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ESR1 – Understanding how bacterial adhesins recruit host ligands
Faculty of Biological Sciences, University of Leeds (UK)
This project will involve identification of binding surfaces, crystallisation and structure solution of trimeric autotransporters adhesins by state of the art techniques, the use of engineered binding partners (scFvs etc.) to stabilise the complex. The structures will also be used as the basis for the development of novel anti-adhesive strategies, based on the structures solved. Crystals will be studied at national and international synchrotrons. This is a structural biology project that focuses on understanding how trimeric autotransporter adhesins (TAAs) recognise and bind host factors, building on our work on YadA, and E. coli immunoglobulin proteins to produce the first structures of a complex between a TAA and its ligand. We will apply state of the art binding studies and binding partners towards this goal, and cryoEM as necessary (e.g. Leo et al. & Goldman, Structure 19, 1021-1030 (2011); Bhattacharjee, et al. & Goldman, J. Biol. Chem. 288, 18685-18695 (2013); Wright et al. & Goldman, Acta Crystallogr F 73, 101-108 (2017)). The project will involve secondments to other network partners to test binding via NMR at the University of Oslo, and to the Centre for Nanotechnology and smart materials (CeNTI) in Portugal to test antiadhesive surfaces.
ESR2 – Adhesins from Burkholderia pseudomallei
Faculty of Biological Sciences, University of Leeds (UK)
This is a structural/functional biology project focused on understanding how trimeric autotransporter adhesins (TAAs) from Bukholderiafunction in virulence, and to identify effective ligands for diagnostics. The work will involve Identification of specific host targets for novel Bukholderia TAAs including BpaC, structures of BpaC with host ligands using crystallography and combined methods including cryoEm, and development of surface-based diagnostics against BpaC. This builds on our work on YadA, and E. coli immunoglobulin proteins (e.g. Leo et al. & Goldman, Structure 19, 1021-1030 (2011); Wright et al. & Goldman, Acta Crystallogr F 73, 101-108 (2017); Szczesny et al. PLoS Pathog. 4: e1000119 (2008); Kaiser et al. & Kempf Cell Microbiol 15, 759-778 (2012); Schmidgen et al. & Kempf, J. Bacteriol. 196, 2155-2165 (2014)). The project will involve secondments to other network partners to identify Bukholderiahost protein targets at the University of Frankfurt, and at bioMérieux to develop antibody-based diagnostics. The structures will also be used as the basis for the development of novel anti-adhesive strategies. Crystals will be studied at national and international synchrotrons.
ESR3 – Nanobiosensors for pathogen analysis
Faculty of Maths & Physical Sciences, University of Leeds (UK)
This is translational project that focuses on fabricating biosensors and accompanying microfluidic lab-on-a-chip devices in which the biosensors will be deployed. Construction of biosensors, both optical and electrochemical, will utilise host factor proteins or their fragments as bioreceptors to sense specific autotransporter adhesins (TAAs). The subsequent readout of the binding event will be by electrochemical impedance (e.g. Caygill et al., 2012; Biosens. Bioelectr. 32, 104-110) or lanthanide fluorescence from up-conversion nanoparticle (Guller et al., 2015; Nano Research 8, 1546-1562) or lanthanide tagged Affimers (Tiede et al., 2017; eLife 2017; 6:e24903) which are synthetic binding proteins. Design of the bioreceptor proteins will be critically informed by the output from other ESR projects, e.g. ESR1. Biosensors developed in this project will be integrated into microfluidic devices for ease of control over small sample volumes, automation of fluid handling and integrated detection methods. The post holder will receive training in the design and fabrication of microfluidic devices for integration with biosensors to produce bespoke, adhesin-based diagnostic platforms. The project will involve secondments to our network partners Centre for Nanotechnology and smart materials (CeNTI) (Portugal) and the University of Hull (UK).
ESR4 – Binding studies of bacterial adhesin heads with extracellular matrix components
Department of Biosciences, University of Oslo (Norway)
This project combines modern structural biology and biophysical techniques (mainly NMR) with classic molecular biology methods (cloning, mutagenesis). The project is focused on understanding the molecular details of a bacterial adhesin and its capacity to bind to extracellular matrix components such as collagen during the infection process. The project builds on previous work of the group on the trimeric autotransporter adhesin YadA (e.g. Mühlenkamp et al., J Innate Immun. 9, 33-51 (2017); Shahid et al., Nat Methods 9, 1212-1217 (2012)) and on related proteins from Salmonella and other species (e.g. Hartmann et al., PNAS 109, 20907-20912 (2012); Schmidgen et al., J. Bacteriol. 196, 2155-65 (2014)). We will apply state of the art binding studies using protein labeling techniques and solution NMR or solid-state NMR spectroscopy as necessary to identify binding surfaces and interfaces. The constructs used in these studies will also be employed as surface coatings in lab-on-a-chip diagnostic assays. To this end, the project will involve secondments in companies working with diagnostic chip development within the network (ELISHA, Eluceda, both located in the UK). Planned secondments also include visits to the Institut Pasteur (Paris, France) for NMR data evaluation.
ESR5 – Developing new diagnostic tests for detection and identification of pathogens using adhesin-ligand interaction
Background: BioMérieux designs, manufactures and markets systems, technologies and diagnostic tests to allow the detection and identification of pathogenic agents responsible for infectious disease. The current project’s overall aim is to use adhesion-ligand couples or other forms of bimolecular interaction toward the efficient detection of microbial pathogens with proven clinical usefulness. All these will be explored in the ViBrANT network. The PhD students in the network will work on measuring and understanding the binding modes of adhesins or other bimolecular interactions, on the structural biology and biophysics of adhesion, and on aspects of pathogen capture including microfluidics and the development of new diagnostic platforms.
Aim of project: The PhD student is expected to develop a genomic and phenotypic database showing variation in adhesion and ligand structures, e.g. penicillin binding protein and beta-lactam antibiotics. A direct mathematic procedure for the translation of genotypes into phenotypes will be pursued. A defined target will be the development of such a translational system that focusses directly onto the definition of an innovative antibiotic susceptibility testing system where also in vitro studies will be performed. The latter will target difference between adhesion and ligands in living, growth-arrested, dying and dead cells. In the end, the development of routine diagnostic systems is the key target. The research will comprise the development of genomic and phenotypic databases, assessing the effect of antibiotics on ligand binding to microbial cells and to develop routine tests for instance by using the bioMerieux VIDAS or BioFire FilmArray platforms.
ESR6 – Analysis of TAA-dependent bacterial adhesion to matrix and host cells
Institute for Medical Microbiology & Infection Control, Goethe University Frankfurt (Germany)
Adhesion to host cells represents the first step in the infection process and one of the decisive features in the pathogenicity of human pathogenic bacteria. The genus Bartonella harbours a variety of trimeric autotransporter adhesins (TAAs) such as the Bartonella adhesin A (BadA) of B. henselae. TAAs mediate many of the biological properties of Bartonella spp., e.g. adherence to endothelial cells and extracellular matrix proteins. The exact molecular binding functions of BadA need to be analysed in detail to understand whether and how ‘antiligands’ as a new class of antibiotics might be produced. In this project we will investigate how bacterial pathogens bind to their respective binding partners (matrix proteins, host cells). This will be done using the human pathogen B. henselae and BadA-mutants. The binding of BadA-expressing bacteria to matrix proteins (e.g. fibronectin) and host cells will be analysed. Peptides will be modelled and produced which shall inhibit bacterial adhesion to matrix and host cell surfaces. Read out will be done using various in vitro and ex vivo infection models. For mutant construction and peptide synthesis, a secondment with our partner in Leeds (UK) is planned. To analyse bacterial binding mechanisms (NMR), a secondment with our partner in Oslo (Norway) is planned. Moreover, a secondment with Biomode (Portugal) is scheduled to analyse bacterial adherence. For details please see: O’Rourke et al. & Kempf, Cell. Microbiol. 17, 1447-63 (2015); Schmidgen et al. & Kempf, J. Bacteriol. 196, 2155-65 (2014); Kaiser et al. & Kempf, Cell. Microbiol. 14, 198-209 (2012); O’Rourke et al. & Kempf, Adv. Exp. Med. Biol. 715, 51-70 (2012); Müller et al. & Kempf, Infect. Immun. 79, 2544-2553 (2011); Kaiser et al. & Kempf, Cell. Microbiol 10, 2223-34 (2008); and Riess et al. & Kempf, J. Exp. Med. 200, 1267-1278 (2004).
ESR7 – TAAs as diagnostic targets
Institute for Medical Microbiology & Infection Control, Goethe University Frankfurt (Germany)
Adhesion to host cells represents the first step in the infection process and one of the decisive features in the pathogenicity of human pathogenic bacteria. The genus Bartonella harbours a variety of trimeric autotransporter adhesins (TAAs) such as the Bartonella adhesin A (BadA) of B. henselae. TAAs mediate many of the biologically properties of Bartonella spp., e.g. adherence to endothelial cells and extracellular matrix proteins. The exact molecular binding functions of BadA need to be analysed in detail to understand whether and how ‘anti-ligands’ as a new class of antibiotics might be produced. In this project we will investigate whether bacterial TAAs (here BadA) are a valuable antigen for serodiagnosis of infections, and whether BadA-specific antibodies will inhibit bacterial adherence (matrix proteins, host cells). This will be done using the human pathogen B. henselae and a quality-certified human serum library. The binding of BadA-expressing bacteria will be inhibited using poly- and monoclonal antibodies which are thought to prevent infections. Read out will be done using various in vitro and ex vivo infection models. For BadA-peptide constructions, a secondment with our partner in Oslo (Norway) is planned. For identifying antibody-specific target sequences, a secondment with our partner in Lund (Sweden) is planned. Moreover, a secondment with bioMeriéux (Lyon, France) is scheduled to analyse inhibition of bacterial adherence. For details please see: Schmidgen et al. & Kempf, J. Bacteriol. 196, 2155-65 (2014); Kaiser et al. & Kempf, Cell. Microbiol. 14, 198-209 (2012); O’Rourke et al. & Kempf, Adv. Exp. Med. Biol. 715, 51-70 (2012); Eberhardt et al. & Kempf, Proteomics 30, 1967-1981 (2009); Wagner et al. & Kempf, Int. J. Med. Microbiol. 298, 579-90 (2008); Kaiser et al. & Kempf, Cell. Microbiol 10, 2223-34 (2008) and Riess et al. & Kempf, J. Exp. Med. 200, 1267-1278 (2004).
ESR8 – Development of Innovative Anti-Adhesive Materials in Diagnosis Tools
Centre for Nanotechnology and Smart Materials (CeNTI) and University of Minho (Portugal)
Pathogen infection represents a great problem affecting millions of people. Currently, there are still a number of challenges that limit the efficacy and specificity of diagnostic tools for pathogen detection. For instance, fouling and bacterial adhesion to diagnosis devices including microfluidic lab-on-chip devices. Hence, new diagnostic tools are eagerly sought after. The development of anti-adhesive materials with minimal fouling to inhibit adhesion based on ‘grafting-from’ approach comprises an interesting and promising solution. The aim of this project is to develop innovative anti-adhesive materials to be used within diagnostics. The post holder will study, engineer and develop new anti-adhesive surfaces by incorporating a variety of agents that can prevent pathogen aggregation and sticking-to-walls in microfluidic lab-on-a-chip devices. These may include essential oils, surface active molecules, bio-surfactants and specific anti-adhesins such as those studied within the ViBrANT network. Incorporation of these agents into polymer-based materials that will be machined into diagnostic tools is expected to improve the tools’ function and enhance the device response/readout, and so be more effective at diagnosing pathogens. The project will involve secondments to other network partners to select the most appropriate anti-adhesive agents and to test anti-adhesive materials with respect to pathogen aggregation at University of Minho (Portugal); and to conduct studies related to pathogens in clinical materials at Goethe University Frankfurt (Germany). This project will involve the selection of the most promising anti-adhesive agents; the establishment of methods to modify several materials (polymer-based); the incorporation of anti-adhesive agents on those materials and their physicochemical characterization; and finally the development of anti-adhesive materials able to prevent pathogens aggregation and sticking-to-walls.
ESR9 – Design of Multifunctional Materials to Capture Pathogens from Biological Samples
Department of Biological Engineering, University of Minho (Portugal)
Pathogen infection is a current concern in both developed and developing world with a great health, social and economic impact. Despite all the developments to diagnose and treat those infections, these still involve complex and lengthy procedures. Therefore, the design of novel solutions that can capture/enrich pathogens from clinical samples is of utmost relevance for clinical diagnostics. Indeed, detecting the interaction between entire bacteria and mixtures of ligands provides an unexplored route for target enrichment. Specific ligands immobilized onto the surface of materials used for fabrication of diagnostic devices such as arrays, microbeads, membranes and electrodes represents a promising approach. The aim of this project is to develop multifunctional materials to capture pathogens from biological samples combining grafting with plasma/UV treatments. Specific ligands (peptides/proteins, biosurfactants and aptamers) that bind to the extracellular matrix (ECM), membrane components or capsids of given pathogen(s) will be immobilized onto the surface of materials used for fabrication of diagnostic devices. These multifunctional materials must provide high and well-controlled binding capacities for ligands, intact cells and cellular extracts; prevent denaturation of the immobilized ligands, and be based on simple and efficient techniques for immobilization. The project will involve secondments to other network partners to explore proteins coupled to materials for enrichment purposes at Eluceda (UK), to use genomic context analysis and other bioinformatics tools to evaluate new targeting molecules at University of Oslo (Norway), and to evaluate the applicability of the multifunctional materials in diagnosis tools at University of Hull (UK).
ESR10 – Understanding and exploiting host-pathogen interactions in tick-borne encephalitis virus
Faculty of Biological and Environmental Sciences, University of Helsinki (Finland)
This is a structural biology project that focuses on understanding how flavivirus envelope proteins recognise and bind host factors. We will apply state of the art cryo-electron microscopy and three-dimensional image reconstruction (3DEM) to the complexes as well as X-ray crystallography (e.g. Shakeel et al. 2017 (http://rdcu.be/pybT); Shakeel et al. 2016 http://dx.doi.org/10.1038/ncomms11387). The project will involve secondments to UK network partners for data collection (University of Leeds) and test laminin binding for preanalytical sample preparation for viruses at Elisha Systems Ltd.
ESR11 – Structural Analysis of Adenovirus Binding to Integrins
Faculty of Science and Faculty of Medicine, University of Tübingen (Germany)
Viruses use highly specific recognition processes to engage the target cell. The initial interaction between a viral capsid protein and the cell determines cell and host tropism as well as pathogenicity of a virus, and it often triggers and enables subsequent interactions that then allow the virus to enter the cell. Adenoviruses are established human pathogens that can cause a variety of serious diseases and are also of interest for gene delivery purposes as they are able to easily package and deliver foreign genomes to a target cell. While the structural basis of adenovirus binding to its protein receptors CAR and CD46 has been established, much less is known about the interactions of some of these virus strains with glycan receptors. In addition, all adenoviruses use integrin receptors for entry, and the structural basis of this interaction is entirely unknown. It is clear, however, that understanding the rules that allow adenoviruses to engage these attachment and entry receptors will greatly facilitate the use of these particles for targeting specific cell types, such as certain tumor cells that for example express high levels of specific glycan receptors. The predominant aim of this PhD project is to investigate structural aspects of the interactions of adenoviruses with both glycan and integrin receptors. To achieve this, the relevant recombinantly expressed virus proteins (fiber, penton) will be complexed with their respective receptors and the structures will be solved by X-ray crystallography in combination with additional biophysical approaches that can serve to support the observed interactions. The interactions will also be probed with site-directed mutagenesis to validate the observed contacts.
ESR12 – Targeted cross-linking mass spectrometry and host-pathogen interactions
Faculty of Science and Faculty of Medicine, University of Lund (Sweden)
Bacterial pathogens can form complex protein interaction networks with human host proteins to facilitate bacterial colonization and survival and to avoid immune detection and bacterial clearance. The ‘universe’ of these protein interactions is however largely unknown. The recent development of quantitative and structural mass spectrometry (MS) techniques has provided new possibilities to measure protein-protein interactions in great detail. One of these techniques relies on chemical cross-linking (XL) between adjacent proteins to determine binding interfaces and mode of binding. In recent work, the Malmström group has developed new XL-MS techniques referred to as targeted cross-linking MS (TX-MS) that enables structural investigations of complete protein interaction networks. The predominant aim of this PhD proposal is to investigate structural aspects of protein interaction networks formed between the human pathogen Streptococcus pyogenes and human host proteins. S. pyogenes is a gram-positive bacterium and one of the most important human pathogens associated with considerable morbidity and mortality. The project will involve identification of protein-protein binding interfaces between human host proteins and bacterial surface proteins using state-of-the art quantitative MS and TX-MS techniques. The identified protein binding interfaces will be used to discriminate between highly protective and potentially detrimental antibody responses to further advance the understanding of the development of a severe and invasive disease. The post holder will be trained in a highly interdisciplinary manner, gaining experience in mass-spectrometry, proteomics and bioinformatics.
ESR13 – An integrative strategy for structure determination of bacterial type IV pili
Institut Pasteur, University of Paris (France)
To interact with their environment, bacteria have acquired a number of proteinaceous appendages that decorate their surface. These appendages, such as pili, secretion systems or flagella, are large macromolecular assemblies that participate in adhesion, cell-cell interactions, auto-aggregation, DNA exchange, motility and virulence factor transport. Type IV pili (T4P) are a class of fimbrial adhesins present in numerous pathogenic bacteria and are crucial for host colonization and virulence of many gram-negative bacteria. T4P assembly system is very closely related to type II protein secretion systems (T2S) that are involved in the transport of folded proteins from the periplasm across the outer membrane. The common architecture of T4P and T2S is based on the assembly of helical fibers in the plasma membrane with membrane protein subunits called pilins. Other proteins called minor pilins are crucial for the initiation of this assembly. However, their molecular complexity, their dynamics, and membrane localization seriously hamper structural characterization of these fibers at atomic resolution with traditional methods (X-ray crystallography, NMR, and EM). It has become clear that a combination of structural information from many sources is the key to success. In this project, we will implement an integrative strategy to determine the structure of a T4P, improving upon our work on T2S (see Campos et al., PNAS 107, 13081-13086 (2010); Campos et al., J Struct Biol. 173, 436-444 (2011); Campos et al., Structure 22, 685-696 (2014). First, the high-resolution structure of the soluble domain of the major pilin will be determined by NMR. In a second stage, helical parameters of pili will be obtained by EM and this data will be then combined with the pilin structure and co-evolutionary data to construct a model of the assembled pilus using a flexible molecular modeling approach. Finally, to better understand the fiber assembly mechanism, molecular interactions between major and minor pilins will be characterized by NMR and other biophysical techniques (Microcalorimetry, SPR and fluorescence). The project involves secondments with other network partners for labeled protein production (Tubingen University), training in cryo-EM data analysis (University of Helsinki) and at the Centre for Nanotechnology and smart materials (CeNTI) in Portugal to design antiadhesive surfaces.
ESR14 – Lab on a Chip for Pathogen Analysis
Background: Pathogen infection is a big burden in the developed as well as developing world affecting millions each year with symptoms ranging from mild discomfort to a threat to life. A large challenge in managing pathogen treatment is that fact that pathogen analysis is hampered by lengthy procedures, often involving selective culturing of samples that may take several days. One promising technique for faster pathogen analysis is fluorescence in-situ hybridization (FISH), which allows the identification of microbial cells by fluorescence microscopy directly in a sample of interest in less than 3 h. However, due to low numbers of cells present in the clinical samples, pre-enrichment steps are required before the FISH procedure can be carried out, which results in full analysis taking one day.
Aim of project: In this project we will investigate microfluidic-based methods for fast pathogen cell pre-enrichment in blood samples and subsequent trapping in defined locations for FISH analysis. Pathogen cell enrichment procedures will include inertial microfluidics, as well as magnetic or acoustic trapping forces. For analysis, we will employ biochemistry developed by one of the ITN partners (Biomode), based on peptide nucleic acid (PNA) or locked nucleic acid (LNA) which are more robust and specific compared to conventional DNA probes. Furthermore, in collaboration with our parnters in Frankfurt, we will develop miniaturised platforms with immobilised adhesins to study binding and interactions to host-receptors. The post holder will be trained in a highly interdisciplinary manner, gaining experience of microfluidic device design and fabrication, physical principles of cell separation and bioanalysis of pathogens.
ESR15 – Translating adhesin-ligand interaction towards existing diagnostic platforms
BioMérieux designs, manufactures and markets systems, technologies and diagnostic tests to allow the detection and identification of pathogenic agents responsible for infectious disease. The aim of the project is to use biochemical, biophysical and molecular methods, next generation sequencing and MALDI TOF mass spectrometry as read out systems for the detection of microbial adhesion. Using synthetic or immobilized ligands and receptors, innovative diagnostic detection assays will be developed. Binding inhibition studies using antibodies will be used to prove the specificity of ligand receptor interaction and to quantify the stability of binding. The effect of ligand-receptor interactions on the metabolomics state of microbial cells will be explored. In addition, the interactions that will be defined as the more stable ones will be used for enrichment of microbes directly from (mock) clinical specimens. The aim of this project is to improve upon the current diagnosis and allow better prediction of patient outcome. This will permit physicians to adapt and/or select the appropriate treatment. The project will use modern analytical-immunological technologies and new “omics” technologies will be integrated as possible read-out systems as well.