Over the millennium, M. tuberculosis complex strains have branched into several lineages and genotypic variations can determine the virulence and transmissibility of clinical M. tuberculosis (Mtb). However, the mechanism behind the variability in transmission of Mtb remains elusive. Therefore, we investigated understanding the pathogenesis and vaccine efficacy between the high, moderate and low transmission Mtb in mice. The study used three Mtb strains based on their transmission within the Kenyan population – high, moderate and low. The project focused on the characterisation of Mtb strains isolated from Kenyan individuals, BCG vaccine efficacy and pathological analysis in mice against Mtb strains.
The causative agent of tuberculosis (TB), Mycobacterium tuberculosis (Mtb), is an intracellular pathogen infecting nearly 10 million people world-wide. Its cousin, Mycobacterium abscessus (MABC), is a major cause of death in immunocompromised individuals. However, despite the availability of multi-drug chemotherapy, the majority of deaths in both are due to drug-sensitive strains. This is often due to the presence of a phenotypically-resistant sub-population of bacteria termed antibiotic persisters in TB, which increase upon intracellular exposure, or antibiotic-tolerant biofilms within the lung airways for MABC. Determining antibiotic penetration and targeting persisters and biofilms can be challenging in highly heterogenous mycobacteria. We aim to use single cell techniques, such as microfluidics, nano-scale secondary ion mass spectrometry and atomic force microscopy, to evaluate antibiotic penetration and survival and to identify novel strategies to curtail these global health threats.
No abstract but I have a poster focused on the BEAT diabetes programme – a consortium between industry, the NHS and the university of surrey to roll-out and evaluate a digital, online, supported self-management programme for people living with type 2 diabetes. We have recruited >600 people to take part in the programme and matched with a control group to evaluate the impact of the programme on glycosylated haemoglobin (HbA1c) and our secondary outcomes, including weight, blood pressure and cholesterol. We have also conducted a qualitative process evaluation to sit alongside this implementation study.
The ability of genetically identical cells to display different phenotypes is a significant obstacle for the treatment of many human diseases. This is especially true for tuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis. Heterogeneity plays a key role in TB’s continued presence as a global health threat, providing a huge reservoir of latent disease in the world and preventing cure of active disease. Genetic diversity of either host or pathogen does not, alone, explain all this heterogeneity. We hypothesize that non-genetic sources of heterogeneity in pathogen populations lead to variation in outcome. Testing this hypothesis with conventional methods is difficult as it requires studying pathogen cells as individuals, rather than populations, at timescales that can capture the dynamics of heterogeneity. In addition, developing therapeutics to target heterogeneity demands a deep understanding of the molecular mechanisms underlying this phenomenon. My lab uses a combination of fluorescent reporters, time-lapse microscopy, and bacterial genetics to understand the mechanisms and consequences of phenotypic heterogeneity in mycobacterial populations.
This presentation explores how the University of Surrey’s first ever artist-in-residence Anna Dumitriu engages with cutting edge research methods in the health sciences to engage and inspire diverse audiences in new technologies that have the potential to affect all our lives.
Dumitriu works hands-on in the lab and the art studio to create sculptural or installation-based works that incorporate diverse materials such as altered historical objects, textiles, bacteria and DNA. Her high impact artworks draw threads across time, from the history of science and medicine to cutting edge fields such as synthetic biology and bacterial genomics and have been shown around the world in prestigious museums and galleries and featured across all forms of media and in numerous publications.
Since 2012 she has been developing artworks that explore tuberculosis, such as her ‘Romantic Disease’ and ‘Susceptible’ projects which have taken audiences on a journey from past superstitions about the disease to the application of cutting edge genomics techniques to combat issues of antibiotic resistance. In the past year she has been working with researchers at the University of Surrey to explore new research and techniques from looking for ancient bovine TB DNA in Iron Age bones, and the development of vaccines, to carbon capturing microbes, and quantum biology.
Her multi-layered artworks enable audiences to explore scientific ideas, as well as the ethical, cultural and societal impacts of new technologies through sensory and aesthetic approaches, inviting viewers to notice important things that have previously gone unnoticed and to think about them in different ways. She peels away layers of complexity and providing glimpses of ‘weak signals’ from the future, and always reflecting on the past.
We are developing open multidimensional fluorescence microscopy instrumentation, including endomicroscopy, high content analysis (HCA), super-resolved microscopy, and optical projection tomography (OPT). We have particularly focused on fluorescence lifetime imaging (FLIM) and Forster resonant energy transfer (FRET) to study molecular interactions and more recently on super-resolved microscopy using single molecule localisation microscopy (SMLM) to probe ultrastructure and molecular clustering. To provide a complementary label-free readout, we are developing semi-quantitative (single-shot) phase contrast imaging for cell segmentation, tracking and morphology quantification.
For our current and future fluorescence microscopy, we are developing a modular open-source microscopy platform based on openFrame, a low-cost, modular, open microscopy hardware platform to be used with open-source software tools, including MicroManager and FIJI, for instrument control, data acquisition, analysis and management, in order to make them practical in lower resource settings. We are particularly focussing on implementing our techniques in an open-source HCA platform for more robust cell biology studies.
For FLIM/FRET HCA, we have developed an automated multiwell plate FLIM platform utilising open-source software for data acquisition and analysis, which we have applied to assay protein interactions, including applications to viral disease processes. For super-resolved HCA, we are developing automated multiwell plate easySTORM, providing low-cost, large FOV SMLM together with accelerated open-source SMLM analysis parallelised on a high-performance computing cluster. We have applied easySTORM in studies of defective phagocytosis, cancer and kidney disease.
For clinical applications we are developing histoSTORM – an implementation of easySTORM with frozen or FFPE tissue sections and clinically-approved antibody labelling. Other open microscopy developments include a low-cost modular OPT platform that can image mm-cm scale samples, including live zebrafish, and can provide single-shot volumetric imaging.
Enteroinvasive pathogens, such as Shigella and Salmonella, induce their uptake into non-phagocytic epithelial cells through the injection of effectors by the type-3-secretion system. The bacteria are ingested in tight bacterial-containing vacuoles (BCVs) that are surrounded by in situ formed infection-associated macropinosomes (IAMs). In contrast to previous reports, we have recently shown via novel 3D imaging techniques that macropinocytosis is not required for the entry of these bacterial pathogens, however the IAMs regulate their subsequent intracellular trafficking. In the case of Shigella, IAMs do not fuse with the BCV, and contact between these two compartments results in the destabilization of the BCV and membrane rupture. In the case of Salmonella two scenarios occur; IAMs either fuse with the BCV, which results in the generation of the Salmonella containing vacuole surrounded by Salmonella induced filaments (Sifs). Simple contact between IAMs and the BCV also promote vacuolar rupture in the case of Salmonella leading to cytoplasmic hyper-replication. Interestingly, BCV contacts with the surrounding compartments also dictates intravuolar bacterial growth or dormancy. We have performed ultra-structural studies, combined with dynamic imaging and proteomics of the involved compartments to identify the molecules that drive these complex interactions. This has shown a regulatoy network of Rab GTPases, the Exocyst complex, and SNAREs that is hijacked by injected bacterial effectors. I will describe how these factors determine the intracellular niche formation for both, Shigella and Salmonella.
Measuring metabolic fluxes in living cells by 13C metabolic flux analysis has become a key technology for improving our quantitative understanding of cellular metabolism. After two and a half decades of development, driven by diverse analytical and computational innovations, a rich set of tools has become available supporting all steps of the 13C MFA workflow, ranging from the assembly of high-fidelity models and their efficient simulation to convenient design of informative tracer compositions. For studying complex biological systems in less defined environments, such as pathogens residing in a host, however, many challenges remain. One critical step is the identification of a “useful” model formulation with which the fluxes are to be inferred from the data at hand. In the talk, I will present new directions in 13C MFA, powered by Bayesian statistics, to go about this hitherto neglected question and show first successful applications for Escherichia coli and Mycobacterium tuberculosis. Following this path keeps promise to strengthen the position of 13C MFA as an epistemic tool for explaining phenotypes and building models of metabolism.
The presence of metals within biological systems has long been associated with physiological and metabolic processes of the body, including synthesis of complex biomolecules, energy production and intracellular signaling. The determination of elemental distributions in cells is therefore critical in advancing our understanding of disease pathogenesis. Of particular interest is the study of cellular responses to external stimuli, specifically for the development of novel drug treatments which minimize or eliminate undesirable side effects. For this purpose, it is necessary to assess how the metabolic state and variations in elemental distributions of a cellular population impact the effectiveness of drugs and susceptibility to infection. In order to carry out this type of research, sensitive instrumentation and accurate analysis methods are required, however current techniques often exhibit severe matrix effects, poor spatial resolution and high cost. Recent advances have focused on the use of Laser Ablation – Inductively Coupled Plasma – Mass Spectrometry (LA-ICP-MS) and Laser Induced Breakdown Spectroscopy (LIBS), with both techniques adapting the physical process of laser ablation in which a short pulsed laser beam is focused on the sample. The emission of light of a specific wavelength by the elements upon excitation by the laser and the removal of fine ablated particles from the surface of the sample are used for qualitative and quantitative analysis.
This project, funded by the Doctoral College at the University of Surrey and the Natural Environment Research Council (NERC/T009187/1), aims to open the door to new possibilities for cellular research by developing novel methods for biochemical elemental analysis. Investigation of the optimum sample preparation and substrate for the analysis of cells using LIBS has been explored, with issues surrounding sensitivity for intrinsic elemental analysis having been identified. Future work will focus on tackling the analysis of exogenous metals associated with Tuberculosis drugs incorporated into THP-1 cells.
Antimicrobial resistance (AMR) is one of the major challenges we are facing this century. Understanding the mechanism behind the rise of AMR is therefore crucial to tackle this global threat. This is the case for Mycobacterium abscessus (Mabs), a fast-growing non-tuberculous mycobacterium considered as a “clinical nightmare” due to difficulties in its treatment and its multiple resistance and tolerance mechanisms to antibiotics. Mabs is an environmental bacterium whose physiology is driven by environmental factors. Among those are transition metals such as copper, cobalt and nickel. How presence of these ions affects Mabs physiology and its drug susceptibility is unknown, and any research addressing this question will considerably increase our knowledge in AMR and help discovery of a potential drug target. During my PhD, I investigated the impact of transition metals on Mabs physiology and how it affects drug susceptibility, using microbiological and bioenergetic techniques in combination with targeted and untargeted metabolomics with liquid chromatography-mass spectrometry (LC-MS). Use of LC-MS flux analysis with stable isotope labelling identified how nickel (Ni2+), a transition metal ion widely present in the water system, perturb Mabs central carbon metabolism and nitrogen metabolism. These results agree with RNA sequencing and bioenergetics studies using Seahorse analyser showing decreased activity of TCA cycle activity and changes in expression of the related enzymes. Mass spectrometry also enabled quantification of intracellular uptake of antibiotics by Mabs in presence of Ni2+ and efflux pump inhibitors, showing that increased uptake of antibiotics, such as clarithromycin upon Ni2+ treatment, could be linked to increased susceptibility of Mabs. In conclusion, this study demonstrates that transition metals in environment interfere with carbon and nitrogen metabolism, which in turn shapes its drug susceptibility. Targeting Mabs metabolic enzymes or the way Mabs senses and responds to trace elements could offer new solutions to tackle AMR.