the toxin export signal and identified translocation stages and intermediates of the toxin export mechanism. We have elucidated high resolution structures of the two common elements of all these machineries, the TolC exit duct and the adaptor, and by combining crystallography and electrophysiology of purified TolC open state variants in lipid bilayers suggested a model for coiled-coil movements underlying TolC opening. By crystallising sequential open states we are defining key movements and interactions of the opening pathway. Combining extensive in vivo site-specific cross-linking and modeling defined interaction interfaces between TolC, adaptor and antiporter, and allowed us to derive the first data-based view of a complete assembled tripartite multidrug efflux machinery, the ?RND-type? AcrA-AcrB-TolC pump of E.coli, Salmonella, P.aeruginosa and N. gonorrheae. We aim to exploit these findings and expertise to establish, by molecular genetics, membrane protein purification, crystallography and site-specific cross-linking, the key elements describing a second type of drug efflux pump, MacA-MacB-TolC, and the toxin export machinery HlyB-HlyD-TolC. This will be underpinned by our additional experience studying toxin export, which has also yielded useful intermediate stalled complexes and a wealth of reagents, bacterial strains and expression constructs. We will generate key structural information and define interaction interfaces central to both systems, providing evidence-based insights into their assembly and operation. This will indicate potential target sites for future anti-infectives.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: A. Diabate, IRSS/Centre Muraz, Bobo-Dioulasso, Burkina Faso - Targeting male mosquito mating behavior for vector control
Funder: Medical Research Council

Abstract: The control of mosquito populations via insecticide-treated bednets and indoor residuals spraying (IRS) are amongst the most effective ways of decreasing malaria incidence in endemic areas of Africa. However, because mosquito populations constantly evolve behavioral and physiological resistance to available insecticides, these approaches progressively loose their effectiveness and there is a constant and urgent need for exploring and developing novel tools for vector control. Here, The African co-PI and its UK partner join their expertise and propose an extensive research program targeting male reproductive behaviour of Anopheles gambiae, the main vector of malaria in Africa. Male mating behaviour is a key aspect of the mosquito life-cycle and an aspect of their biology that is crucial for the development of several promising vector control strategies. Anopheles gambiae mates in flying aggregations or swarms in which they choose their mate and copulate. Swarms would constitute an ideal target for vector control because their elimination would prevent or delay female mosquito mating, hence decrease population growth. Swarm elimination would particularly be effective in combination with IRS or the release of sterile or genetically-modified mosquitoes. We propose to build on recent advances in our understanding of swarming behaviour by the co-PI and thoroughly explore the feasibility of predicting and manipulating swarm location for mass killing. Importantly, the impact of this intervention on mosquito populations combined or not with IRS will be directly evaluated. Male mating behaviour is also crucial for vector control programs aiming to release sterile male mosquitoes that mate with wild females and induce their sterility and for programs aiming to introduce genes of refractoriness to malaria in mosquito populations via genetically-modified mosquitoes. Evidence from sterile mosquito releases and competition experiments suggests that laboratory-reared males do not mate with wild females very effectively. The second aim of the research program will therefore investigate the determinants of reproductive success and mate recognition within swarms which is the key for improving breeding strategies and mating performance of laboratory-reared male mosquitoes. Experiments on swarms will take place in large outdoor cages and selected sites in Burkina Faso making use of ecological techniques developed by the Co-PI, PI and others in previous studies. A significant Burkina-UK molecular biology and population dynamics training component is build into the program and will be based in the UK partner institution. The new approaches proposed here are expected to strongly impact this research field and future vector control strategies.


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: J GYAPONG, UNIVERSITY OF GHANA, FILARIASIS ELIMINATION IN AFRICA: REFINING THE STRATEGIES THROUGH RESEARCH
Funder: Medical Research Council

Abstract: My track record, over the past 10 years in the management of disease intervention programmes, conducting operational research, and successful collaborative initiatives with international partners demonstrate strong leadership capabilities. I have the expertise, leadership and motivation necessary to successfully carry out the proposed work. I have a broad background in epidemiology and control of infectious diseases (particularly neglected tropical diseases), with specific training and expertise in conducting research on lymphatic filariasis. Lymphatic filariasis is targeted for elimination in 81 endemic countries by 2020 through mass drug administration (MDA). Many endemic countries requiring treatment have implemented MDA. In Africa 14 out of 38 endemic countries have effective programmes. The Ghana LF programme which was initiated in the year 2000 as one of the first in Africa has had several successes as well as challenges. LF is endemic in about half of the country. This places approximately 10 million people at risk of infection. The objectives of the national LF programme are in line with the global objectives. With over 60 implementation units in different epidemiological zones, this presents as good an opportunity for addressing the challenges of the programme through operational research with Africa wide implications for the findings. Research proposed here will address challenges related to health systems strengthening to augment MDA scale-up and impact assessment in resource constrained settings. It will develop novel approaches to the community-directed treatment strategy for lymphatic filariasis in urban settings and the criteria and interventions that facilitate interruption of LF transmission. We will aim at: 1. Improving treatment coverage focusing on social and behavioural factors affecting community treatment compliance and understanding the dynamics of urban populations in uptake of these interventions. 2. Developing innovative ways of improving reporting from the community through to the national level using mobile telephony platforms for reporting as a means of strengthen the health system. 3. Verifying absence of transmission, when to stop mass drug administration (MDA) and effective post-MDA surveillance to prevent recrudescence. 4. Studying the added benefit of vector control on LF elimination especially in areas where reduction in prevalence and intensity of infection has been ineffective with MDA only 5. Documenting the effect of integrated control with other neglected tropical diseases on the elimination of LF and the health system. 6. Developing research capacity in the field of epidemiology and the social sciences at the Ghana School of Public Health.


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Statistical outbreak detection methods for large multiple surveillance systems
Funder: Medical Research Council

Abstract: This project is to develop statistical methods to detect outbreaks of infectious diseases as they arise, with application to large surveillance systems in the UK. Over the past decade much new statistical work has been undertaken on statistical outbreak detection, in response to public concern about infectious disease outbreaks, emerging infections, and the potential threat of bioterrorism. This research has been facilitated by the wide availability of computerised syndromic and laboratory data. Much of this methodological effort has been to develop relatively complex models for surveillance of a single or a small number of infections. These include, for example, wavelet decomposition models, hierarchical time series models, two-component changepoint models, and Markov switching models. However, large surveillance systems tracking hundreds or thousands of different infections on a weekly basis ? as is the case with the laboratory-based outbreak detection systems in use at the Health Protection Agency (HPA) and Health Protection Scotland (HPS) ? require simple, robust methods needing minimal user intervention and no data-intensive manipulation. The detection system at HPA and HPS was designed in the early 1990s by teams which included the PI and the collaborators. It uses a simple algorithm based on a quasi-Poisson generalized linear model, adjusted to correct for non-stationarity and overdispersion. The purpose of the present project is to elaborate this model as necessary, correct some of its shortcomings, and evaluate its performance against suitable alternatives. Specifically, we will study more robust methods for estimating the alarm thresholds, using a range of methods including quantile regression. We will investigate new methods for correcting for outbreaks in the historical baselines. We will seek to improve identification of outbreaks with insidious onset, by exploiting information from recent weeks. We will move from monitoring counts by date of report, to counts by date of specimen, incorporating an adjustment for reporting delay. We will investigate methods of handling multiplicities. We will study the evaluation metrics proposed in the literature and adapt them to our purpose. We will conduct a comprehensive evaluation of this and other outbreak detection systems based on laboratory data from England and Wales. The application originates from a 1-year NIHR project funded under the Research Methods Opportunity Funding Scheme. The applicants and collaborators are senior academics and scientists, with extensive experience of outbreak detection and statistical methodology. In view of the large amount of data processing envisaged, funding is sought over three years for two post-doctoral researchers.


Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: INFECTION
Title: Probing the mechanism of protein export by the bacterial Tat transport system
Funder: Medical Research Council

Abstract: The Tat system is a highly unusual protein export pathway that transports folded proteins across the cytoplasmic membranes of bacteria. It is required for the virulence of many important human pathogens including Pseudomonas aeruginosa , Legionella pneumophila and Escherichia coli 0157, and it is essential for viability of pathogenic Mycobacteria. The Tat system therefore represents a novel target for development of antibacterial compounds, particularly since it is absent in animal cells. Proteins substrates are targeted to the Tat system by means of N-terminal signal peptides that contain a conserved twin arginine motif. In E. coli TatA, TatB and TatC are the essential components of the Tat protein export machinery and they interact in a dynamic manner to form the membrane-located protein complexes necessary for substrate transport. TatB and TatC form an equimolar complex that has previously been purified. This complex binds Tat substrates, probably solely through their twin arginine signal peptides. No high resolution structural information is available for the TatBC complex and there is little information about how it is organised. We have used cysteine mapping experiments to identify a patch on the transmembrane helix of TatB that specifically interacts with one of the six transmembrane helices in TatC. We wish to build on these observations, taking both in vivo and in vitro approaches to find out (i) whether the disulphide cross-linking is sensitive to different stages of the Tat protein transport cycle, the presence of substrate or signal peptide and dissipation of the protonmotive force. (ii) To identify whether a second patch on TatB contacts other regions of TatC and (iii) to ascertain whether TatA can bind in a similar manner to TatC. Taken together the experiments will reveal important molecular details about the contact sites between Tat components and the conformational changes that occur during protein transport.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Safer aminoglycoside therapeutics by biosynthetic engineering
Funder: Medical Research Council

Abstract: Gentamicin and related aminoglycosides are valuable broad-spectrum antibiotics, active against both Gram positive and Gram negative bacteria. Gentamicin itself is being used increasingly to combat hospital-acquired sepsis, despite the well-known nephrotoxicity and ototoxicity associated with its use. Unfortunately, most studies aimed at clarifying the mechanisms of toxicity and alleviating these side effects have not taken into account that gentamicin as used clinically is a mixture of four main components with subtle structural differences. In a recent study, component C2 was reported to be non-nephrotoxic (in rats) while retaining the antibacterial effectiveness of the other components. Meanwhile, sequencing of multiple aminoglycoside biosynthetic gene clusters has made available a wealth of information on the number and type of enzymes involved. The first aim of this project is to analyse each enzyme in the late stages of the pathway and define its specificity, role and mechanism of action. A combined genetic and biochemical approach will be required: specific gene deletion will be used to gain insight into the exact sequence of biochemical events and identify branchpoints. The putative intermediates accumulated in such blocked mutants will be identified and characterised. These compounds will also serve as potential substrates for individual recombinant enzymes expressed from the genes of the pathway. Expression of sets of late-stage gentamicin biosynthetic genes in heterologous host strains will be used to complement the in vitro studies. The pathway contains unusual enzymes, including ones catalysing ring deoxygenation, and a potentially methylcobalamin-dependent C-methyltransferase of the so-called radical SAM family. We aim to clarify the mechanism of action of these enzymes. As the detailed gentamicin pathway is elucidated, we aim to design and test strains that produce, as far as possible, single component gentamicins instead of mixtures.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: Large-effect genetic variants in susceptibility to invasive pneumococcal disease
Funder: Medical Research Council

Abstract: The aim of the project is to utilise next-generation sequencing technology to identify novel, large-effect genetic susceptibility variants for invasive pneumococcal disease (IPD) in European and African populations. Streptococcus pneumoniae is a leading cause of death in children world-wide and the most common cause of pneumonia in the UK. Nasopharyngeal colonisation by the pneumococcus is widespread, yet only a minority of individuals develop invasive disease. Host genetic factors play an important role in explaining this inter-individual variation in susceptibility, but the genes involved are largely unknown. We are currently performing genome-wide association (GWA) studies for IPD in UK and Kenyan populations. Whilst this approach may detect common polymorphisms that associate with IPD, it will not identify large-effect, rare variants. Increasing evidence suggests that individually rare mutations of large effect are collectively responsible for a significant portion of susceptibility to common disease. This interest in rare variants is particularly relevant to infectious disease, as it parallels a paradigm shift in the concept of primary immunodeficiency (PID), with recent descriptions of ?selective? PIDs which underlie single episodes of severe infectious disease in otherwise healthy adults. The enormous selective pressure exerted by pneumococcal disease further supports the concept that most major IPD susceptibility variants will be rare. Advances in sequencing technology now permit the systematic study of rare variants in susceptibility to IPD and related bacterial disease phenotypes. This project represents a collaboration between European and African centres to assemble the largest sample collection available world-wide for the study of IPD (n=1970) and related phenotypes of bacterial respiratory disease (n=6100). Two approaches will be taken to evaluate the burden of rare variants in IPD cases compared with controls: sequencing of candidate genes (those underlying GWA signals and monogenic PIDs), and whole-exome sequencing in individuals with extreme pneumococcal disease phenotypes. These sequencing approaches will be followed by replication and functional studies. Parallel study of European and African individuals is particularly valuable as it may reveal population-specific susceptibility variants and pathways. Combined use of sequencing and GWA data will enable dissection of the genetic architecture of IPD susceptibility, and for the first time define the relative contributions of rare and common variants both in individuals and at the population level. This approach will establish the frequency of known PIDs, and potentially characterise novel PID states. Identification of large-effect variants may also have direct clinical implications, for example the use of prophylactic interventions in high-risk individuals.


Research activity codes: 2.6 RESOURCES AND INFRASTRUCTURE (AETIOLOGY);2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: ROLE OF SURFACE PROTEINS IN CLOSTRIDIUM DIFFICILE INFECTION
Funder: Medical Research Council

Abstract: Clostridium difficile is a highly problematic pathogen that causes serious enteric infections, largely associated with antibiotic therapy in hospital environments. Treatment relies primarily on administration of metronidazole and/or vancomycin, but these are often ineffective and relapse is common, contributing to thousands of deaths annually in the UK. We know little of how the bacterium colonises the gut - an essential process for growth and release of toxins that cause tissue damage and severe diarrhoea. There are no vaccines at present, and those in development target the toxins and not the bacterium. We will use a recently described mouse model of infection in combination with a panel of defined C. difficile mutants in cell wall proteins (CWPs) to identify proteins that contribute to successful colonisation of the gut. Studies of colonisation are best performed in the mouse, as the hamster model of acute infection is very rapid and does not parallel the typical infections seen in the clinic. Our preliminary data in mice indicate that at least two CWPs, CwpV and Cwp66, are involved in either maintenance of colonisation in mice or in re-establishment of infection in the presence of inciting antibiotic. We will refine the mouse model to facilitate co-infection of wild-type and mutant bacteria, which will allow us to determine the relative fitness of defined mutants compared to wild-type bacteria. We will examine a number of mutants, focussing on a family of 30 CWPs and the type IV pili, together with other proteins that may be identified through on-going genome sequencing projects. Finally we will construct experimental vaccines based on promising cell wall proteins and examine their ability to induce an immune response that can reduce or prevent colonisation of mice with wild-type strains. Knowledge of which CWPs are involved in infection is crucial to further our understanding of the pathogenesis of C. difficile and in the design of novel therapeutic strategies for detection, prevention and treatment of disease. Our experience with Clostridial cell walls, genetics and vaccine development means this project has a high chance of success.


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