NIHRDataInsights/HRCSData huggingface dataset:
Title: Macrophage-induced drug tolerant persisters in tuberculosis
Funder: Medical Research Council

Abstract: Aim 1. To identify the Mtb trigger gene(s) required for MIP formation we will use the next generation deep-sequencing based method transposon sequencing (Tnseq). Stage 1) Our library (10e10 with ~10e5 mutants) will be inoculated into either A) cell culture media without macrophages, B) human monocyte-derived macrophage (MDMs) or C) IFN-gamma activated MDMs. After 2 hours the intracellular bacilli will be isolated by differential lysis. Stage 2) Anti-mycobacterial drug will be administered (rifampicin 60ug/ml) for each treatment followed by CFU plating and library recovery (we estimate a recovery of 10e 7bacilli, sufficient to maintain reasonable library coverage). Libraries recovered immediately prior to (T0) and 48h after the drug treatment will be compared for all three treatments to identify trigger genes for conditions A-C separately. The genes identified will then be compared across treatments to identify genes specific to each condition. Mutant abundance will be determined by sequencing transposon junctions using Illumina Hiseq sequencing, followed by bioinformatic and statistical analysis carried out by the Faculty's designated bioinformatics officer. Up to 10 candidate genes will be chosen, and gene knockouts and gene overexpressing strains generated using vectors and the defined mutant library from our collaborator Professor Jacobs Jnr's laboratory, and their antibiotic tolerances characterised. Reporter gene fusions will also be created and observed in single cell studies in our microfluidics system imaged using confocal microscopy. Aim 2: To determine the specific macrophage environment that induces MIPs, we will expose Mtb cultures to stress conditions associated with the macrophage environment, alone or in synergy, prior to an antibiotic kill to determine the level of persisters. A drug testing scaffold will be designed following identification of the host trigger in a 384-well format with controls including a positive anti-mycobacterial drug.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Tuning the immune response in tuberculosis
Funder: Medical Research Council

Abstract: Understanding the molecular determinants that tune immune responses to provide effective control of mycobacterial growth without immunopathology is a key priority in tuberculosis (TB) research. I will combine transcriptional profiling of the tuberculin skin test (TST) for molecular level assessment of human anti-mycobacterial immune responses, with studies using the genetically tractable zebrafish Mycobacterium marinum (Mm) infection model to evaluate the role of a key immunoregulatory cytokine, interleukin (IL)10, in tuning the immune response in TB. I will also adopt a genome-wide discovery approach to identify novel regulators of human immune responses to Mycobacterium tuberculosis (Mtb), on the basis that variability in anti-mycobacterial responses is responsible for differences in pathogenesis. I will cross-reference genes within the TST that exhibit highly variable expression levels between different individuals, with data from recent functional genomic studies that describe putative genetic interactions, in order to identify key regulatory hubs. Functional effects of potential master-regulators on anti-mycobacterial immune responses will be investigated by genetic manipulation in the zebrafish Mm model. By selecting candidates based on variable human anti-mycobacterial immune responses and testing them using the zebrafish, which has a successful track record for modelling human TB, this cutting-edge strategy promises to elucidate novel pathways that tune immune responses to determine clinical outcome in TB. Importantly, this will inform development of adjunctive host-directed therapies to significantly shorten duration of standard anti-tuberculous chemotherapy, risk stratification of active disease in latent TB infection and strategies for design of novel anti-TB vaccines.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Role of chromatin dynamics in regulation of intestinal innate immunity gene expression
Funder: Medical Research Council

Abstract: How chromatin dynamics in the intestinal epithelium orchestrates innate immunity is an important question but little is known about it. Our hypothesis is that chromatin dynamics, especially changes of histone variant H2AZ occupancy over CpG islands, regulate innate immunity gene expression in intestinal epithelium cells. We propose that histone H2AZ occupancy relates to cellular stress and the innate immunity response, e.g., during exposure to microbes, may reflect such a stress. To test this, we will exploit a very well established colitis model in the mouse, Dextran Sodium Sulfate (DSS) mediated colitis, and focus on the role of two chromatin remodeling factors that promote (EP400) and counteract (Smarcad1) histone H2AZ occupancy. We will examine an exciting new histone modification, crotonylation, which we find is affected by chromatin remodeling and may be linked to H2AZ occupancy and possible the microbiota. We will use chromatin immunoprecipitation coupled to high-throughput sequencing (ChIPseq) approaches to map histone H2AZ, histone crotonylation and chromatin remodeling factor occupancy during normal and colitis conditions and link these occupancies to a detailed analysis of the transcriptome, including the noncoding transcriptome. We will systematically examine CpG island chromatin using mass spectrometry approaches under all these conditions. Our work will test if chromatin remodeling factors are involved in coordinating an innate immunity response. Together, data generated in this proposal will paint a detailed picture how the transcriptome in colon epithelial cells is affected during colitis and how chromatin dynamics prime these cells to respond to an insult.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: A molecular basis for autoimmune sequelae of streptococcal infections
Funder: Medical Research Council

Abstract: Viral and bacterial pathogens have been suggested to trigger autoimmune diseases, but the molecular bases for such long-term consequences of infections are elusive. We propose to study the interaction of the M3 protein from Streptococcus pyogenes with the most abundant human protein, collagen. Binding to collagen is likely to mediate bacterial adhesion, invasion and immune evasion. The M protein:collagen interaction may also form the molecular basis for the induction of anti-collagen antibodies that is observed in the devastating autoimmune disorder rheumatic heart disease. We propose that this autoimmune response is a collagen conformeropathy, caused by exposure of neoepitopes of collagen upon M3 binding. Due to their architecture (fibrillar triple helical and dimeric coiled coil proteins, respectively) collagens and M proteins have largely been beyond the reach of conventional structural biology. We propose to combine long-range restraints obtainable by PELDOR with short and long-range NMR restraints to map the in-solution conformation of the collagen-binding hypervariable region of the M3 protein. We will use isopep-tagging as a novel strategy to covalently link proteins to generate differentially labelled M3 homodimers for NMR and EPR. Collagen constructs for M3 binding studies will be designed using Toolkits, libraries of overlapping triple-helical collagen peptides, in particular a new collagen XIII library. The collagen-binding site in M3 will be mapped, and conformational changes in both binding partners will be identified by PELDOR and NMR. Collagen neoepitopes exposed as a result of M3 binding will be identified using scFv antibody phage libraries. These scFvs will be compared to rheumatic heart disease autoantibodies and used in cell-based studies and bacterial adhesion and invasion assays (electron microscopy) aimed at elucidating the biological role of M3-collagen binding for bacterial infections.


Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFECTION
Title: Elucidating functions of a specialised staphylococcal secretion system during infection
Funder: Medical Research Council

Abstract: The human pathogen Staphylococcus aureus is a leading cause of healthcare-associated infections. The advent of new antibiotic resistant strains and higher recurrence rates of staphylococcal infections have placed a significant cost burden on healthcare systems worldwide. Chronic infections have been associated with intracellular S. aureus, although intracellular mechanisms mediating bacterial persistence are poorly studied. Esat-6 secretion system (Ess), a specialised type VII secretion system of S. aureus, is required for staphylococcal virulence and persistence. Proteins secreted by the Ess, including EsxA and EsxB, are crucial virulence factors and putative vaccine candidates. The molecular mechanisms underlying Ess functions during persistent staphylococcal infection are currently unclear. Our recent work demonstrates that the Esx proteins play a role in intracellular staphylococcal infection by interfering with host cell death pathways. The goal of this project is to understand the mechanism of action of Ess during staphylococcal infection. Employing isogenic mutants, high-resolution microscopy and biochemical methods we will elucidate host cellular pathways that Ess proteins interfere with during intracellular infection. We will examine Ess-associated local immune and cellular responses in ex vivo and in vivo models of acute and chronic infection. Deciphering the biological functions of the Ess proteins will further our understanding of these intriguing bacterial systems and unravel novel staphylococcal mechanisms for intracellular survival and persistence.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Unveiling the protein landscape of the African trypanosome cell surface and chasing down potential targets for therapeutic intervention
Funder: Medical Research Council

Abstract: The cell surface is the major point of interaction between an extracellular parasite like Trypanosoma brucei and its human host. This interface also represents the primary target for host immune attack. As a result, stable and accessible surface antigens that are unique to the parasite represent promising targets for both drug and vaccine development. With the availability of a validated, high-confidence cell surface proteome for the T. brucei bloodstream-form, testing for such surface antigens that are necessary for parasite survival becomes possible. Here, I will use established methods for gene silencing by RNA interference coupled with target DNA sequencing to screen a surface protein-specific library for growth in vitro and in the animal model of disease. This will identify those surface-exposed molecules essential to parasite survival in the host and discriminate between those effecting uptake of nutrient, resistance to innate immunity and clearance of antibodies. I will use similar methods, plus tools for endogenous-locus tagging of membrane protein genes I have developed, to identify in a genome-wide RNAi-based screen the membrane barrier components that, when ablated, cause surface proteins to escape from specialised membrane domains. Finally, I will use a piloted in vitro assay to test if parasite surface proteins are accessible to antibodies and effectively killed by host serum components. This will inform an in vivo immunisation study to test for potential as vaccines.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;3.4 VACCINES
Health categories: INFECTION
Title: Functional analysis of Epstein-Barr Virus genome variation in relation to cell growth and disease
Funder: Medical Research Council

Abstract: Epstein-Barr virus causes infectious mononucleosis and contributes to 1.5% of human cancers, yet the vast majority of humans carry EBV as an asymptomatic lifelong infection. Most EBV research has focussed on a small number of laboratory strains, many of them defective, and has neglected the diversity of circulating strains. We recently published the first major sequence analysis of worldwide EBV strains; we shall now investigate how this sequence diversity affects virus function. Our first aim is to identify viruses from among the sequenced strains that best represent wild-type (circulating) viruses and clone them in bacterial artificial chromosome (BAC) vectors. This will allow us to generate virus and investigate their behaviour in vitro (activation, proliferation and transformation of B cells; infection of epithelial and T cells; EBV gene expression differences) and in vivo - using mice with human immune system components. We shall seek to produce an in vivo model for EBV infection that more closely reflects human infection. We expect these new EBV clones will become standard strains for EBV research worldwide. Our second aim is to investigate the two major specific variations that we have previously identified through the sequencing project. First is the linkage between EBNA2 and EBNA3s in defining type1 and type 2 EBVs. Our preliminary data imply some functional compatibility of the EBNA2 and EBNA3 variants that affects B cell transformation and growth. We shall test this by generating viruses with additional combinations of EBNA2/3 types and assessing their transforming ability. Second, we shall assess the function of specific polymorphisms in EBV strains associated with nasopharyngeal carcinoma (NPC) in southern China. We shall test whether specific polymorphisms in NPC-associated EBV strains in the BART miRNA region of EBV result in a more profound suppression of p53, such that they increase susceptibility to NPC.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Understanding the transmission of tuberculosis using Mycobacterium tuberculosis sequence data
Funder: Medical Research Council

Abstract: Tuberculosis, caused by Mycobacterium tuberculosis, is an important global public health issue. Understanding the factors underlying transmission is essential for disease control, but surprisingly little is known about the role of pathogen genomic variation, where most infections occur, the importance of host factors, or who transmits to whom. M. tuberculosis can be grouped into seven lineages or phylogenetic clades, and further into sub-lineages, which may vary in propensity to transmit and cause disease. By sequencing M. tuberculosis in a population-based setting it is possible to construct transmission chains using phylogenetic-based algorithms: strains with near identical genomes are most likely to be due to a transmission event, and will appear on the same branch of the phylogenetic tree. However, there are few large population-based studies in high tuberculosis prevalence areas that can apply long-term large-scale whole genome sequencing. Here we focus on the Karonga study in northern Malawi where substantial epidemiological data have been collected and ~2500 sequenced samples are available spanning a 20-year period. Sequencing studies to date have been limited to single nucleotide polymorphisms and have ignored insertions and deletions and highly variable regions such as the pe/ppe gene families. By including all these variations and considering all the tuberculosis cases in the Karongan population, it will be possible to build a probabilistic model of transmissions, and hence assess effects of pathogen variation and host factors (age, sex, HIV status, contact patterns, proximity) on transmissibility using regression-based methods. Further, it will be possible to adopt a genome-wide approach to identify loci in the bacteria that are associated with or under evolutionary selective pressure from a transmissibility phenotype. Loci identified as being associated with transmissibility will be validated using sequence data from other populations.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Quorum sensing and virulence in Gram positive pathogens: structure, function and inhibition of the agr system
Funder: Medical Research Council

Abstract: In Staphylococcus aureus and related Gram positive pathogens, virulence is regulated via the agr-dependent quorum sensing (QS) system that is an attractive target for novel anti-infective agents. With previous MRC funding, the successful development of in vitro assays for processing the AgrD pro-peptide by AgrB enabled us to uncover a new pathway for the generation and export of autoinducing peptide (AIP) signal molecules. In addition to AgrB/D, this pathway includes a novel CPBP transmembrane protein and an ABC transporter. In the present proposal we plan to adopt a multidisciplinary approach combining bacterial physiology and genetics with structural biology, biophysics and medicinal chemistry to: (1) elucidate the molecular basis and specificity of the interaction between AgrB and AgrD, (2) use NMR and crystallography to determine the high resolution structures of AgrB alone and in complex with AgrD, (3) employ medicinal chemistry approaches using ambuic acid as a structural template to develop novel AgrB inhibitors with activity against staphylococci, clostridia, enterococci and listeria, (4) elucidate the physiological function(s), selectivity, structure, contribution to virulence and regulation of the CPBP transmembrane protein, using techniques applied in (1) and (2). We discovered that mutations in a key ABC transporter operon resulted in the loss of extracellular AIP and we will therefore (5) elucidate the contribution of this ABC transporter to AIP export. By using super resolution microscopy, we propose to (6) define the cellular localization and spatial organization of agr system components and the impact of exogenously supplied agonists or antagonists. We have developed new tools for in vivo imaging that will be used to gain new insights into (7) the dynamics of agr expression in vivo and the efficacy of agr pathway inhibitors active against AgrB and AgrC.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Mechanistic analysis of RNA-replication elements involved in early stages of chikungunya virus replication.
Funder: Medical Research Council

Abstract: Chikungunya virus (CHIKV) is a mosquito-transmitted RNA virus that causes incapacitating chronic joint pain in humans. Having re-emerged as an epidemic in 2004 around the Indian Ocean the virus has been imported into many countries, with >1,135,000 cases reported in the Americas and Europe by 2015. There remains no vaccine or specific antiviral therapy, with development hampered by lack of insight into its biology. Given the scale and expanding geographic distribution of these outbreaks, more research on this virus is urgently needed. In preliminary studies we used a combination of structural and reverse genetic analysis to investigate stem-loop RNA replication elements (RREs) conserved in coding and non-coding regions of the CHIKV genome. Results presented in the 'Case For Support' demonstrate that each of the individual RREs functions during the early stages of CHIKV replication, through a host dependent mechanism. This application seeks support to define genetic, structural and trans-activating interactions essential to RRE function during CHIKV replication, through complimentary in vitro and physiological approaches. Roles in translation and genome replication will be defined by reverse genetic analyses of RRE mutants and through comparison of contrasting host-specific vs. mutant phenotypes we will dissect functional mechanisms. We expect these results to have direct application in identification of novel therapeutic targets. Furthermore, mechanistic understanding of RRE-mutant attenuation will have direct application in studies towards a genetically stable attenuated vaccine, which we would aim to exploit through future studies involving both academic and industrial partners.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Control of gene expression in trypanosomes: Defining the nuclear lamina
Funder: Medical Research Council

Abstract: Trypanosomatids evade the host immune response by multiple mechanisms, but in all species the parasite surface is central to virulence. In Leishmania and the American trypanosome, mechanisms are focused on invasion and survival within host cells. For African trypanosomes, where the parasite resides in the host bloodstream, the major mechanism is antigenic variation. All of these parasites rely on specific expression of highly complex gene cohorts encoding surface molecules, and significantly many of these molecules are expressed from genes that are at sub-telemetric loci. Our recent identification of the protein NUP-1, that is an analog to the lamins from mammalian cells, and which has a role in maintaining heterochromatin, provides a major insight into mechanisms of gene regulation. Given that NUP-1 is present across the trypanosomatids, but is restricted to them, it is likely that common mechanisms pertain to this entire group of pathogens. In African trypanosomes, antigenic variation facilitates long term infection, and is predicated upon the monoallelic expression of a single variant surface glycoprotein (VSG) gene, and which involves inactivation within heterochromatin of other VSG genes. In most eukaryotes heterochromatin is maintained by the lamina, a proteinaceous network subtending the nuclear envelope. In mammals the lamina is comprised of 60kDa lamin proteins, but these are absent from trypanosomes and replaced by NUP-1. Proteomics has identified additional components of the trypanosome lamina, including proteins we have designated as NUP-2 and NUP-3, together with connections to the nuclear pore complex. We propose to gain insights into composition, location and interactions between NUP-1 and 2 and other nuclear structures including DNA by a range of state of the art techniques, and specifically super-resolution microscopy, proteomics and next generation sequencing, applied to both transcriptional analysis and chromatin IP.


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