Mcfadden J, Al-Khalili J (2015) Good Vibrations, SCIENTIST 29 (8) pp. 62-62 LABX MEDIA GROUP
Lamprecht C, Liashkovich I, Neves V, Danzberger J, Heister E, Rangl M, Coley HM, McFadden J, Flahaut E, Gruber HJ, Hinterdorfer P, Kienberger F, Ebner A (2009) AFM imaging of functionalized carbon nanotubes on biological membranes, NANOTECHNOLOGY 20 (43) ARTN 434001 IOP PUBLISHING LTD
Double-walled carbon nanotubes (DWNTs) prepared by catalytic chemical vapour deposition were functionalized in such a way that they were optimally designed as a nano-vector for the delivery of small interfering RNA (siRNA), which is of great interest for biomedical research and drug development. DWNTs were initially oxidized and coated with a polypeptide (Poly(Lys:Phe)), which was then conjugated to thiol-modified siRNA using a heterobifunctional cross-linker. The obtained oxDWNT-siRNA was characterized by Raman spectroscopy inside and outside a biological environment (mammalian cells). Uptake of the custom-designed nanotubes was not associated with detectable biochemical perturbations in cultured cells, but transfection of cells with DWNTs loaded with siRNA targeting the green fluorescent protein (GFP) gene, serving as a model system, as well as with therapeutic siRNA targeting the survivin gene, led to a significant gene silencing effect, and in the latter case a resulting apoptotic effect in cancer cells.
Beste DJV, Hooper T, Stewart G, Bonde B, Avignone-Rossa C, Bushell M, Wheeler P, Klamt S, Kierzek AM, McFadden J (2007) GSMN-TB: a web-based genome scale network model of Mycobacterium tuberculosis metabolism, GENOME BIOLOGY 8 (5) ARTN r89
BIOMED CENTRAL LTD
We have designed a drug delivery system for the anti-cancer drugs doxorubicin and mitoxantrone based on carbon nanotubes, which is stable under biological conditions, allows for sustained release, and promotes selectivity through an active targeting scheme. Carbon nanotubes are particularly promising for this area of application due to their high surface area, allowing for high drug loading, and their unique interaction with cellular membranes. We have taken a systematic approach to PEG conjugation in order to create a formulation of stable and therapeutically effective CNTs. The presented drug delivery system may be a means of improving cancer treatment modalities by reducing drug-related side effects.
An in-depth study of a novel functionalization of carbon nanotubes for their application as protein and DNA carriers is presented. First, the optimum conditions for the dispersion of singlewalled carbon nanotubes (SWCNTs) with amphiphilic polypeptides were obtained, and the SWCNT-polypeptide complexes were characterized by different techniques (UV-Vis-NIR, CD, and AFM). Based on the properties of the SWCNT-polypeptide complexes, a model that characterizes the adsorption of natural proteins onto SWCNT was described for the first time. This model predicts the adsorption of natural proteins on SWCNTs based on the protein structure and composition, and therefore, allows the design of methods for the preparation of SWCNT-protein complexes. Besides, the use of cationic-designed amphiphilic polypeptides to disperse SWCNTs is applied for subsequent and efficient binding of DNA to carbon nanotubes by a bilayer approach. Therefore, in this article, we develop procedures for the use of SWCNTs as protein and DNA carriers. The systems were delivered into cells showing that the efficiency of delivery is affected by the charge of the complexes, which has important implications in the use of SWCNT as platforms for protein and DNA binding and subsequent use as delivery systems. © Springer Science+Business Media B.V. 2012.
Golchin SA, Stratford J, Curry RJ, McFadden J (2012) A microfluidic system for long-term time-lapse microscopy studies of mycobacteria., Tuberculosis (Edinb) 92 (6) pp. 489-496
Phenotypic heterogeneity in bacterial populations is thought to contribute to a number of important phenomena including sporulation and persistence. The latter has clinical implications in many diseases such as tuberculosis, where persistence of Mycobacterium tuberculosis within the human host is believed to be the root cause of latent tuberculosis and the ability of a minority population of cells to survive antibiotic exposure, despite being genetically identical to the bulk population that are killed. However, phenotypic variations caused by non-genetic mechanisms are difficult to study because of the transient nature of the persistent state and thereby the requirement to observe individual cells in real-time. Recently, microfluidics, combined with time-lapse microscopy, has become a powerful tool for studying population heterogeneity in bacteria. However, growth and replication of mycobacterial cells provide particular problems for the development of microfluidic systems due to their tendency to grow in three dimensions. We here describe a novel microfluidic device for the observation of growth and antibiotic killing in individual mycobacterial cells. We constructed a microfluidic device suitable for studying single cell behavior in mycobacteria. The growth of single cells of Mycobacterium smegmatis expressing green fluorescent protein was monitored using a confocal laser scanning microscope. Within the device M. smegmatis cells were tightly confined within a hydrogel matrix thus promoting planar growth. Cell growth and killing was observed in the device with dead cells highlighted by uptake of propidium iodide. Conclusions/Significance. We demonstrate that our device allows real-time analysis and long-term culture of single cells of mycobacteria, and is able to support the study of cell death during the application of antibiotics. The device will allow observation of individual cells' cell genealogy to be determined and direct observation of rare states, such as persistence.
Neves V, Gerondopoulos A, Heister E, Tîlmaciu C, Flahaut E, Soula B, Silva SRP, McFadden J, Coley HM (2012) Cellular localization, accumulation and trafficking of double-walled carbon nanotubes in human prostate cancer cells, Nano Research 5 (4) pp. 223-234
Carbon nanotubes (CNTs) are at present being considered as potential nanovectors with the ability to deliver therapeutic cargoes into living cells. Previous studies established the ability of CNTs to enter cells and their therapeutic utility, but an appreciation of global intracellular trafficking associated with their cellular distribution has yet to be described. Despite the many aspects of the uptake mechanism of CNTs being studied, only a few studies have investigated internalization and fate of CNTs inside cells in detail. In the present study, intracellular localization and trafficking of RNA-wrapped, oxidized double-walled CNTs (oxDWNT-RNA) is presented. Fixed cells, previously exposed to oxDWNT-RNA, were subjected to immunocytochemical analysis using antibodies specific to proteins implicated in endocytosis; moreover cell compartment markers and pharmacological inhibitory conditions were also employed in this study. Our results revealed that an endocytic pathway is involved in the internalization of oxDWNT-RNA. The nanotubes were found in clathrin-coated vesicles, after which they appear to be sorted in early endosomes, followed by vesicular maturation, become located in lysosomes. Furthermore, we observed co-localization of oxDWNT-RNA with the small GTP-binding protein (Rab 11), involved in their recycling back to the plasma membrane via endosomes from the trans-golgi network. © 2012 Tsinghua University Press and Springer-Verlag Berlin Heidelberg.
McFadden J, Beste DJV, Kierzek AM (2012) Systems Biology of Tuberculosis, Springer
The book starts with a general introduction into the relevance of systems biology for understanding tuberculosis.
Several theories of consciousness first described about a decade ago, including the conscious electromagnetic information (CEMI) field theory, claimed that the substrate of consciousness is the brain's electromagnetic (EM) field. These theories were prompted by the observation, in many diverse systems, that synchronous neuronal firing, which generates coherent EM fields, was a strong correlate of attention, awareness, and consciousness. However, when these theories were first described there was no direct evidence that synchronous firing was actually functional, rather than an epiphenomenon of brain function. Additionally, any EM field-based consciousness would be a 'ghost in the machine' unless the brain's endogenous EM field is also able to influence neuron firing. Once again, when these theories were first described, there was only indirect evidence that the brain's EM field influenced neuron firing patterns in the brain. In this paper I describe recent experimental evidence which demonstrate that synchronous neuronal firing does indeed have a functional role in the brain; and also that the brain's endogenous EM field is involved in recruiting neurons to synchronously firing networks. The new data point to a new and unappreciated form of neural communication in the brain that is likely to have significance for all theories of consciousness. I describe an extension of the CEMI field theory that incorporates these recent experimental findings and integrates the theory with the 'communication through coherence' hypothesis.
Casey R, McFadden J, Newcombe J, Bodman-Smith M, Bodman-Smith K (2007) C-reactive protein binds to Neisseria meningitidis and affects macrophage responses to infection, IMMUNOLOGY 120 pp. 20-20 BLACKWELL PUBLISHING
Brian D. Robertson and Brendan W. Wren
Polyethyleneimine-coated double-walled carbon nanotubes (DWCNTs) were used for dual gene and drug delivery, after loading the DWCNTs with the drug chloroquine, a lysosomotropic compound that is able to promote escape from the lysosomal compartment. Different forms of functionalization of the DWCNTs were examined in order to optimize this system. They included the testing of different treatments on DWCNTs to optimize the loading and delivery of chloroquine and the selection of a cationic polymer for coating the DWCNTs for optimum DNA binding and delivery. An acid oxidation treatment of DWCNTs was selected for optimum chloroquine loading together with polyethyleneimine as optimum cationic coating agent for plasmid DNA binding. Optimization of the conditions for choroquine-enhanced gene delivery were developed using luciferase expression as a model system. We have demonstrated that chloroquine-loading increases the ability of polyethyleneimine-coated DWCNTs to deliver functional nucleic acid to human cells. Cell viability tests have shown no cytotoxicity of the functionalized DWCNTs at the concentrations needed for optimum gene delivery. These results support the potential applications of this methodology in gene therapy.
Beste DJ, Nöh K, Niedenführ S, Mendum TA, Hawkins ND, Ward JL, Beale MH, Wiechert W, McFadden J (2013) (13)C-Flux Spectral Analysis of Host-Pathogen Metabolism Reveals a Mixed Diet for Intracellular Mycobacterium tuberculosis., Chem Biol 20 (8) pp. 1012-1021 Elsevier
Whereas intracellular carbon metabolism has emerged as an attractive drug target, the carbon sources of intracellularly replicating pathogens, such as the tuberculosis bacillus Mycobacterium tuberculosis, which causes long-term infections in one-third of the world's population, remain mostly unknown. We used a systems-based approach-(13)C-flux spectral analysis (FSA) complemented with manual analysis-to measure the metabolic interaction between M. tuberculosis and its macrophage host cell. (13)C-FSA analysis of experimental data showed that M. tuberculosis obtains a mixture of amino acids, C1 and C2 substrates from its host cell. We experimentally confirmed that the C1 substrate was derived from CO2. (13)C labeling experiments performed on a phosphoenolpyruvate carboxykinase mutant revealed that intracellular M. tuberculosis has access to glycolytic C3 substrates. These findings provide constraints for developing novel chemotherapeutics.
Michelon A, Conceicao FR, Binsfeld PC, da Cunha CW, Moreira AN, Argondizzo AP, McIntosh D, Armoa GRG, Campos AS, Farber M, McFadden J, Dellagostin OA (2006) Immunogenicity of Mycobacterium bovis BCG expressing Anaplasma marginale MSP1a antigen, VACCINE 24 (37-39) pp. 6332-6339 ELSEVIER SCI LTD
The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.
Karlyshev AV, Snyder LA, McFadden J, Griffin R (2015) Insight into proteomic investigations of Neisseria meningitidis serogroup C strain L91543 from analysis of its genome sequence., FEMS Microbiol Lett 362 (9)
Here, we describe the draft sequence of a virulent isolate of Neisseria meningitidis strain L91543, belonging to serogroup C. The findings from previous proteomic and metabolomic studies of this strain can now be further interpreted with genomic analysis. Comparative analysis of the genome sequence revealed close similarity and localized synteny with the genome sequence of N. meningitidis serogroup C strain, FAM18. Polymorphisms were identified in the signal peptide sequence of factor H binding protein, a target for new meningococcal vaccines, which may result in its inefficient translocation across the cytoplasmic membrane affecting its processing (lipidation and cleavage of the signal peptide) and transportation to the outer membrane in strain L91543. This would explain the unusual proteomic data for factor H binding protein for this strain. NadA, another target for new vaccines, and the MtrR regulator, which controls expression of NadA, both contain SNPs between strains L91543 and FAM18. The genome sequence data were generated using Ion Torrent PGM sequencing, assembled into 50 contigs with 64× coverage and annotated with 2262 genes, 14 rRNAs and 56 tRNAs. The availability of the genome of N. meningitidis strain L91543 will aid our understanding of the proteome of this organism, importantly its vaccine antigens.
Shi L, Sohaskey CD, Pfeiffer C, Datta P, Parks M, McFadden J, North RJ, Gennaro ML (2010) Carbon flux rerouting during Mycobacterium tuberculosis growth arrest, MOLECULAR MICROBIOLOGY 78 (5) pp. 1199-1215 WILEY-BLACKWELL
McFadden J (2013) The CEMI Field Theory Gestalt Information and the Meaning of Meaning, JOURNAL OF CONSCIOUSNESS STUDIES 20 (3-4) pp. 152-182 IMPRINT ACADEMIC
In earlier papers I described the conscious electromagnetic information (CEMI) field theory, which claimed that the substrate of consciousness is the brain's electromagnetic (EM) field. I here further explore this theory by examining the properties and dynamics of the information underlying meaning in consciousness. I argue that meaning suffers from a binding problem, analogous to the binding problem described for visual perception, and describe how the gestalt (holistic) properties of meaning give rise to this binding problem. To clarify the role of information in conscious meaning, I differentiate between extrinsic information that is symbolic and arbitrary, and intrinsic information, which preserves structural aspects of the represented object and thereby maintains some gestalt properties of the represented object. I contrast the requirement for a decoding process to extract meaning from extrinsic information, whereas meaning is intrinsic to the structure of the gestalt intrinsic information and does not require decoding. I thereby argue that to avoid the necessity of a decoding homunculus, conscious meaning must be encoded intrinsically -- as gestalt information -- in the brain. Moreover, I identify fields as the only plausible substrate for encoding gestalt intrinsic information and argue that the binding problem of meaning can only be solved by grounding meaning in this field-based gestalt information. I examine possible substrates for gestalt information in the brain and conclude that the only plausible substrate is the CEMI field.
Borsuk S, Seixas FK, Ramos DF, Mendum T, McFadden J, Dellagostin O (2012) Rational design of diagnostic and vaccination strategies for tuberculosis., Braz J Infect Dis 16 (1) pp. 68-73
The development of diagnostic tests which can readily differentiate between vaccinated and tuberculosis-infected individuals is crucial for the wider utilization of bacillus Calmette-Guérin (BCG) as vaccine in humans and animals. BCG_0092 is an antigen that elicits specific delayed type hypersensitivity reactions similar in size and morphological aspects to that elicited by purified protein derivative, in both animals and humans infected with the tubercle bacilli. We carried out bioinformatics analyses of the BCG_0092 and designed a diagnostic test by using the predicted MHC class I epitopes. In addition, we performed a knockout of this gene by homologous recombination in the BCG vaccine strain to allow differentiation of vaccinated from infected individuals. For that, the flanking sequences of the target gene (BCG_0092)were cloned into a suicide vector. Spontaneous double crossovers, which result in wild type revertants or knockouts were selected using SacB. BCG_0092 is present only in members of the Mycobacterium tuberculosis complex. Eight predicted MHC class I epitopes with potential for immunological diagnosis were defined, allowing the design of a specific diagnostic test. The strategy used to delete the (BCG_0092) gene from BCG was successful. The knockout genotype was confirmed by PCR and by Southern blot. The mutant BCG strain has the potential of inducing protection against tuberculosis without interfering with the diagnostic test based on the use of selected epitopes from BCG_0092.
Senaratne RH, Sidders B, Sequeira P, Saunders G, Dunphy K, Marjanovic C, Reader JR, Lima P, Chan S, Kendall S, McFadden J, Riley LW (2008) Mycobacterium tuberculosis strains disrupted in mce3 and mce4 operons are attenuated in mice, JOURNAL OF MEDICAL MICROBIOLOGY 57 (2) pp. 164-170 SOC GENERAL MICROBIOLOGY
Jenson JS, Casey R, Newcombe J, Smith M, McFadden JJ, Bodman-Smith K (2008) Dendritic cell responses to C-reactive protein-opsonised Neisseria meningitidis, IMMUNOLOGY 125 pp. 124-124 WILEY-BLACKWELL PUBLISHING, INC
Sanz V, Coley HM, Silva SRP, McFadden J (2012) Protamine and chloroquine enhance gene delivery and expression mediated by RNA-wrapped single walled carbon nanotubes, Journal of Nanoscience and Nanotechnology 12 (3) pp. 1739-1747
The use of non-viral vectors as delivery systems in gene therapy has been extensively studied recently owing to their advantages over viral vectors. Here, we propose a new gene delivery system based on the use of RNA-wrapped single-walled carbon nanotubes (SWCNTs) complexed with the cationic protein, protamine and the drug chloroquine. Protamine was selected as a cationic protein acting as bridge between negatively charged RNA-wrapped SWCNTs and plasmid DNA. Protamine also contains a nuclear localization signal which enhances the expression of the transfected gene. The drug chloroquine, a lysosomotropic compound which has been reported to increase the transfection efficiency, was attached to RNA-wrapped SWNTs by ionic interactions. The simultaneous delivery of the drug chloroquine with plasmid DNA clearly showed an enhanced gene delivery and expression. The levels of gene expression were quantified using the luciferase reporter gene as model. Optimal conditions for transfection and gene expression were obtained and cytoxicity of the carbon nanotube complexes measured. The optimal complexes were shown to efficiently deliver plasmid DNA for efficient gene expression and may thereby be useful as gene delivery systems for gene therapy. Copyright © 2012 American Scientific Publishers.
Borsuk S, Mendum TA, Fagundes MQ, Michelon M, Cunha CW, McFadden J, Dellagostin OA (2007) Auxotrophic complementation as a selectable marker for stable expression of foreign antigens in Mycobacterium bovis BCG, TUBERCULOSIS 87 (6) pp. 474-480 CHURCHILL LIVINGSTONE
Despite decades of research, many aspects of the biology of Mycobacterium tuberculosis remain unclear, and this is reflected in the antiquated tools available to treat and prevent tuberculosis and consequently this disease remains a serious public health problem. Important discoveries linking the metabolism of M. tuberculosis and pathogenesis has renewed interest in this area of research. Previous experimental studies were limited to the analysis of individual genes or enzymes, whereas recent advances in computational systems biology and high-throughput experimental technologies now allows metabolism to be studied on a genome scale. In the present article, we discuss the progress being made in applying system-level approaches to study the metabolism of this important pathogen.
Santangelo MP, McIntosh D, Bigi F, Armoa GRG, Campos ASD, Ruybal P, Dellagostin OA, McFaddend J, Mendum T, Gicquel B, Winter N, Farber M, Cataldi A (2007) Mycobacterium bovis BCG as a delivery system for the RAP-1 antigen from Babesia bovis, VACCINE 25 (6) pp. 1104-1113 ELSEVIER SCI LTD
McFadden J (2013) Making the Quantum Leap., The Biologist 60 (2) pp. 13-16
Professor Johnjoe McFadden reveals how quantum physics could help explain some of biology?s most mysterious phenomena
Slayden RA, Jackson M, Zucker J, Ramirez MV, Dawson CC, Crew R, Sampson NS, Thomas ST, Jamshidi N, Sisk P, Caspi R, Crick DC, McNeil MR, Pavelka MS, Niederweis M, Siroy A, Dona V, McFadden J, Boshoff H, Lew JM (2013) Updating and curating metabolic pathways of TB, TUBERCULOSIS 93 (1) pp. 47-59 CHURCHILL LIVINGSTONE
Rustam T, McClean S, Newcombe J, McFadden J, Eales-Reynolds LJ (2006) Reduced toxicity of lipo-oligosaccharide from a phoP mutant of Neisseria meningitidis: an in vitro demonstration, JOURNAL OF ENDOTOXIN RESEARCH 12 (1) pp. 39-46 MANEY PUBLISHING
Heister E, Lamprecht C, Neves V, Tilmaciu C, Datas L, Flahaut E, Soula B, Hinterdorfer P, Coley HM, Silva SRP, McFadden J (2010) Higher Dispersion Efficacy of Functionalized Carbon Nanotubes in Chemical and Biological Environments, ACS NANO 4 (5) pp. 2615-2626
AMER CHEMICAL SOC
Wu Huihai, von Kamp A, Leoncikas Vytautas, Mori W, Sahin N, Gevorgyan A, Linley C, Grabowski M, Mannan AA, Stoy Nicholas, Stewart Graham, Ward LT, Lewis David, Sroka J, Matsuno H, Klamt S, Westerhoff HV, McFadden Johnjoe, Plant NJ, Kierzek Andrzej (2016) MUFINS: Multi-Formalism Interaction Network Simulator, NPJ Systems Biology and Applications 2 16032
Nature Publishing Group
Systems Biology has established numerous approaches for mechanistic modelling of molecular networks in the cell and a legacy of models. The current frontier is the integration of models expressed in different formalisms to address the multi-scale biological system organisation challenge. We present MUFINS software, implementing a unique set of approaches for multiformalism simulation of interaction networks. We extend the constraint-based modelling (CBM) framework by incorporation of linear inhibition constraints, enabling for the first time linear modelling of networks simultaneously describing gene regulation, signalling and whole-cell metabolism at steady state. We present a use case where a logical hypergraph model of a regulatory network is expressed by linear constraints and integrated with a Genome Scale Metabolic Network (GSMN) of mouse macrophage. We experimentally validate predictions, demonstrating application of our software in an iterative cycle of hypothesis generation, validation and model refinement. MUFINS incorporates an extended version of our Quasi Steady State Petri Net approach to integrate dynamic models with CBM, which we demonstrate through a dynamic model of cortisol signalling integrated with the human Recon2 GSMN and a model of nutrient dynamics in physiological compartments. Finally, we implement a number of methods for deriving metabolic states from ~omics data, including our new variant of the iMAT congruency approach. We compare our approach with iMAT through analysis of 262 individual tumour transcriptomes, recovering features of metabolic reprogramming in cancer. The software provides graphics user interface with network visualisation, which facilitates use by researchers who are not experienced in coding and mathematical modelling environments.
Lofthouse EK, Wheeler PR, Beste DJV, Khatri BL, Wu H, Mendum TA, Kierzek AM, McFadden J (2013) Systems-Based Approaches to Probing Metabolic Variation within the Mycobacterium tuberculosis Complex, PLOS ONE 8 (9) ARTN e75913 PUBLIC LIBRARY SCIENCE
Beste DJ, Bonde B, Hawkins N, Ward JL, Beale MH, Noack S, Nöh K, Kruger NJ, Ratcliffe RG, McFadden J (2011) ¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation., PLoS Pathog 7 (7)
Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using ¹³C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with ¹³C labeled glycerol or sodium bicarbonate. Through measurements of the ¹³C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate--oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO into biomass. As the human host is abundant in CO this finding requires further investigation in vivo as CO fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using ¹³C-MFA.
Newcombe J, Mendum TA, Ren CP, McFadden J (2013) Identification of the immunoproteome of the meningococcus by cell surface immunoprecipitation and mass spectrometry (CSIP-MS)., Microbiology
Most healthy adults are protected from meningococcal disease by the presence of naturally acquired anti-meningococcal antibodies; however, the identity of the target antigens of this protective immunity remains unclear, particularly for protection against serogroup B disease. To identify the protein targets of natural protective immunity we developed an immunoprecipitation and proteomics approach to define the immunoproteome of the meningococcus. Sera from 10 healthy individuals showing serum bactericidal activity against both a meningococcal C strain (L91543) and the B strain MC58, together with commercially available pooled human sera were used as probe anti-sera. Immunoprecipitation was performed with each serum sample and live cells from both meningococcal strains. Immunoprecipitated proteins were identified by mass spectrometry. Analysis of the immunoproteome from each serum demonstrated both pan-reactive antigens that were recognised by most sera as well as subject-specific antigens. Most antigens were found in both meningococcal strains but a few were strain-specific. Many of the immunoprecipitated proteins have previously been characterised as surface antigens including adhesins and proteases, several of which have been recognised as vaccine candidate antigens e.g. fHBP, NadA and NHBA. The data clearly demonstrates the presence of meningococcal antibodies in healthy individuals with no history of meningococcal infection and a wide diversity of immune responses. The identification of the immuno-reactive proteins of the meningococcus provides a basis for understanding the role of each antigen in the natural immunity associated with carriage and may help to design vaccination strategies.
Sroka J, Bieniasz-Krzywiec L, Gwózdz S, Leniowski D, L?cki J, Markowski M, Avignone-Rossa C, Bushell ME, McFadden J, Kierzek AM (2011) Acorn: a grid computing system for constraint based modeling and visualization of the genome scale metabolic reaction networks via a web interface., BMC Bioinformatics 12 (196)
Constraint-based approaches facilitate the prediction of cellular metabolic capabilities, based, in turn on predictions of the repertoire of enzymes encoded in the genome. Recently, genome annotations have been used to reconstruct genome scale metabolic reaction networks for numerous species, including Homo sapiens, which allow simulations that provide valuable insights into topics, including predictions of gene essentiality of pathogens, interpretation of genetic polymorphism in metabolic disease syndromes and suggestions for novel approaches to microbial metabolic engineering. These constraint-based simulations are being integrated with the functional genomics portals, an activity that requires efficient implementation of the constraint-based simulations in the web-based environment.
One of the most challenging problems in microbiology is to understand how a small fraction of microbes that resists killing by antibiotics can emerge in a population of genetically identical cells, the phenomenon known as persistence or drug tolerance. Its characteristic signature is the biphasic kill curve, whereby microbes exposed to a bactericidal agent are initially killed very rapidly but then much more slowly. Here we relate this problem to the more general problem of understanding the emergence of distinct growth phenotypes in clonal populations. We address the problem mathematically by adopting the framework of the phenomenon of so-called weak ergodicity breaking, well known in dynamical physical systems, which we extend to the biological context. We show analytically and by direct stochastic simulations that distinct growth phenotypes can emerge as a consequence of slow-down of stochastic fluctuations in the expression of a gene controlling growth rate. In the regime of fast gene transcription, the system is ergodic, the growth rate distribution is unimodal, and accounts for one phenotype only. In contrast, at slow transcription and fast translation, weakly non-ergodic components emerge, the population distribution of growth rates becomes bimodal, and two distinct growth phenotypes are identified. When coupled to the well-established growth rate dependence of antibiotic killing, this model describes the observed fast and slow killing phases, and reproduces much of the phenomenology of bacterial persistence. The model has major implications for efforts to develop control strategies for persistent infections.
Jeynes JCG, Mendoza E, Chow DCS, Watts PCR, McFadden J, Silva SRP (2006) Generation of chemically unmodified pure single-walled carbon nanotubes by solubilizing with RNA and treatment with ribonuclease A, ADVANCED MATERIALS 18 (12) pp. 1598-+ WILEY-V C H VERLAG GMBH
Neves V, Heister E, Costa S, Tilmaciu C, Borowiak-Palen E, Giusca CE, Flahaut E, Soula B, Coley HM, McFadden J, Silva SRP (2010) Uptake and Release of Double-Walled Carbon Nanotubes by Mammalian Cells, ADVANCED FUNCTIONAL MATERIALS 20 (19) pp. 3272-3279 WILEY-V C H VERLAG GMBH
Heister E, Neves V, Tilmaciu C, Lipert K, Beltran VS, Coley HM, Silva SRP, McFadden J (2009) Triple functionalisation of single-walled carbon nanotubes with doxorubicin, a monoclonal antibody, and a fluorescent marker for targeted cancer therapy, CARBON 47 (9) pp. 2152-2160
PERGAMON-ELSEVIER SCIENCE LTD
Beste DJV, McFadden J (2013) Metabolism of Mycobacterium tuberculosis, In: Systems biology of tuberculosis 4
The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such asMycobacterium bovisBCG orM. smegmatiswas made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinantM. smegmatisbacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in bothM. smegmatisandM. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of theSchistosoma mansoniantigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response.
BACKGROUND: Neisseria meningitidis is an important human commensal and pathogen that causes several thousand deaths each year, mostly in young children. How the pathogen replicates and causes disease in the host is largely unknown, particularly the role of metabolism in colonization and disease. Completed genome sequences are available for several strains but our understanding of how these data relate to phenotype remains limited. RESULTS: To investigate the metabolism of N. meningitidis we generated and selected a representative Tn5 library on rich medium, a minimal defined medium and in human serum to identify genes essential for growth under these conditions. To relate these data to a systems-wide understanding of the pathogen's biology we constructed a genome-scale metabolic network: Nmb_iTM560. This model was able to distinguish essential and non-essential genes as predicted by the global mutagenesis. These essentiality data, the library and the Nmb_iTM560 model are powerful and widely applicable resources for the study of meningococcal metabolism and physiology. We demonstrate the utility of these resources by predicting and demonstrating metabolic requirements on minimal medium such as a requirement for PEP carboxylase, and by describing the nutritional and biochemical status of N. meningitidis when grown in serum, including a requirement for both the synthesis and transport of amino acids. CONCLUSIONS: This study describes the application of a genome scale transposon library combined with an experimentally validated genome-scale metabolic network of N. meningitidis to identify essential genes and provide novel insight to the pathogen's metabolism both in vitro and during infection.
Cell growth experiments with a microfluidic device produce large scale time-lapse image data, which contain important information on cell growth and patterns in their genealogy. To extract such information, we propose a scheme to segment and track bacterial cells automatically. In contrast to most published approaches, which often split segmentation and tracking into two independent procedures, we focus on designing an algorithm that describes cell properties evolving between consecutive frames by feeding segmentation and tracking results from one frame to the next one. The cell boundaries are extracted by minimising the Distance Regularised Level Set Evolution model. Each individual cell was identified and tracked by identifying cell septum and membrane as well as developing a trajectory energy minimisation function along time-lapse series. Experiments show that by applying this scheme, cell growth and division can be measured automatically. The results show the efficiency of the approach when testing on different datasets while comparing with other existing algorithms. The proposed approach demonstrates great potential for large scale bacterial cell growth analysis.
For over 50 years, drug combinations have been the gold standard in the therapy of tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb). Clinically, synergistic combinations of drugs are favoured over monotherapy as they allow the use of lower doses and also suppress the development of drug resistance. However, multiple drug-resistant TB (MDR-TB) is on the rise, and is threatening the effective control of the disease globally. Prior to this thesis, drug synergy studies in mycobacteria have largely been determined empirically with little or no mechanistic understanding of the mechanisms of synergy. Also, although it has been assumed that mutation rates for drug combinations are multiples of the mutation rates for single drugs, this assumption has not been thoroughly tested.
In this thesis, drug synergy was demonstrated in Mycobacterium smegmatis (Msm), a model TB organism, using Isoniazid (INH) and Rifampicin (RIF), the two most effective bactericidal drugs used in TB therapy. A novel indirect approach of cell inhibition was employed, whereby the single drugs on their own were utilized at sub-lethal concentrations that alone were only weakly inhibiting, but in combination resulted in synergy. The hypothesis investigated in this study was that synergy was a consequence of the interaction between antibiotics inducing a cryptic perturbation state (AICPs), such that each single antibiotic produced a synergistic effect in combination. To investigate changes induced in the AICPs, RNA-sequencing (RNA-seq) was performed on RNA extracted from cells in the AICPs. Perturbed genes were mapped onto Kyoto Encyclopaedia for Genes and Genomes (KEGG) pathway maps, to identify affected metabolic pathways and identify differences between perturbations due to INH and RIF. Differential Producibility Analysis (DPA) was also used to highlight metabolite differences in states induced by each antibiotic. Mutation rates were also measured to INH and RIF singly and in combination using the fluctuation assay.
Mapping of the differentially expressed genes from RNA-seq onto KEGG pathway analysis identified perturbation of a significant number of genes encoding proteins involved in sulphur metabolism and ribosome synthesis pathways, exclusively in the AICPs to INH, but not in the AICPs to RIF. Perturbations to genes encoding ABC transporters were identified in the AICPs to both antibiotics, but the specific transporters involved differed to the two antibiotics. DPA revealed stronger metabolite signals in the AICPs in response to INH, affecting putrescine, iron, malonate, and ²-alanine that affected several areas of metabolism. However, only one metabolite involved in potassium ion transport was significantly perturbed by DPA analysis in the AICPs induced by RIF. The action of these metabolites and genes acting in parallel pathways provides a novel explanation for drug synergy in mycobacteria.
Mutation rates to resistance to INH and RIF were estimated separately and, also, mutations to resistance to both drugs present in combination. Surprisingly, mutations to resistance to both drugs were found at a rate four orders of magnitude higher than expected from the mutation rate to single drugs. This result needs to be confirmed by further studies but, if confirmed, would suggest that drug combinations may, paradoxically, enhance the mutation rate to two or more drugs.
These results have provided novel insights into the mechanism of drug synergy in mycobacteria.
The use of carbon nanotubes as a gene delivery system has been extensively studied in recent years owing to its potential advantages over viral vectors. To achieve this goal, carbon nanotubes have to be functionalized to become compatible with aqueous media and to bind the genetic material. To establish the best conditions for plasmid DNA binding, we compare the dispersion properties of single-, double- and multi-walled carbon nanotubes (SWCNTs, DWCNTs and MWCNTs, respectively) functionalized with a variety of surfactants by non-covalent attachment. The DNA binding properties of the functionalized carbon nanotubes were studied and compared by electrophoresis. Furthermore, a bilayer functionalization method for DNA binding on SWCNTs was developed that utilized RNA-wrapping to solubilize the nanotubes and cationic polymers as a bridge between nanotubes and DNA.
Neisseria meningitidis causes meningococcal disease, a global life threatening illness with annual incidences of between 1 and 1000 per 100,000 population. Humans are the only known host with approximately 10% of people having asymptomatic nasopharyngeal carriage at any one time. Thus, the ability of meningococci to attach, invade, and grow in the epithelium is crucial for both its commensal and pathogenic properties. In the rare event that meningococci cross the epithelium into the bloodstream, disease may occur. In order to better understand the mechanisms of meningococcal pathogenesis, transposon mutagenesis was used to identify bacterial genes involved in epithelial cell adherence and internalization as well as traversal of the epithelial barrier.
Three epithelial cell lines of respiratory origin, A549 cells, 16HBE14o- cells and Detroit 562 cells were used to examine N. meningitidis L91543 (C:2a:P1.2, ST-11; ET-37) pathogenesis. First, adhesion, invasion and traversal assays were optimized for bacterial uptake to enable the maximum number of mutants to be tested and to avoid stochastic loss from the transposon library. Since the highest level of meningococci adherence and invasion was observed using 16HBE14o- cells, this cell line was chosen for subsequent traversal assays, where an intact epithelial barrier was established on Transwell® membrane inserts. Epithelial barrier integrity was assessed by measuring transepithelial electrical resistance (TEER), permeability of the marker protein, 70 kDa Dextran, and by examining the distribution of the tight junction proteins, occludin and ZO-1, by immunofluorescence.
Next, transposon mutagenesis libraries comprising of approx. 14,500 N. meningitidis L91543 mutants, were used to probe meningococcal interactions with 16HBE14o- epithelial cells. Illumina sequencing of amplified transposon junctions was performed on DNA extracted from both input and output pools obtained from the various assays. Comparative analysis of input/output pools showed reduced fitness, not only of genes associated with type IV pili, but mainly of genes involved in metabolism especially nucleotide and amino acid metabolism. Genes involved in membrane transport, regulatory functions and cellular processes also showed reduced fitness. The function of putative genes of interest was validated by generating insertion knockout mutants and testing them independently for their ability to alter meningococcal-epithelial cell interactions. The knockout mutant assays showed 67-100% agreement to the Tn-Seq analysis prediction. Based on the knock out mutant assays as well the Tn-Seq prediction we can conclude that type IV pili, nucleotide biosynthesis, glucose and amino acid metabolism, as well as resistance to antimicrobial peptide are critical for meningococcal interaction with epithelial cells.
Basu Piyali, Sandhu Noor, Bhatt Apoorva, Singh Albel, Balhana Ricardo, Gobe Irene, Crowhurst Nicola A, Mendum Thomas, Gao Liang, Ward Jane L, Beale Michael H, McFadden Johnjoe, Beste Dany (2018) The anaplerotic node is essential for the intracellular survival of Mycobacterium tuberculosis, Journal of Biological Chemistry 293 (15) pp. 5695-5704
American Society for Biochemistry and Molecular Biology
Enzymes at the phosphoenolpyruvate (PEP)?pyruvate?oxaloacetate or anaplerotic (ANA) node control the metabolic flux to glycolysis, gluconeogenesis, and anaplerosis. Here we used genetic, biochemical, and 13C isotopomer analysis to characterize the role of the enzymes at the ANA node in intracellular survival of the world's most successful bacterial pathogen, Mycobacterium tuberculosis (Mtb). We show that each of the four ANA enzymes, pyruvate carboxylase (PCA), PEP carboxykinase (PCK), malic enzyme (MEZ), and pyruvate phosphate dikinase (PPDK), performs a unique and essential metabolic function during the intracellular survival of Mtb. We show that in addition to PCK, intracellular Mtb requires PPDK as an alternative gateway into gluconeogenesis. Propionate and cholesterol detoxification was also identified as an essential function of PPDK revealing an unexpected role for the ANA node in the metabolism of these physiologically important intracellular substrates and highlighting this enzyme as a tuberculosis (TB)-specific drug target. We show that anaplerotic fixation of CO2 through the ANA node is essential for intracellular survival of Mtb and that Mtb possesses three enzymes (PCA, PCK, and MEZ) capable of fulfilling this function. In addition to providing a back-up role in anaplerosis we show that MEZ also has a role in lipid biosynthesis. MEZ knockout strains have an altered cell wall and were deficient in the initial entry into macrophages. This work reveals that the ANA node is a focal point for controlling the intracellular replication of Mtb, which goes beyond canonical gluconeogenesis and represents a promising target for designing novel anti-TB drugs.
Mendum Tom A., Chandran Aneesh, Williams Kerstin, Vordermeier H. Martin, Villarreal-Ramos Bernardo, Wu H., Singh Albel, Smith Alex A., Butler Rachel E., Prasad Aravind, Bharti Neeraj, Banerjee Ruma, Kasibhatla Sunitha M., Bhatt Apoorva, Stewart Graham R., McFadden Johnjoe (2019) Transposon libraries identify novel Mycobacterium bovis BCG genes involved in the dynamic interactions required for BCG to persist during in vivo passage in cattle, BMC Genomics 20 431 pp. 1-13
BCG is the most widely used vaccine of all time and remains the only licensed vaccine for use against tuberculosis in humans. BCG also protects other species such as cattle against tuberculosis, but due to its incompatibility with current tuberculin testing regimens remains unlicensed. BCG?s efficacy relates to its ability to persist in the host for weeks, months or even years after vaccination. It is unclear to what degree this ability to resist the host?s immune system is maintained by a dynamic interaction between the vaccine strain and its host as is the case for pathogenic mycobacteria.
To investigate this question, we constructed transposon mutant libraries in both BCG Pasteur and BCG Danish strains and inoculated them into bovine lymph nodes. Cattle are well suited to such an assay, as they are naturally susceptible to tuberculosis and are one of the few animal species for which a BCG vaccination program has been proposed. After three weeks, the BCG were recovered and the input and output libraries compared to identify mutants with in vivo fitness defects. Less than 10% of the mutated genes were identified as affecting in vivo fitness, they included genes encoding known mycobacterial virulence functions such as mycobactin synthesis, sugar transport, reductive sulphate assimilation, PDIM synthesis and cholesterol metabolism. Many other attenuating genes had not previously been recognised as having a virulence phenotype. To test these genes, we generated and characterised three knockout mutants that were predicted by transposon mutagenesis to be attenuating in vivo: pyruvate carboxylase, a hypothetical protein (BCG_1063), and a putative cyclopropane-fatty-acyl-phospholipid synthase. The knockout strains survived as well as wild type during in vitro culture and in bovine macrophages, yet demonstrated marked attenuation during passage in bovine lymph nodes confirming that they were indeed involved in persistence of BCG in the host.
These data show that BCG is far from passive during its interaction with the host, rather it continues to employ its remaining virulence factors, to interact with the host?s innate immune system to allow it to persist, a property that is important for its protective efficacy.
McFadden Johnjoe, Al-Khalili Jim (2018) The origins of quantum biology, Proceedings of the Royal Society A: Mathematical, Physical and Engineering Sciences 474 (2220) 20180674 pp. 1-13
Quantum biology is usually considered to be a new discipline, arising from recent research that suggests that biological phenomena such as photosynthesis, enzyme catalysis, avian navigation or olfaction may not only operate within the bounds of classical physics but also make use of a number of the non-trivial features of quantum mechanics, such as coherence, tunnelling and, perhaps, entanglement. However, although the most significant findings have emerged in the past two decades, the roots of quantum biology go much deeper?to the quantum pioneers of the early twentieth century. We will argue that some of the insights provided by these pioneering physicists remain relevant to our understanding of quantum biology today.
New approaches are needed to control leprosy, but understanding of
the biology of the causative agent Mycobacterium leprae remains rudimentary, principally because the pathogen cannot be grown in axenic culture. Here, we applied
13C isotopomer analysis to measure carbon metabolism of M. leprae in its primary
host cell, the Schwann cell. We compared the results of this analysis with those of a
related pathogen, Mycobacterium tuberculosis, growing in its primary host cell, the
macrophage. Using 13C isotopomer analysis with glucose as the tracer, we show that
whereas M. tuberculosis imports most of its amino acids directly from the host macrophage, M. leprae utilizes host glucose pools as the carbon source to biosynthesize
the majority of its amino acids. Our analysis highlights the anaplerotic enzyme phosphoenolpyruvate carboxylase required for this intracellular diet of M. leprae, identifying this enzyme as a potential antileprosy drug target.
Nitrogen metabolism of Mycobacterium tuberculosis(Mtb) is crucial for the survival of this important pathogen in its primary human host cell, the macrophage, but little is known about the source(s) and their assimilation within this intracellular niche. Here, we have developed 15N-flux spectral ratio analysis(15N-FSRA) to explore Mtb?s nitrogen metabolism; we demonstrate that intracellular Mtb has access to multiple amino acids in the macrophage, including glutamate, glutamine, aspartate, alanine, glycine,and valine; and we identify glutamine as the pre-dominant nitrogen donor. Each nitrogen source is uniquely assimilated into specific amino acid pools,indicating compartmentalized metabolism during intracellular growth. We have discovered that serine is not available to intracellular Mtb, and we show that a serine auxotroph is attenuated in macrophages. This work provides a systems-based tool for exploring the nitrogen metabolism of intracellular pathogens and highlights the enzyme phosphoserine transaminase as an attractive target for the development of novel anti-tuberculosis therapies.
Bovine tuberculosis (BtB) caused by Mycobacterium bovis remains a major problem in both the developed and developing countries. control of BtB in the UK is carried out by test and slaughter of infected animals, based primarily on the tuberculin skin test (ppD). Vaccination with the attenuated strain of the M. bovis pathogen, BcG, is not used to control bovine tuberculosis in cattle at present, due to its variable efficacy and because it interferes with the PPD test. Diagnostic tests capable of Differentiating Infected from Vaccinated Animals (DIVA) have been developed that detect immune responses to M. bovis antigens absent in BCG; but these are too expensive and insufficiently sensitive to be used for BtB control worldwide. to address these problems we aimed to generate a synergistic vaccine and diagnostic approach that would permit the vaccination of cattle without interfering with the conventional ppD-based surveillance. the approach was to widen the pool of M. bovis antigens that could be used as DiVA targets, by identifying antigenic proteins that could be deleted from BcG without affecting the persistence and protective efficacy of the vaccine in cattle. Using transposon mutagenesis we identified genes that were essential and those that were non-essential for persistence in bovine lymph nodes. We then inactivated selected immunogenic, but non-essential genes in BcG Danish to create a diagnostic-compatible triple knock-out BCG TK strain. The protective efficacy of the BcG tK was tested in guinea pigs experimentally infected with M. bovis by aerosol and found to be equivalent to wild-type BcG. A complementary diagnostic skin test was developed with the antigenic proteins encoded by the deleted genes which did not cross-react in vaccinated or in uninfected guinea pigs. this study demonstrates the functionality of a new and improved BcG strain which retains its protective efficacy but is diagnostically compatible with a novel DIVA skin test that could be implemented in control programmes.
Hingley-Wilson Suzie, Ma Nan, Hu Yin, Casey Rosalyn, Bramming Anders, Curry Richard J., Tang Hongying Lilian, Wu Huihai, Butler Rachel, Jacobs Jr. William R., Rocco Andrea, McFadden Johnjoe (2020) Loss of phenotypic inheritance associated with ydcI
mutation leads to increased frequency of
small, slow persisters in Escherichia coli, Proceedings of the National Academy of Sciences
National Academy of Sciences
Whenever a genetically homogenous population of bacterial cells is exposed to antibiotics, a tiny fraction of cells survives the treatment,the phenomenon known as bacterial persistence [G.L. Hobby et al.,
Exp. Biol. Med. 50, 281?285 (1942); J. Bigger, The Lancet 244, 497?
500 (1944)]. Despite its biomedical relevance, the origin of the phenomenon is still unknown, and as a rare, phenotypically resistant subpopulation, persisters are notoriously hard to study and define.
Using computerized tracking we show that persisters are small at birth and slowly replicating. We also determine that the highpersister mutant strain of Escherichia coli, HipQ, is associated with the phenotype of reduced phenotypic inheritance (RPI). We identify the gene responsible for RPI, ydcI, which encodes a transcription
factor, and propose a mechanism whereby loss of phenotypic inheritance causes increased frequency of persisters. These results provide insight into the generation and maintenance of phenotypic
variation and provide potential targets for the development of therapeutic strategies that tackle persistence in bacterial infections.