NIHRDataInsights/HRCSData huggingface dataset:
Title: Characterisation of disulfide bond formation in bacterial pathogens: Unravelling the adaptation of a classical pathway into a virulence aid.
Funder: Medical Research Council

Abstract: In Gram-negative bacteria the Disulfide bond (DSB) system is the master regulator of disulfide bond formation, a process which is required for extracytoplasmic proteins to fold and operate. It also has a prominent role in the pathogenesis of Gram-negative bacteria as it is involved in the assembly of numerous virulence factors and it is essential for processes like adhesion, toxin/enzyme secretion, capsule biogenesis etc. I aim to explore the role of this protein pathway in mechanisms of bacterial pathogenesis, with the long-term goal of contributing to the development of novel antibacterial strategies. My proposed research will combine a comprehensive bioinformatic approach with experiments on the DSB systems of two human Gram-negative bacterial pathogens, Neisseria meningitidis and Pseudomonas aeruginosa. The DSB systems of pathogenic bacteria often diverge from the classical paradigm by containing more than one analogue of key DSB proteins. I will use bioinformatics to identify the full set of analogues of each DSB protein across all Gram-negative bacteria. This will allow me to pinpoint underlying common traits between the DSB systems of pathogens and to understand the evolution of this pathway into a virulence aid. I will work on N. meningitidis, which encodes three analogues of the oxidase DsbA, in order to elucidate the contribution of these proteins to the assembly of virulence factors. I will identify the full set of DsbA substrates via, amongst other in vivo techniques, differential proteomics. Finally I will use P. aeruginosa, a pathogen that encodes two analogues of the reductase DsbD, to understand the connection between this duplication and the protection of the bacterium against oxidative stress. I will also crystallise and determine the structure of the unique transmembrane reductant conductor DsbD. These studies could offer new ways of developing vaccines against a multi-drug resistant pathogen like P. aeruginosa.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFECTION
Title: Persistent Salmonella infection: characterization of non-replicating bacteria and Toxin-Antitoxin module function
Funder: Medical Research Council

Abstract: Many bacterial pathogens cause persistent infection despite exposure to multiple courses of antibiotics, which are thought to contribute to the emergence of stable antibiotic resistance. Persisters are multidrug-tolerant bacteria that could account for the relapse of infections. Serovars of Salmonella enterica are important intracellular bacterial pathogens that cause a variety of human diseases, including gastroenteritis and typhoid fever. We developed a method allowing tracking of single cells and discovered that upon macrophage phagocytosis, a significant proportion of the Salmonella population forms non-replicating persisters. Then, I provided the first direct evidence that non-replicating persisters are also formed in infected mice where they survive extended periods of antibiotic treatment. I also showed that a family of genes, named class II Toxin/Antitoxin (TA) modules, is involved in the formation of Salmonella persisters. Class II TA operons encode a non-secreted toxin, which is an inhibitor of an essential cellular function and an antitoxin that neutralizes the toxin. The antitoxin is degraded under various stress conditions leading to growth arrest of the bacterial cell. How growth-arrested bacteria can establish a long-term viable but non-replicative state in host cells is unknown. The overall aim of the project is to characterize Salmonella persisters through 3 specific objectives. First, by undertaking an in-depth characterization of Toxin-Antitoxin modules I intend to understand how these proteins function collectively to govern entry into the persister state. Second, I will study the role of TA modules in the context of natural infection of mice. Third, I will investigate the basis for maintenance of a non-replicating viable state within host cells. The knowledge gained has great potential to offer opportunities for targeting persistent bacterial infections to improve antibiotic efficacy and counteract the generation of antibiotic resistance.


Research activity codes: 1.1 NORMAL BIOLOGICAL DEVELOPMENT AND FUNCTIONING
Health categories: INFECTION
Title: Recognition by the type II secretion system and how it enables Legionella pneumophila to thrive
Funder: Medical Research Council

Abstract: Legionella pneumophila is an opportunistic Gram-negative bacterium, ubiquitous in natural and anthropogenic freshwater environments. It is the causative agent of Legionnaires' disease, an often-fatal pneumonia, and Pontiac fever, a milder flu-like disease. Man-made infrastructures that store and distribute water are omnipresent in Western society and these bacteria can be found in many large buildings, including hospitals and hotels. The high prevalence of Legionella within the environment is due to its ability to survive within biofilms or as an intracellular parasite of biofilm associated protozoa. L. pneumophila uses a type II secretion system (T2SS) to translocate substrates across its outer membrane, where they support biofilm formation, replication in the host, dampening of cytokine output and survival in mammalian lungs. A number of these have novel amino acid sequences and have been implicated in promoting broad host tropism and persistence of disease. As these are exclusive to Legionella I have called them Unique Legionella Proteins (ULPs). The T2SS is also used by other human pathogens (e.g. Chlamydia trachomatis, Vibrio cholerae, Escherichia coli, Pseudomonas aeruginosa, Klebsiella spp., Yersinia enterocolitica). Translocation involves a number of recognition events with substrates identified via a 'conformational' motif; but how can a single transport apparatus identify multiple unique substrate structures? I plan to (i) use these ULPs to investigate the molecular details of their recognition in the periplasm prior to export; and (ii) ascertain which ULPs are involved in disease or ecology. Whilst these studies will provide insight into new mechanisms related to Legionella fitness, the major outcome of this research will be to advance of our limited knowledge of T2SS/substrate recognition. In turn this may provide the bedrock for targeted development of sustainable prevention and control strategies for a range of infectious diseases.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Characterisation of the Ess protein secretion system of Staphylococcus aureus, a key virulence factor.
Funder: Medical Research Council

Abstract: The Type VII/Ess protein secretion system is a general protein export pathway found in many Gram positive bacteria including the serious pathogens Mycobacterium tuberculosis and Staphylococcus aureus. Previous studies have shown that this secretion pathway is essential for the virulence of both of these organisms. Indeed the loss of one of these systems from a strain of M. bovis is the basis for the attenuated BCG vaccine. Although the Type VII/Ess export pathway shares a number of common components between bacterial species there are clear differences in the composition of the machinery between Actinobacteria (including M. tuberculosis) and firmicutes (S. aureus). We have been working on the characterisation of the Ess secretion system from S. aureus. We have shown that EssC is essential for Ess secretion system function in S. aureus. We have obtained diffracting crystals of the large C-terminal domain of EssC and have shown that it binds ATP. Proteomic analysis of our essC mutant strain has led to the identification of several novel candidate substrates of the Ess machinery. Finally we have shown that The Ess secretion system of S. aureus RN3690 is essential for virulence in the mouse pneumonia model of infection. We now seek to build on our preliminary data to obtain high-resolution crystal structures for EssC and for one of the secreted substrate proteins. In addition we will probe the organisation of the membrane-bound secretion complex in S. aureus using cross-linking and native level protein purification coupled with yeast two-hybrid studies and co-purification approaches. Finally we will determine whether substrate proteins interact with EssC during export by the machinery.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Mechanism of EGFR activation by a novel fungal pore-forming toxin
Funder: Medical Research Council

Abstract: The ability of mucosal tissues to discriminate between commensal and pathogenic microbes is essential to human health. Recently, we have identified several epithelial signalling mechanisms instrumental in identifying the commensal-pathogen switch of Candida albicans, a major life-threatening fungal pathogen of humans. These activation mechanisms target the hyphal form of the fungus, resulting in the induction of epithelial immunity and protection against damage. We have now identified a C. albicans hyphal protein (Ece1p) and an internal 32 aa sequence (Ece1-III) as a novel pore-forming toxin (PFT), which induces cellular damage and activates epithelial immunity. Furthermore, we have identified EGFR as the target receptor for this newly discovered fungal PFT and provide evidence that this PFT-EGFR interaction event appears critical in the epithelial recognition of the commensal-pathogen switch of C. albicans. We demonstrate that C. albicans Ece1-III can activate EGFR directly and indirectly. Direct activation is independent of pore formation and occurs via direct binding of Ece1-III to EGFR. Indirect EGFR activation is dependent on pore formation but is the result of a complex series of surface-mediated signalling events. Given that EGFR is a key receptor mediating both epithelial immunity and damage protection during C. albicans infection, we aim to identify precisely how this novel fungal PFT indirectly activates EGFR. Murine experiments will also be performed as a basis for future studies to demonstrate that the Ece1-III-mediated EGFR activation mechanism that we have demonstrated in vitro has a bearing on fungal infection and mucosal protection in vivo. This project will provide substantial new insights to our understanding of fungal PFTs, epithelial-microbial interactions, and will also identify new microbial and epithelial targets for the development of new drugs, immune-based treatments, vaccines and adjuvants to prevent mucosal diseases.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Characterising the Mode of Action of VP22 - a Novel Herpes Simplex Virus Virulence Factor Important for In Vivo Replication.
Funder: Medical Research Council

Abstract: Herpes simplex virus (HSV) is an often-disregarded human infection that nonetheless causes considerable disease, infecting 24 million annually, and resulting in significant morbidity and mortality. No vaccine has yet been developed for HSV, and although HSV antivirals have existed for decades, their use has made no impact on HSV infection rates. This proposal concerns a major structural protein of HSV named VP22, and its role in pathogenesis. Work in animal models has shown that HSV replication in the host is severely attenuated in the absence of VP22, suggesting that this protein is important for disease. Paradoxically, VP22 is not required for HSV replication in tissue culture, and despite much work, its role in virus infection has remained elusive. However, we have recently found that VP22-negative HSV is restricted for growth in primary human fibroblasts, and this cell type is therefore an excellent model to uncover the activity of VP22. Unlike most immortalised cells, primary fibroblasts are fully competent for antiviral responses, leading us to hypothesize that VP22 is required to counteract an antiviral response. We propose to use transcriptomic analysis to determine how human fibroblasts respond to HSV infection in the absence of VP22. We will also use proteomic analysis of VP22-containing complexes from infected fibroblasts to identify candidate factors through which VP22 may act to overcome cellular restriction. Potential partners of VP22 will be confirmed by in vitro interaction studies, and their role in virus restriction determined by RNAi depletion studies. Finally, we will assess the ability of varicella zoster virus and bovine herpesvirus 1 homologues of VP22 to rescue the restriction of VP22-negative HSV, and test the species specificity of VP22. By determining the mode of action of VP22 and enhancing our understanding of the HSV disease process, this work will contribute to future rational approaches to vaccine and antiviral design for HSV.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: The Apical Complex: a Targeted Investigation of the Molecular Functions of this Structure Essential to Apicomplexan Parasite Invasion and Replication.
Funder: Medical Research Council

Abstract: This project will use the genetically amenable Toxoplasma gondii as a model to study the architecture and function of the apical complex. T. gondii can be readily genetically modified by targeted recombination, allowing gene-tagging with reporters, inducible protein knockdowns by promoter swaps, and gene knockouts. Detailed phenotypic investigations of cell morphology, ultrastructure, growth, invasion and replication will be undertaken to characterise the behaviour and functions of individual apical complex structures. These procedures are all established in the Waller laboratory. Identification of novel apical complex proteins will use three independent approaches. 1) Comparative bioinformatics using known peripheral cytoskeletal proteins from related ciliates. 2) Proximity-dependant biotinylation, using either promiscuous biotin ligase (BirA*) or modified peroxidases (APEX) fused to known apical complex proteins, to tag and identify further new components of this structure. 3) Immuno pulldowns of interaction partners of known apical complex proteins. Protein locations will be validated, and a detailed structural and behavioural model of the apical complex will be made using reporter-tagged proteins and live cell imaging, immuno-fluorescence (including with 3D-SIM super-resolution microscopy) and electron microscopy. Functional dissection of apical complex proteins will be undertaken by inducible knockdown (tetracycline-responsive promoter replacement and controllable degradation domains), and gene knockouts using modified versions of the Cre/Lox recombinase system, and CRISPR gene excision. The effects of selective protein depletion will be measured and characterised by high-resolution microscopy and sensitive assays for parasite growth, replication and invasion processes (e.g. invasion efficiency, cell motility, regulated microneme and rhoptry protein secretion). These approaches will allow thorough interrogation of apical complex assembly and function.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Probing the translational dynamics of influenza virus infection.
Funder: Medical Research Council

Abstract: In order to produce progeny virions, the major human and animal pathogen influenza A virus (IAV) uses the host cell translational machinery to synthesize viral proteins. Simultaneously, the virus attempts to inhibit cellular macromolecular synthesis; both to free up the translation machinery and to block the synthesis of antiviral response proteins. IAV has evolved several seemingly redundant shutoff mechanisms, highlighting the importance of this aspect of virus infection. However, whilst this reinforces this subject as an area for potential therapeutic intervention, the key molecular events from either virus or host perspective are obscure. Our objectives are to: 1. Utilise the recently developed technique of ribosomal profiling in conjunction with RNAseq to define the cellular and viral 'translatome' in permissive (293T and A549 cells) and non-permissive (monocyte-derived macrophages) cell types and with human-adapted (A/PR/8/34, A/Udorn/72) and avian (A/mallard/NL/10/99) strains of virus. 2. To define the key viral mechanisms and cellular targets that decide the outcome of the battle for control of the cellular translation machinery, by comparing the translatomes of wild type and mutant strains of IAV disabled for specific shut-off functions; PA-X mutants, PB2/PA mutants defective in RNA Pol II degradation and various NS1 mutants defective for CPSF, PAF1, eIF4GI binding. 3. Test the hypothesis that IAV specifically manipulates the biogenesis and/or composition of ribosomes to bias them towards viral translation, using an unbiased affinity purification method followed by mass spectrometry as well as testing specific hypotheses involving viral interactions with the NOH61 helicase and the PELP1, LAS1L, TEX10 and WDR18 complex. The project will define the key events from both virus and cellular perspective that define the output of progeny virions from an individual infected cell; data that will aid of new therapeutic intervention strategies.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Use of EGF-R antagonists for the treatment of infections and tumour growth
Funder: Medical Research Council

Abstract: We recently have found that activated regulatory T-cells (Tregs) require the expression of the EGF-R for their optimal functioning. So lack for example Tregs that fail to express the EGF-R the capacity to prevent the development of colitis in a T-cell transfer-based mouse model; and can treatment with EGF-R antagonists induce the clearance of a tumour by tumour-specific immune responses. In this project, we would like to determine how Tregs limit the success of tumour immuno-therapy and how they contribute to persistence of chronic infections. Our hypothesis is that EGF-R antagonists diminish Treg function and thereby substantially enhance the efficiency of both tumour- and pathogen-specific immune responses. We will test this proposition in the context of i) genetic defects in EGF-R expression in the regulatory T cell lineage, ii) lack of Tregs and iii) in context of treatment with EGF-R antagonists. Based on our findings, we will develop a new type of EGF-R inhibitor that specifically targets Tregs. In this way, we establish an EGF-R inhibitor that most optimally modulates immune responses, while keeping the function of the EGF-R for other physiological functions, such as tissue homeostasis, intact. We expect that the outcome of these studies could contribute to a wider application of EGF-R inhibitors, in particular in combination with tumour immunotherapies, to enhance clinical response rates and could create novel possibilities to treat diseases, such as chronic viral infections, for which yet no treatments exist at all.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: INFECTION
Title: The cellular mechanisms underpinning the host restriction of Salmonella Typhi
Funder: Medical Research Council

Abstract: The human-restricted bacterial pathogen Salmonella Typhi causes a severe systemic disease of global importance called typhoid fever. S Typhi initiates infections by directing intracellular replication within Salmonella-containing vacuoles (SCVs). S.Typhi is incapable of establishing infection in animal cells where the pathogen is killed. I want to understand the cellular mechanisms behind S.Typhi's destruction in animal cells and the pathogen's human host restriction. The small GTPase Rab32 localises to SCVs where it drives S.Typhi degradation in mouse but not human macrophages. This shows Rab32 determines host-specificity and reveals a vital difference in Rab32 pathways. Rabs recruit cognate mammalian effector proteins to direct membrane transport but how mouse Rab32 destroys S.Typhi, and through which effectors, is unknown, as is the reason why human Rab32 is ineffective. Understanding Rab32 would unveil a new pathogen killing mechanism and provide a molecular key for combating S.Typhi in human cells. Fundamental to Rab function is their cooperation with the membrane to which they are anchored but studying this presents formidable challenges. I aim to build a new approach that focuses on the key relationship between Rabs and their membrane to unveil disease mechanisms. By immobilising phospholipid bilayers on silica microspheres I will engineer membrane platforms anchored with host-specific Rab32 that mimic SCVs. Rab32 platforms will enable me to reconstitute the operation of Rab32 in mouse and human cell extracts. I will capture and identify the pivotal Rab32-machinery, characterise its regulation and cooperation with lipids, and reconstitute processes underlying S.Typhi degradation. In parallel, Rab32 effectors and pathway components will be identified via SCV proteomics and infection-based screens. Cell biology studies will dissect the Rab32 network, and resolve cellular and virulence mechanisms that are decisive in the host restriction of S.Typhi.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT
Health categories: INFECTION
Title: Peri-domestic behaviour of African malaria vectors and the impact of insecticides
Funder: Medical Research Council

Abstract: Limited knowledge of malaria vector behaviour in the peri-domestic environment is a major obstacle to the development of better tools for vector control. We have developed robust mosquito tracking technology that overcomes many of those obstacles. The project will deploy the purpose-built and unique system in an experimental hut system at a rural setting in Tanzania, to record and analyse the behaviour of wild vector populations. The first aim is to track and fully characterize endophilic and endophagic behaviour of Anopheles gambiae and other mosquitoes. In a specially constructed experimental hut, activity of mosquitoes responding to human volunteers will be recorded and analysed. Using human volunteers as 'bait', we will capture and describe the complete sequence of mosquito behaviour from arrival to exit, including the key events involved in orientation to, and bloodfeeding at humans. Data will be produced on entry and exit (location, timing), flight (flight type, 3D spatial location, velocity, tortuosity, saccades and other parameters) and resting (timing, location, duration) activities in periods of <3 days. We will use human volunteers of both sexes and a range of ethnicities and ages. We anticipate numerous valuable insights and the primary deliverable is detailed characterisation of Anopheles gambiae behaviour in the human home. The second aim is to investigate behaviour in response to insecticide treatments. Here we will examine how insecticide-based interventions (long-lasting insecticide-treated bednets and durable wall linings) affect behaviour, to explore modes of action of existing tools. Fundamental to our study is the eventual translation of basic behaviour findings to design new tools; hence we will test mosquito response to a new, patent pending, LLIN design. The findings will deliver important evidence for understanding mechanisms of existing vector control tools, whilst potentially identifying targets for new tools.


Research activity codes: 2.2 FACTORS RELATING TO PHYSICAL ENVIRONMENT;3.2 INTERVENTIONS TO ALTER PHYSICAL AND BIOLOGICAL ENVIRONMENTAL RISKS
Health categories: INFECTION