NIHRDataInsights/HRCSData huggingface dataset:
Title: Making it through the life cycle: Motility for pathogenicity in Leishmania parasites
Funder: Wellcome Trust

Abstract: Trypanosomatid parasites are major human pathogens which are transmitted by an insect vector and infect humans and other mammals. They have a single flagellum and swim, while also using the flagellum to interact with their environment. Motility is known to be vital for some life cycle stages, based on Trypanosoma brucei, but how motility is controlled is essentially unknown. This egregious gap in understanding is particularly clear for Leishmania. These parasites presumably need to swim to navigate through the sandfly vector, find surfaces to adhere to and select host cells to infect. For Leishmania to control its motility it must a) biomechanically regulate how the flagellum moves to control swimming b) use signalling molecules to modulate flagellum movement and c) do so using appropriate environmental cues in diverse host environments. I will address these questions; answering how eukaryotic flagella movement is controlled, which secondary signalling molecules are responsible, and how this basic biology contributes to the ability for parasites to find their way through complex host environments. Leishmania provides new opportunities to analyse this basic cell biology of flagellum function. It is also likely to be critical for the parasite and contribute to the pathology of the disease.

Lay abstract: For both bacteria and single-cell eukaryotic (nucleated) parasites the ability to move or swim is extremely important in causing disease. Leishmania parasites are single-cell microbes which causes a major human disease; Leishmaniasis. In the tropics they are the most deadly eukaryotic microbe after Plasmodium, which causes malaria. Leishmania movement may be important to find an environment in a person's body where they thrive, or to navigate through the organs of the sandfly which transmits the disease. However precisely how and why they move, and what chemical or physical cues guide their direction, is not known. My research aims to discover the internal processes which control how Leishmania swim, then use this to learn which signals from the cell's surroundings are important for finding their way through the sandfly or finding cells to infect in a person. Ultimately this will show how Leishmania movement is important for causing human disease.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: How cells convert cytosol-invading bacteria into anti-bacterial signalling platforms
Funder: Wellcome Trust

Abstract: Our cytosol could provide ample resources for invasive bacteria, yet is maintained in a remarkably sterile state. We recently discovered that cells deposit polyvalent protein arrays at bacterial surfaces to convert cytosol-invading bacteria into anti-bacterial signalling platforms. The deposition of M1-linked ubiquitin chains by the E3 ubiquitin ligase LUBAC recruits and activates IKK complexes, resulting in NF-kB signalling, while the deposition of guanylate-binding proteins (GBPs) recruits and is required for the LPS-dependent activation of Caspase-4, resulting in pyroptotic cell death and the release of IL-18. Here, we will explore this novel principle of cell-autonomous immunity to better understand cytosolic anti-bacterial defence. We will therefore investigate 1. The origin and function of ubiquitin-dependent signalling platforms, with special emphasis on the ubiquitylation of bacterial lipopolysaccharide (LPS) by the E3 ubiquitin ligase RNF213, which represents the unprecedented ubiquitylation of a non-proteinaceous substrate 2. The origin and function of the GBP-dependent signalling platform, specifically its assembly mechanism and how it promotes activation of caspase-4 and other downstream effectors.

Lay abstract: Our ability to withstand infections relies on the capacity of professional immune cells to defend our body and the skills of individual cells to defend themselves. The contributions of professional immune cells to innate and acquired immunity have received much attention. Much less is known about the principles governing cell-autonomous immunity. We have discovered that human cells deposit cellular proteins such as poly-ubiquitin and guanylate-binding proteins onto the surface of cytosol-invading bacteria, thereby generating poly-valent signalling platforms for the recruitment of further cellular proteins with anti-bacterial functions. We will investigate how cells identify bacterial surfaces as the ‘right’ place to build such signalling platforms, analyse the machinery involved and the effector proteins recruited. Knowledge from these studies will inform on targets for potential novel therapies.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Dissecting the host response underlying recognition of a diverse group of pathogens
Funder: Wellcome Trust

Abstract: Multicellular organisms use systemic signals to transmit biological information across tissues in order to co-ordinate responses to stress or infection. Discovering such signals, how they spread and what responses they trigger is therefore of paramount importance for organismal development. We have previously introduced the first tractable animal system to investigate a diverse group of eukaryotic pathogens, the oomycetes, which cause deadly infections in humans, as well as the model organism Caenorhabditis elegans. We have now discovered that C. elegans is able to sense these pathogens in neurons and mount a protective transcriptional response, the hallmark of which is the induction of conserved chitinase-like proteins in its epidermis. We propose to use comprehensive molecular genetics, cell biology, biochemistry, metabolomics and developmental imaging to determine the host response underlying pathogen recognition and elucidate the molecular gene network underlying the mounting of this response via neuron-to-epidermis signal exchange. Studying intercellular signalling requires a system-wide approach and, to this end, C. elegans offers a great opportunity to investigate this fundamental problem at the whole organism level. The proposed work will expand our knowledge on core cell and developmental biology mechanisms of intercellular signal exchange and immune response in the context of oomycete recognition.

Lay abstract: Oomycetes are fungal-like pathogens causing disease in humans, such as the deadly tropical pythiosis for which no drugs are currently available. Infections by oomycetes have been so far difficult to study due to the paucity of tractable experimental systems. We have recently discovered that oomycetes are natural pathogens of the model organism Caenorhabditis elegans, a microscopic animal that is easy to culture in the lab allowing us to now model these understudied infections. We propose here to dissect how animals sense these pathogens in order to mount defence responses. Our preliminary results indicate that communication between different tissues is required, which highlights the need to work at the whole-organism level as opposed to isolated cell lines. This work will allow us to gain insights into oomycete recognition in animals and understand core mechanisms of cross-tissue communication, which may have key implications for systemic human disease and immunity.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Uncovering the roles of dynamic RNA interactions during assembly and assortment of multi-segmented viral genomes
Funder: Wellcome Trust

Abstract: Rotaviruses (RV) comprise a diverse and rapidly evolving group of segmented double-stranded (ds)RNA viruses of the Reoviridae family that cause deadly gastroenteritis in children worldwide. A hallmark of rotavirus morphogenesis is the stoichiometric assembly of eleven distinct genome ssRNA segment precursors prior to packaging. Elucidating the mechanism of accurate ‘molecular counting’ of RNAs is essential to understanding RV replication and their evolution. Our recent studies suggest that segmented genome assembly in RVs, and potentially other Reoviridae, requires the formation of an RNA assortment complex, whose assembly is facilitated by the non-structural proteins that accumulate in viroplasms. The key goals of this proposal are: a) to investigate how the dynamic RNA structures control accurate assembly of a multi-segmented genome; b) to directly visualize the assembly of distinct ssRNAs and to identify pathways for disrupting this process. To achieve these goals, I will combine structural probing of ssRNAs, tools for detecting inter-molecular RNA-RNA contacts and structural modeling. To visualize multiple interacting ssRNAs in situ, I will employ recently developed quantitative super-resolution multiplexed RNA imaging approaches. These fundamental studies will pave the way for identification of routes for controlling RV assembly, having potential uses in the development of antivirals and rationally designed vaccines.

Lay abstract: Rotaviruses are highly contagious viruses that infect children worldwide, causing > 200,000 deaths annually, mostly in low-income countries. In order to develop new treatments to combat rotaviruses, it is essential to understand their molecular underpinnings. A single infectious rotavirus particle contains 11 unique RNA segments used to store its genetic material, serving as a multi-page instruction manual for building new viruses. It is a mystery how rotaviruses select and package 11 distinct segments, ensuring that each newly built virus has a complete set of instructions for infecting cells. I will use a combination of extremely sensitive microscopy and molecular biology tools to crack the code of how rotaviruses select and package a complete set of segments. Understanding the mechanism of segment counting will accelerate our efforts to design improved rotavirus vaccines that offer greater protection, and identify new targets for developing antiviral drugs.

Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: PubMLST: Disseminating and exploiting bacterial diversity data for public health benefit
Funder: Wellcome Trust

Abstract: Multilocus sequence typing (MLST) has been the principal approach to bacterial strain characterization for >20 years and is increasingly employed for whole genome sequence (WGS) analyses (whole genome and core-genome MLST). PubMLST (https://pubmlst.org) provides a major international resource for the storage, analysis, and dissemination of assembled microbial WGS data and hosts >100 databases that provide MLST nomenclature and link curated genetic, provenance, phenotype, and population data. The PubMLST resource, which has existed in various guises since 1998, operates from an Oxford-based web server and uses the open-source and open-access software platform BIGSdb, which was specifically developed for this purpose. PubMLST provides services to a wide variety of other resources, some of which employ the BIGSdb software. PubMLST also hosts the ribosomal MLST (rMLST) database, which enables bacterial species identification by cross-referencing >1.5M ribosomal allele sequences to a validated dataset of ~250,000 genomes representing 9500 bacterial species. Ongoing development of BIGSdb facilitates complex data querying and comparative analyses through a web interface. Data can also be retrieved and queried by other external (e.g. public health) databases via a programming interface. This application seeks to continue the operation and development of PubMLST, specifically enhancing the reuse of data is a major goal.

Lay abstract: MLST is the gold-standard DNA-sequence based method for identifying and naming microbes. Uses include: identifying disease outbreaks; understanding how pathogens change; and tracking disease spread globally. By changing the number of genes used, from a handful to thousands, the user can get best level of detail for their particular needs. Essential open-access catalogues of information are held in public, web-accessible, databases and PubMLST hosts most of these ( > 100 databases), employing our bespoke software (BIGSdb), which links DNA data to other information including: location; disease; patient age; etc. PubMLST also hosts a very large genome database, containing > 250,000 genomes from about 9,500 bacterial species, specifically developed to facilitate accurate bacterial identification from sequence data. Support is essential for continued availability of these unique resources. Further, we shall renew, upgrade, and develop the infrastructure and software, with the particular goal of making these rich datasets even more accessible to an increasingly-wide audience.

Research activity codes: 1.5 RESOURCES AND INFRASTRUCTURE (UNDERPINNING)
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: CTP switches in bacterial chromosome segregation and physiology: structures, functions, and mechanisms
Funder: Wellcome Trust

Abstract: ATP and GTP switches are extensively used to control conformations and functions of proteins in a wide range of biological processes. However, CTP switches have rarely been found in biology. Recent work from our laboratory and others has shown that ParB is a founding member of a CTPase protein family that uses a CTP switch to regulate bacterial chromosome segregation. We hypothesize that CTP switches are currently unappreciated and may be widespread. In the proposed work we will exploit the tripartite ParABS system from Caulobacter crescentus to elucidate the structure, function, and mechanism of the CTP switch that ensures faithful chromosome segregation. Next, we will investigate how other CTP switches regulate membrane association and gene expression by employing noc and KorB (proteins crucial for chromosome integrity and plasmid transmission, respectively) as models. Lastly, we will identify and characterize other CTP switch proteins in C.crescentus and systematically discover putative CTP-binding/CTPase proteins across all sequenced bacterial genomes. Altogether, this work will provide fundamental knowledge on the mechanism and evolution of CTP switches and open new and unexpected horizons in numerous fields beyond bacterial chromosome segregation. Moreover, our immediate research on plasmid/chromosome segregation and transmission may inform strategies to combat plasmid-borne antibiotic resistance.

Lay abstract: The binding and degradation of high-energy small molecules, such as ATP and GTP, is widely exploited in biology to switch on/off biological functions. Surprisingly, CTP, another abundant building block of life, is rarely employed as a switch. A recent discovery showed that ParB is the founding member of a CTPase protein family and can bind and break down CTP to control inheritance of genetic material (chromosomes and plasmids) in bacteria; it is possible that CTP switches are ubiquitous in biology but currently under-appreciated. We will investigate this possibility by (i) investigating how CTP switches operate at the molecular level, using three proteins (ParB, noc, and KorB) as research models, and (ii) discovering other CTP switch proteins in bacteria. Altogether, we will gain fundamental knowledge of CTP switches, open new frontiers in biology and contribute to the effort to combat the transmission of antibiotic-resistance plasmids by directly interfering with CTP switches.

Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Understanding and inducing immunity against rabies and Epstein Barr virus Class III fusion proteins
Funder: Wellcome Trust

Abstract: Viral fusion proteins are important targets for vaccine-induced neutralising antibody. Structure-guided design of such antigens in pre-fusion conformation has created leading vaccine candidates against viruses with Class I and II fusion proteins, such as respiratory syncytial virus and dengue virus. Use of pre-fusion stabilised Class III proteins for vaccination has not yet been reported, but these too are major vaccine targets for rabies virus and the herpesviruses (the gB proteins). I propose to build upon preliminary data demonstrating pre-fusion stabilisation of rabies glycoprotein. I will evaluate pre-fusion-stabilised immunogens for vaccines against rabies and a chosen herpesvirus, Epstein Barr virus (EBV). By isolating monoclonal antibodies against EBV gB, I will ascertain whether herpesvirus gB proteins contain pre-fusion-specific neutralising epitopes. In healthy EBV carriers, I will explore whether salivary EBV shedding can be used as a marker of immune control of virus reactivation, to dissect mechanisms of natural immunity, and to rapidly detect efficacy in future early-phase vaccine trials. A low-cost single-dose vaccine based on stabilised rabies glycoprotein could help reduce the 60,000 deaths due to rabies each year. An effective EBV vaccine could reduce the >100,000 deaths per year due to EBV-driven cancers and, many believe, could also prevent multiple sclerosis.

Lay abstract: The project focuses upon the structurally-related viral fusion proteins of rabies and Epstein Barr virus (EBV). Antibodies against these proteins can neutralise the viruses. Such proteins can adopt distinct pre-fusion and post-fusion forms. The project will investigate whether it is possible to design vaccines to induce pre-fusion-specific antibodies and whether such antibodies will neutralise the viruses more potently. A further aspect of the project will examine whether the level of shedding of EBV in carriers’ saliva could serve as a marker of immunity against the virus. This work aims to address the need for new vaccines against rabies and EBV. Rabies kills about 60,000 people every year: a cheap, single-dose vaccine would be a major step forward. Most adults carry EBV without suffering serious problems, but it causes 100,000 deaths due to cancer each year and there is increasing evidence it contributes to development of multiple sclerosis.

Research activity codes: 3.4 VACCINES
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Ringing the changes: how bacterial enzymes convert ribosomally-synthesised peptides into antibiotics
Funder: Wellcome Trust

Abstract: The project aims to investigate the mechanism of peptide transformations carried out by YcaO enzymes and to use this knowledge to create novel antimicrobials. Bacterial YcaO proteins are essential for the biosynthesis of multiple classes of peptide natural products through their ability to introduce important structural modifications including heterocycles, macrocycles, and thioamides. YcaO repurposing and swapping between different pathways offers exciting possibilities to create novel artificial peptides bearing such modifications. Despite much recent progress, we still do not really understand how these proteins work, and how their activities are coordinated within multisubunit enzyme complexes. I will use cryo-EM in combination with X-ray crystallography to solve the structures of YcaOs within their native complex assemblies trapped at different catalytic stages. In parallel, I will discover and characterise new YcaO proteins to further expand their known catalytic repertoire. Finally, the promiscuity of YcaOs allows for the production of large genetically encoded peptide antibiotic libraries. I will generate these by co-expressing randomised precursor genes along with modification systems. A high-throughput fluorescence-based version of a Waksman platform will be used for screening, where nano-sized “Petri dishes” (hydrogel beads) will be inoculated with both producer and reporter strains and sorted by FACS.

Lay abstract: Rapid spreading of resistance to commonly used antibiotics is a major healthcare problem. Scientists are constantly searching for new antibiotics, but they are increasingly difficult to find using current knowhow. My study focusses on bacterial enzymes, called YcaOs, which can convert peptides into antibiotics, for example, by introducing various shaped rings into peptides. I will look closely at how YcaOs function by determining their 3D structures. In parallel, I will dig into the huge variety of YcaO-modified peptides predicted to be made by bacteria associated with humans or plants. Finally, I will produce artificial nature-inspired antibiotics using already characterised YcaO enzymes. Cells producing such peptides will be embedded into miniature agar (jelly-like) beads which will serve as tiny Petri dishes. With this system, it is possible to screen millions of peptides over a single day to find new antibiotics, ensuring that the future drug pipeline does not run dry.

Research activity codes: 5.1 PHARMACEUTICALS
Health categories: GENERIC HEALTH RELEVANCE;INFECTION
Title: Genetic and chemical validation of sugar nucleotide biosynthesis as a target against Candida albicans
Funder: Wellcome Trust

Abstract: "Fungi" are types of bugs that are widespread and most of us carry some of these organisms. Normally our immune system is perfectly capable of suppressing their growth. However, under conditions where the immune system is weakened, for instance during chemotheraphy, organ transplants or HIV infection, these microbes are able to establish lethal infections that are not uniformly/well treatable with currently available drugs - there is a significant unmet medical need. This proposal aims to give us an understanding of proteins that are important for building the "cell wall" of the fungal pathogen Candida albicans - a structure that protects this microbe from its environment. This work will ultimately lead to the identification of opportunities for the discovery of novel anti-fungal drugs by the pharmaceutical industry.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: GENERIC HEALTH RELEVANCE;INFECTION