NIHRDataInsights/HRCSData huggingface dataset:
Title: Dynamics of Susceptibility and Transmission of Campylobacter jejuni in Chickens
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Modelling of the transmission dynamics of C. jejuni in chickens has been limited by the lack of suitable data. Likewise, the role that transmission dynamics play in the empirical assessment of differences in production methods or application of preventative treatments has been neglected. An important example of this is the routine use of groups of co-housed birds in dose-response studies for C. jejuni in chickens. We have developed a framework that combines classical dose-response models with simple transmission models that has the potential to estimate both susceptibility and subsequent rates of transmission of an infectious agent simultaneously within a single experimental design. Building on our previous work we propose to derive optimal experimental designs to achieve this objective using likelihood-based optimal design theory. We will use stochastic individual-based transmission models to produce simulated datasets to assess these designs in the face of incomplete observational data - e.g. due to imperfect sensitivity and specificity of cloacal swabs. We also propose to apply mathematical models to understand the dynamics of colonisation at the flock level. A unique data set quantifying the prevalence and strain diversity of C. jejuni within a single free-range flock suggests that clearance may occur within individual hosts over a long-enough time-scale of observation. Supporting this hypothesis requires a systematic exploration of the competing mechanisms consistent with these observed epidemic dynamics. We will develop a hierarchy of nested, stochastic transmission models and perform model selection using information theoretic methods to select the most parsimonious model consistent with the data. In addition we propose to use this data set to inform the development of next-generation strain competition models for C. jejuni, adapting stochastic meta-population models originally developed for macro-parasite transmission through the faeco-oral route.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: INFECTION
Title: Integrating microbiology and modelling to determine the source of Campylobacter infection in the broiler house and develop interventions
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Poultry is the most important source of campylobacteriosis and over two-thirds of chicken at retail are contaminated by Campylobacter. A reduction in the Campylobacter loads and ideally a reduction in their prevalence in poultry at production would be the most effective measure for reducing human disease incidence. However to date, biosecurity has not proved to be entirely successful and it is thought that this may be due to the diversity of reservoirs that Campylobacter resides in. Our, and others, work has indicated that some Campylobacter strains and some Campylobacter reservoirs are probably much more important than others in this process and that it is only by quantitating their relative importance and their interaction with each other that it will be possible to robustly identify the sources of Campylobacter in the broiler house and hence introduce effective measures to prevent colonisation. It will be parameterised by data collected in the study. Up to eight Campylobacter strains will be selected from the top strains isolated from human cases and from chicken, cattle, sheep, pigs and wild birds. The efficacy of different strains to move from reservoir species (cattle, sheep, pigeon, chicken) to chickens will be quantitated to determine the dose response of each strain. The environmental survival of the Campylobacter strains will be estimated in a range of matrices associated with the broiler environment (faeces from cattle, sheep, pigeons and chickens; soil; water) and survival tested at two temperatures (4C and 15C) over a period of up to 3 months. This proposal will use an agent-based modelling approach, which identify how macroscale dynamics emerge from microscale interactions, to simulate the transmission of Campylobacter from the environment to the broiler house and potentially back to the environment on real farms This will be used to quantify which interventions would be most likely to reduce broiler colonisation.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Interventions effects on Campylobacter populations in poultry and poultry meat
Funder: Biotechnology and Biological Sciences Research Council

Abstract: This proposal seeks to assess the efficacy of practical interventions aimed to reduce the levels of Campylobacter entering the food chain from poultry and thereby the incidence of human Campylobacterosis. The efficacy of the interventions will be assessed against empirical determinations of the incidence of chicken colonization and carcass contamination in commercial poultry production and include an assessment of the diversity of the Campylobacter populations present. Strain diversity will be addressed using a combination of molecular and traditional culture methods from chickens present on farm pre and post thinning, upon transport and slaughter, through processing and on the final carcasses as they are scheduled for retail distribution. These data will be used to construct epidemiological models to estimate the impact of interventions on Campylobacter population levels and diversity. This will be achieved through collaboration with the UK poultry industry scheduling a series of on-farm interventions to be implemented with parallel operational changes in processing.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: INFECTION
Title: Deciphering The Molecular Basis of Environmental Persistence in Campylobacter Using a Systems Approach
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Campylobacter jejuni (Cj) remains a leading cause of food borne infection in the UK with most cases arising from the ingestion of contaminated poultry. Cj is notoriously fastidious and survives poorly under laboratory conditions, yet it appears to be ubiquitous in the environment, forming reservoirs of infection. In addition, it is clear that the bacterium can tolerate abiotic stresses encountered during food processing. Recently, we have shown that this remarkable ability of Cj to survive unfavourable conditions is underlined by the formation of metabolically inactive cells (persister cells) that are analogous to the spores of Gram-positive pathogens. Once conditions improve, these persister cells switch back to a normal-growth state and re-establish a colony and potential subsequent infection. Thus, a full understanding and control of food borne infection mediated by Cj would require an understanding of the formation and regulation of the persister cell phenotype. We will undertake an integrated analysis of persister cell formation and its molecular basis in Cj by combining our expertise in experimental characterisation of pathogenic bacteria and in evolutionary theoretical analysis of bacterial behaviour at network level. This integrated, system-level approach will identify the molecular basis and evolution of persistence in Cj. Ultimately, this will enable us to design novel approaches towards reducing Cj levels in the food chain and that are based on knowledge of the behaviour of the persister population in this bacteria.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Modelling Campylobacter survival and spread through poultry processing: a population genomics approach.
Funder: Biotechnology and Biological Sciences Research Council

Abstract: By identifying the genetic elements and associated phenotypes that enable some chicken-associated genotypes to survive poultry processing and infect humans, the aim of this proposal is to inform targeted interventions to reduce Campylobacter. A modelling approach based upon comparative functional genomics data will address fundamental questions about the genetic basis of genotypic variation from farm chickens to retail poultry. We will exploit recent advances in Illumina GA resequencing technologies, specifically high through-put isolate multiplexing, to sequence multiple genomes in individual and pooled samples to identify genomic differences associated with source. Data from association mapping will feed into the stochastic evolutionary genetics simulation model that incorporates many of the features involved in the generation and maintenance of sequence diversity at a population level including: initial number of genotypes, population size, mutation and recombination rate, growth rate, the fitness of particular genes and niche structure. Variables will be changed to reflect input from real systems enabling hypotheses based on the genome association studies to be tested. Specifically, fitness functions for particular genes will be derived from real data and selective sweeps acting on genes identified as important for survival will simulate the effect of various stages in poultry processing. Predictions from simulations will be tested in replicate experiments by tracking the changes in the genetic structure of Campylobacter populations in the same batch of chickens leaving the farm and at points through processing. Where the relative abundance of particular genes or alleles, identified by PCR, deviates from predictions in silico the model parameters will be adjusted (eg. allele fitness). Simulations will then be run using the validated model to identify points in processing were the Campylobacter population is vulnerable to targeted interventions.


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS
Health categories: INFECTION
Title: Production systems bird welfare and endemic disease affect the susceptibility of chickens to Campylobacter
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Chicken is the source of ~80% of human Campylobacter infections. The bacteria are found on carcass surfaces at high levels and in muscle and liver tissues. Undercooked chicken meat is an important vehicle of Campylobacter infection. It is essential that the number of contaminated chickens is reduced. The proposed research will examine different intensive systems in which UK chickens are grown and determine reasons for the observed differences in Campylobacter status. This project results from a direct request from the UK poultry industry. This proposal brings together modellers, microbiologists, molecular biologists, retailers and poultry producers in a unique combination to address global risks to human health associated with interactions between broiler production systems, endemic disease and physiological state of birds. Our past work showed that chickens reared under higher welfare systems are less likely to have Campylobacter than standard birds. The higher welfare systems use birds that grow more slowly and which are stocked at a lower density. As well as lower Campylobacter levels, the higher welfare birds had better health and this may protect them against Campylobacter. We seek to determine, through field studies, which of bird genotype/growth and/or the in-house environment determine Campylobacter status and to identify the mechanisms by which Campylobacter leave the chicken gut and infect edible tissues. We will use a combined modelling and molecular approach to investigate factors that improve resistance to Campylobacter, reduce risk of colonization and extra-intestinal spread in UK broilers. We will undertake laboratory studies to determine the susceptibility of commonly used broiler chickens to C. jejuni and the major endemic poultry pathogen avian pathogenic E. coli (APEC). Infection with E. coli not only increases the risk of broilers having Campylobacter, but is also strongly correlated with spread from the gut.


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS;2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Predictive modelling to optimise phage intervention against Campylobacter in poultry
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The use of bacteriophage targeted against Camplyobacter infection in poultry is a potentially powerful form of biocontrol. Optimal use of phage therapy will require good understanding of phage-Campylobacter interactions at all stages of poultry farming and processing. This proposal will build a uniquely comprehensive understanding of Campylobacter-bacteriophage interactions in poultry from infection to carcass. It will combine laboratory interventions and predictive modelling in three strands. We will: 1. Build a comprehensive understanding of in vivo phage-Campylobacter kinetics in broiler chickens. 2. Determine how post-slaughter kinetics contributes to phage therapy efficacy. 3. Use our predictive models to design protocols for optimal practical phage therapy.


Research activity codes: 3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS
Health categories: INFECTION
Title: The importance of the intracellular energy metabolism of Salmonella Typhimurium within epithelial cells and macrophages
Funder: Biotechnology and Biological Sciences Research Council

Abstract: In humans S. Typhimurium causes a severe self-limited gastroenteritis, however in mice it becomes systemic resulting in typhoid like fever. In order to become systemic, S. Typhimurium first colonises the specialised antigen sampling epithelial cells lining the Peter's patches of the small intestine, then invades phagocytic cells such as macrophages in the mesenteric lymph nodes. Within macrophages, Salmonella resides within a specialised compartment (the Salmonella containing vacuole (SCV). We recently showed that glycolysis and glucose are required for Salmonella to replicate and survive in macrophages. However, this and other data from our group suggests Salmonella generates ATP for replication via different mechanisms in macrophages and epithelial cells. We hypothesise that S. Typhimurium generates ATP via oxidative phosphorylation in infected epithelial cells, but has to resort to substrate level phosphorylation for ATP generation in macrophages. We also hypothesise that the necessity to use substrate level phosphorylation for ATP generation in infected macrophages is a consequence of the inhibition of the electron transport chain due to defensive reactive oxygen species produced by the host. The mode of energy generation for intracellular growth of Salmonella may be important since the syntheses of virulence determinants, such as flagella and secretion apparatus in intracellular Salmonella are ATP-dependent. In this proposal we will also address a major question that has proved difficult to answer in the past: to what extent does the host contribute to replication and survival of Salmonella within the SCV? We will use a novel application of a proteomic approach (stable-isotope labelling by amino acids in cell culture - SILAC) to determine the contribution of amino acids from host proteins and peptides to the replication of S. Typhimurium within macrophage and epithelial cells


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Investigation of stochastic variations in growth rate as the mechanism of drug tolerance in Mycobacterium tuberculosis
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Mycobacterium tuberculosis (Mtb) is a major pathogen of man and animals. Drug treatment takes six months or more because the pathogen exhibits phenotypic tolerance to the antibiotic regime in vivo. The key signature of antibiotic tolerance is the biphasic kill curve which is obtained when growing bacteria are exposed to antibiotic. The killing rate is initially rapid but then slows down. This tail of slowly killed bacteria is responsible for the phenomenon of drug tolerance. However, the mechanism by which bacteria become tolerant is completely unknown. The most popular theory involves phenotypic switching of cells between two or more states (bistability) corresponding to normal and antibiotic-tolerant cells. However, there are many problems with this theory, for instance, that it has not been possible to engineer cells in pure normal or persistent states. We have developed an alternative persistence hypothesis whose starting point is the well-established fact that antibiotic killing of bacteria is growth rate-dependent. We then consider cells in which growth rate is controlled by a single gene and demonstrate that such a system would generate biphasic kill curves if the gene is expressed at low level and thereby subject to stochastic noise. The aim of this project is to investigate this hypothesis for the TB bacillus. We will engineer cells whose growth rate is controlled by a gene with a range of promoter strengths. We will investigate population variation in gene expression, growth rate and antibiotic killing by single cell studies and differences in metabolism by 13C-metabolic flux analysis. The studies may lead to develop of new therapeutic strategies that target the ability of the pathogen to enter or maintain itself in the persistent (drug-tolerant) state.


Research activity codes: 5.1 PHARMACEUTICALS
Health categories: INFECTION
Title: The role of the Cdc28 Cbk1 and Tpk1 kinases in the formation of hyphae of the human fungal pathogen Candida albicans
Funder: Biotechnology and Biological Sciences Research Council

Abstract: The polymorphic fungus Candida albicans is a major human pathogen. Hyphae, which grow in a highly polarized fashion from their tip, are required for virulence. The project focuses on the role of three kinases, Cdc28, Cbk1 and Tpk1, and two regulatory subunits Mob2 and Hgc1 that are known to be essential for hyphal growth. Low throughput studies in the PI's laboratory has already identified Sec2 as a target of Cdc28. Preliminary data has led to the identification of a number of putative possible targets for Cdc28 and Cbk1 and uncovered a complex pattern of phosphorylation regulating the activity of Cbk1. The first objective is complete these preliminary studies by unambiguously identifying the targeted residues by Mass Spectrometry, and determining the physiological relevance by mutation of the targeted residues. The second objective seeks to extend the search for targets of the three kinases by an unbiased survey of the proteome. This will adopt the approach successfully pioneered in Saccharomyces cerevisiae of using mass spectrometry to identify proteins that co-immuneprecipitate with the kinases. Rapid magnetic bead capture, minimal washes and on-bead protein digestion will be used to address the transient nature of the interaction between kinases and their targets. To eliminate non-specific binders a metabolic labeling approach will be adopted whereby the lysate from a kinase-tagged strain, grown on heavy medium will be mixed with an equal quantity of lysate from a non-tagged strain grown on light medium. Deviation from 1:1 peptide ration will identify proteins that genuinely interact with the kinases. Hits from this screen will be verified by appropriate tests and the targeted residues of selected proteins mutagenised to phosphomimetic and non-phosphorylatable residues to determine the physiological role of the phosphorylation events. Finally, the data will be used to construct a kinase interaction map required for hyphal growth and development.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Biophysical and physiological characterisation of potassium channels from pathogenic fungi
Funder: Biotechnology and Biological Sciences Research Council

Abstract: Fungal genomes express a gene encoding a potassium channel (TOK1) which has no known structural or functional homologues in either mammals or plants and is emerging as a potential therapeutic target for disrupting normal fungal cell physiology. TOK1 channel proteins are distinctive in topology with 2 pore-forming P domains and 8 transmembrane spans (2P/8TM) and unique in their strictly outwardly-rectifying function. I have recently cloned TOK1 homologues from multiple species of pathogenic fungi that represent the primary sources of fatal infections in immunosuppressed individuals (Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans). Preliminary results establish that the TOK1 channel family can be functionally defined by their K+ selectivity, strict outward rectification above EK, voltage-dependence, time-dependent and -independent components to activation, non-inactivating current phenotype and pharmacology. Despite these advances, many key questions concerning the strict gating, physiological regulation and role of TOK1 channels in fungi remain to be addressed. The aim of the present study is to uncover the molecular mechanisms underlying the strict outward rectification of TOK1 channels using a combined molecular and electrophysiological strategy targeting specific residues lining the ion conduction pathway. I have also recently identified a pHO-sensing residue in the S1-S2 loop of the Candida albicans TOK1 homologue that modulates K+ selectivity. I will probe further the molecular and structural basis for this key finding and the role of CaTOK in morphological transitions of Candida albicans, a key virulence trait. These results, describing biophysical, regulatory, and physiological properties of TOK1 channels will provide a suitable platform for developing therapeutic strategies to reduce the prevalence of dangerous mycotic infections.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Single-molecule assays of the assembly/disassembly mechanisms of ssRNA viruses - Tools for screening novel anti-virals
Funder: Biotechnology and Biological Sciences Research Council

Abstract: We are seeking support to develop single molecule (SM) fluorescence assays for the study of ssRNA virus assembly and disassembly mechanisms Our recent work on a range of such viruses suggests that these pathways are novel anti-viral drug targets. The SM assays will provide novel insights into RNA virus assembly mechanisms, and are ideal for future drug screening programmes. In addition, they will be widely applicable to other problems involving the structure and function of long RNAs, many of which are now being identified as potential drug targets. In recent major contributions to understanding the assembly mechanisms of ssRNA viruses, we have shown that the lack of drugable targets in this area may be a consequence of neglecting the contributions of the viral RNA to these processes. We have shown that at least in one model system these include: triggering conformational change within viral coat proteins , switching them between quasi-equivalent conformers; controlling the precise pathway to the final capsid & setting up a capsid that can productively uncoat as the first step in disassembly. These genomic RNAs must be compacted from their solution conformation(s) during assembly and we have shown that small molecules that bind and inhibit such events block assembly, thus porviding proof of principle that these pathways are drug targets. As an example of the utility of the single molecule assays, we have demonstrated a rapid, unexpected collapse in the hydrodynamic radius of the RNA when coat proteins are added to it. This species slowly rearranges to form the final capsid suggesting it is an on-pathway intermediate, and obvious drug target.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION