NIHRDataInsights/HRCSData huggingface dataset:
Title: Unravelling the intracellular interactions and signalling of Apical membrane antigen-1 (AMA1) in malaria parasites.
Funder: Wellcome Trust

Abstract: Apical membrane antigen-1 (AMA1) is a type-I integral membrane protein of malaria parasites, required for host cell invasion. Invasion requires phosphorylation of the AMA1 cytoplasmic domain (CD), suggesting a signalling role. Exactly what process(es) the AMA1 CD regulates, and how it does so, is unknown. This project will examine the role of the AMA1 CD in regulating cellular events during invasion, using the recently culture-adapted malarial species Plasmodium knowlesi. A conditional mutagenesis system (DiCre) will be used to switch expression of wild-type AMA1 to mutant alleles (e.g. non-phosphorylated forms) to examine the role of the CD in invasion. The impact of mutagenesis on discharge of secretory organelles containing invasion ligands will be examined using immunofluorescence and electron microscopy. Proteins that interact with the AMA1 CD will be identified using directed biotinylation by an APEX2 tag fused to the CD, followed by pulldown and mass spectrometry. The role of interacting proteins in invasion will then be explored using DiCre-mediated knockout. A high-throughput Cas9 mutagenesis screen of the AMA1 CD will also be performed to identify crucial residues involved in mediating interaction of partner proteins with the AMA1 CD. This project will elucidate the long-hypothesised signalling role of AMA1.

Lay abstract: AMA1 is a protein on the surface of invasive forms of the malaria parasite, and is required for parasite invasion of red blood cells (RBCs). AMA1 spans the parasite cell membrane, with part of it, the cytoplasmic domain (CD), present inside the cell. The AMA1 CD has an essential role in invasion and is modified through a process called phosphorylation, typically associated with the transduction of signals within cells. I hypothesise that when the external portion of AMA1 contacts a RBC, the CD transmits a signal to the parasite. This project aims to understand how this signal is produced and how the parasite responds to the signal. To do this, the AMA1 CD will be specifically modified to examine how this affects signalling to the parasite, and to identify other parasite proteins that interact with it. These parasite proteins will then be studied for their importance in host cell invasion.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Evaluation of congenital infections, social and economics influences on child health: a population-based record linkage study
Funder: Wellcome Trust

Abstract: Congenital infections are a major cause of adverse pregnancy outcomes. The extent to which they increase the risk of adverse birth outcomes is known for some infections, e.g. syphilis, but uncertainty remains about the effects of other infections and longer term childhood outcomes. The evidence available are controversial and limited to a range pathogens. My fellowship focuses on four diseases (syphilis, toxoplasmosis, chikungunya and Zika) and aims to generate evidence on health and social burden of congenital infections, by linking data from Brazil’s 100 Million Cohort. Large social inequalities and poverty exists in Brazil, so I will take into consideration the social and economic factors to understand the role of social determinants and evaluate protection policies. To achieve my aim: first, I will quantify the association between congenital infections and adverse outcomes. Second, I will determine the extent to which variation in risk of adverse outcomes is explained by social/economic factors. Third, I will determine how families are financially or socially affected by congenital infections and associated complications, and compare mortality rates in mothers living with and without impaired children. Finally I will evaluate the impact of social protection programmes on birth/childhood outcome of women exposed during pregnancy.

Lay abstract: Little is known about the long life outcomes of children who are exposed before birth to maternal infection. Reported cases of negative outcomes are not new; however, the evidence on lifelong impairment is limited, controversial and available for few pathogens. My fellowship will generate new evidence on health and social burden of four congenital infections (syphilis, toxoplasmosis, chikungunya and Zika), by linking administrative collected data from Brazil for over 100 million people with records of births, infectious disease, deaths and hospitalisations. I will also link these with social and economic data to estimate the role of social determinants; evaluate if social protection policy can mitigate the risk of birth/childhood outcome of women exposed during pregnancy, and determine the impact of congenital infections on family living conditions. The outputs of my fellowship will provide evidence for strategies to prevent congenital infections and identify the healthcare and social needs of those affected.

Research activity codes: 2.4 SURVEILLANCE AND DISTRIBUTION
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Dissecting the molecular basis of the anti-phage defence system Zorya II
Funder: Wellcome Trust

Abstract: Bacteria protect themselves against bacteriophage (phage) infection using defensive systems, with phages co-evolving counter-resistant strategies. Several anti-phage systems have been identified and their modes of action determined. Additionally, nine novel systems were recently reported, and thus remain to be characterised. My proposed research builds upon this recent discovery and aims to dissect the molecular and structural details of the novel anti-phage system named Zorya. Two types of Zorya systems with distinct host range have been described, sharing two conserved components, ZorA and ZorB. These two proteins have been proposed to act through a conserved mechanism, based on formation of a putative ion-channel, to neutralise phage infection. I am to test this hypothesis with the following aims: 1. To probe the organisation and function of Zorya II in vivo 2. To define the mechanism by which Zorya II protects from phage infection 3. To identify the trigger of Zorya II anti-phage activity Elucidating the molecular basis of interactions between bacteria and their viral predators and characterising the molecular mechanism of anti-phage systems is vital to understand the nature of bacteria-phage co-evolution and its capacity to shape the composition and dynamics of polymicrobial environments, with implication for therapeutic and biotechnological applications.

Lay abstract: Bacteriophages (phages) are small, non-living entities that depend on their bacterial host for survival.Bacteria and phages are involved in cycles of co-evolution, where bacteria develop mechanisms to prevent infection from their predators and phages adapt to overcome these new strategies. Of recent discovery is a novel anti-phage system, named Zorya. It has been observed that bacterial strains that carry a Zorya system, when infected by a phage, can initiate early cell death to avoid its replication. Nevertheless, little is known about the exact mechanism through which the system is activated and can cause growth cessation, to arrest the phage infection. I will explore the mechanism by which the Zorya system leads to bacterial death, addressing how the single components of the Zorya system can assemble to bring about the death of infected host bacteria and which components of the phage invader is fundamental to determine the initiation of this pathway.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Formation of the outer surface of the mosquito cuticle and its role in determining the fitness of African malaria vectors
Funder: Wellcome Trust

Abstract: The mosquito cuticle provides a barrier to the external environment, protecting from toxins, pathogens and desiccation. The outer epi-cuticular layer consists predominately of cuticular hydrocarbons (CHCs) and the composition of this wax layer affects many traits that, in turn, influence the ability of the mosquito to transmit pathogens. For example, CHCs play a critical role in mate recognition, penetration of insecticides, and survival in arid conditions. Variation in the CHC composition of malaria vectors is well documented but very little is known about the genetic and environmental factors generating this diversity. This project will identify the genes controlling the hydrocarbon biosynthetic pathway in the malaria mosquito Anopheles coluzzii. Transgenic approaches will be employed to perturb this pathway enabling studies on a wide range of different phenotypes to be investigated, including resistance to desiccation, penetration of insecticides and mating success. The influence of humidity fluctuations on the CHC profiles and the role of steroid hormones in regulating the CHC biosynthetic pathway will be determined. The project will provide new knowledge on the physiology of mosquitoes leading to a greater understanding about how they are adapting to climate change and provide new avenues for effective strategies to control these major disease vectors.

Lay abstract: Controlling malaria relies on targeting the Anopheles mosquito. Sustaining, let alone improving vector control is threatened by insecticide resistance and by knowledge gaps on mosquitoes’ biology. Cuticular hydrocarbons (CHC) can affect many traits, like desiccation tolerance, insecticide resistance and mating, that influence mosquitoes’ ability to transmit pathogens. Although variation in CHC composition of malaria vectors is well documented little is known about the genetic and environmental factors generating this diversity. I aim to identify genes controlling CHC biosynthesis in Anopheles by isolating the cells synthesizing hydrocarbons and determining their genetic composition. I will employ transgenic approaches to disrupt this pathway and investigate the effect on traits that determine mosquitoes’ fitness. This will enable us to understand how natural variations in CHC impact on mosquitoes’ adaptability to climate change and survival to vector control interventions. Ultimately, these insights into mosquitoes’ biology will aide in the design of efficient vector control strategies.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: The evolution of mobile genetic elements in Gram-negative bacteria
Funder: Wellcome Trust

Abstract: I will explore how structural variation can be used to understand the evolution of antimicrobial resistance (AMR) on mobile genetic elements (MGEs). Most previous research is biased towards nucleotide-level variations in the core genome (high-effect SNPs). now, the availability of complete genomes means that resolving the larger structural variation of MGEs is both possible and increasingly relevant for the spread of AMR. I will develop a framework to model the non-mutational processes which generate this variation, then apply this to the linked evolution of MGEs and their ‘host’ genomes. During this fellowship, I will sequentially: • Aim 1. Develop a tool to quantify structural variation and resolve common blocks around the flanking region of AMR genes, which I have previously shown can be used for phylogenetic reconstruction. • Aim 2. Apply this to longitudinal datasets and obtain rate estimates for non-mutational processes. • Aim 3. Investigate the compensatory evolution of MGEs as they move between genomic backgrounds. Two major applications will be: • Application 1. Small transposons, leading on from my pioneering work on the mcr-1 transposon. • Application 2. Clinical class 1 integrons, which carry multiple AMR genes. This proposal tackles questions of fundamental importance for the evolution of flexible genomes.

Lay abstract: Bacterial genomes can adapt by small changes in individual genes and by structural rearrangements, including new genes which travel between genomes on mobile genetic elements. Structural variation is of particular importance for antimicrobial resistance (AMR). With older DNA sequencing technologies, genomes were incomplete and fragmented. now, complete genomes are common: the challenge is to understand structural variation. I will use structural variation in bacterial genomes to help us understand their recent evolutionary history. I have developed a promising approach to extract shared sequence patterns from diverse regions around AMR genes. I will extend this and develop models of the underlying processes. I will test specific hypotheses about natural selection in bacterial genomes: between mobile ‘parasites’ and their genome ‘hosts’. By using my computational methods for clinically relevant examples, I will help to understand the evolution and spread of AMR. Making software freely available will allow others to do the same.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Understanding host restriction following bacterial phagosome rupture
Funder: Wellcome Trust

Abstract: Professional phagocytes engulf bacteria inside phagosomes that mediate cargo degradation via lysosome fusion. Some intracellular pathogens evade this fate by disrupting the phagosomal membrane, which triggers antibacterial autophagy. While autophagy may eliminate bacteria for host defence, more recent studies have demonstrated that autophagy can also promote intracellular bacterial survival or be irrelevant for bacterial restriction. Recently, Endosomal Sorting Complex Required for Transport (ESCRT) proteins have been shown to repair small membrane disruptions, potentially providing an alternative pathway for bacterial restriction. The interplay between autophagy and ESCRT machinery during phagosomal damage, the factors triggering each pathway and their contribution to intracellular bacterial killing are poorly understood. Using the important human pathogen Staphylococcus aureus, I will: (1) investigate the interplay between autophagy and ESCRT machinery; (2) identify novel bacterial components that modulate autophagy and ESCRT recruitment; and (3) uncover unknown host regulators of autophagy, ESCRT and cell-autonomous immunity. To complete these objectives, I will use high-resolution microscopy to follow bacteria recruiting autophagy and / or ESCRT machinery at the single bacterial cell level. Additionally, I will use high-content genetic screens to discover novel bacterial (using transposon libraries) and host factors (using CRISPR/Cas9) shaping host-pathogen interactions.

Lay abstract: Immune cells engulf and kill microorganisms inside phagosomes. However, some microorganisms avoid their destruction by damaging the phagosomal membrane. This activates autophagy, which can sometimes clear the infection, but might also be irrelevant or beneficial for bacterial survival. Potentially providing a restriction alternative is the ESCRT machinery, which has been proposed to repair membrane disruptions. I want to investigate the interplay between ESCRTs and autophagy during Staphylococcus aureus infection. Advanced optical microscopy will enable me to follow both machineries in high resolution and may help to understand factors triggering each pathway and their contribution to intracellular bacterial killing. It will also be important to identify bacterial factors that can modify autophagy and / or ESCRT recruitment. Moreover, I aim to discover unknown host regulators of autophagy, ESCRT and cellular immunity that enhance bacterial restriction and might offer potential targets for new therapeutic regimes for infection control.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Bridging the gap between cell and structural biology at the University of Leeds
Funder: Wellcome Trust

Abstract: We request funds to purchase equipment to perform correlative light and electron microscopy (CLEM) and electron microscopy of cells & tissues at the University of Leeds. This will include: a high-pressure freezer to allow rapid freezing of large and/or labile specimens whilst minimizing structural deformation; a freeze-substitution unit for low-temperature substitution of water by dehydrating agents and fixatives, avoiding the damaging effects of room-temperature dehydration; a modern cryo-ultramicrotome, for room-temperature and cryo-sectioning of samples for (cryo-)EM imaging; and a cryo-CLEM system, to image cryo-fixed samples by fluorescent microscopy, identifying interesting biological events for further analysis at high-resolution by cryo-EM. This equipment will build upon the outstanding infrastructure for super-resolution light microscopy and cryo-EM in Leeds. The combination of new and existing equipment will give us an unprecedented ability to integrate high-resolution structural information into its cellular/tissue context. This will help us to tackle the most challenging projects in biomedical discovery, and understand the molecular basis of diseases such as infections, cardiovascular disease, neurodegeneration and cancer. We request £429k of Wellcome Trust funds, complementing the University’s contribution of ~£192k in capital and support salary costs, towards a total project cost of ~£621k.

Lay abstract: Structural Biology, a major research area in the biomedical sciences, aims to understand life in molecular detail. Within structural biology, electron microscopy can study samples at a wide range of resolutions allowing the integration of atomic resolution information into its native cellular and/or tissue environment. The University of Leeds has a strong community of structural biologists and is committed to support this field, as evidenced by the recent ~£10M investment in high-end cryo-electron microscopes. To complement this investment, we request ~£429k of Wellcome Trust funds for equipment that will allow researchers at Leeds to study cells and tissues by electron microscopy. Complemented by other imaging approaches, like (super-resolution) fluorescence microscopy or soft X-ray tomography, we will be able to combine the strengths of each technique to advance biomedical research at Leeds. These capabilities will allow us to make advances in fields such as infection, cardiovascular disease, neurodegeneration and cancer.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Marginalisation and the microbe: how to mobilise around antimicrobial resistance without increasing social inequalities
Funder: Wellcome Trust

Abstract: This research will examine mobilisation in response to antimicrobial resistance (AMR), asking how scientists, clinicians and publics relate to issues of inequality in infectious disease, and raising questions about the stigma that may accompany resistant infections. The project draws on theories of scientific and clinical classification work to examine where and how different vulnerabilities are made visible and actionable, using the concept of marginalisation to understand risks developing at the intersection of age, gender, sexuality, ethnicity/race, poverty and class. We will consider the role of the state using critical contrasts between the UK and US and analyse carefully selected cases in sexual and reproductive health. Mapping histories and geographies of resistance in gonorrhoea, Mycoplasma genitalium, and E.Coli urinary tract infections (UTIs) will illuminate how the problem of AMR is known in policy, microbiology, epidemiology and clinical medicine. We will document practices to manage bacterial resistance and possible stigma through comparative ethnographic work on sexual health settings – exploring how different forms of marginality are addressed in the clinic. We will analyse observed and reported patient experiences of resistance and antibiotic use and non-use, engaging with women discussing recurrent/persistent UTIs online, and those mobilising around Group B streptococcus risks in childbirth.

Lay abstract: Antimicrobial resistance (AMR) is increasingly appearing in common bacterial infections. Responses include more careful use of antibiotics, but attempts to preserve their effectiveness could exacerbate existing health inequalities. In the short term the unintended victims could be people already disadvantaged in relation to healthcare systems. This research will compare past, present and evolving responses to infections in sexual and reproductive health, including those more likely to affect groups made marginal due to a combination of age, gender, sexuality, ethnicity, race, poverty and class. The research offers a novel discussion of responses to AMR in scientific and clinical practice in the UK and US, contrasting more and less publicly visible infections. Careful comparisons of action by policy makers, scientists, clinical professionals – and patients where relevant – will give a critical edge to analysis of political and social factors shaping this field, and different ways of responding to inequality and stigma.

Research activity codes: 8.3 POLICY, ETHICS AND RESEARCH GOVERNANCE
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Putting genomic surveillance at the heart of viral epidemic response.
Funder: Wellcome Trust

Abstract: This proposal is to develop an end-to-end system for processing samples from viral outbreaks to generate real-time epidemiological information that is interpretable and actionable by public health bodies. Fast evolving RNA viruses (such as Ebola, MERS, SARS, influenza etc) continually accumulate changes in their genomes that can be used to reconstruct the epidemiological processes that drive the epidemic. Based around a recently developed, single-molecule portable sequencing instrument, the MinION, we will create a 'lab-in-a-suitcase' that will be deployed to remote and resource-limited locations. These will be used to sequence viral genomes from infected patients which will then be uploaded to a central database for rapid analysis. We will develop methods for a wide-range of emerging viral diseases. Novel molecular biology methods will allow us to sequence individual viruses within a patient. Bioinformatics tools will be developed simple enough for non-bioinformaticians to use, without reliance on Internet connectivity. We will develop software to integrate these data and associated epidemiological knowledge to reveal the processes of transmission, virus evolution and epidemiological linkage. Finally we will develop a web-based visualization platform where the outputs of the statistical analyses can be interrogated for epidemiological insights within days of samples being taken from patients.

Lay abstract: In recent viral outbreaks including Ebola and MERS-CoV, molecular sequencing of the viral pathogen has revealed critical insights into the evolution and transmission of the disease, and offered tantalizing examples of the potential value of this approach to future outbreak control efforts. However, the scale and impact of new DNA technologies in these outbreaks has largely been stunted, due in part to the complexities and logistics of shipping samples for sequencing at existing centres, and the resulting delays in the production of the sequence data meaning that the resulting inferences have limited direct utility for the response efforts. This proposal aims to overcome these challenges by producing a cheap, mobile virus sequencing system, supported by statistically rigorous analysis frameworks, and information sharing platforms, to prepare for the next outbreak and ensure that viral genome sequencing is positioned to have full impact on the public health response.

Research activity codes: 2.5 RESEARCH DESIGN AND METHODOLOGIES (AETIOLOGY)
Health categories: GENERIC HEALTH RELEVANCE;INFECTION