Dany Beste

Dr Dany Beste

Senior Lecturer in Microbial Metabolism
Bsc (Hons) FIBMS MSc PhD
+44 (0)1483 686785
02 AX 01

Academic and research departments

School of Biosciences and Medicine, Faculty of Health and Medical Sciences.

Biography

University roles and responsibilities

  • Programme Leader for MSc Medical Microbiology

    Affiliations and memberships

    Acid Fast Club
    Member
    Society of Microbiology
    Elected Member of the Prokaryotic Division

    Research

    Research interests

    Indicators of esteem

    • Editorial Board of Microbiology

      Supervision

      Postgraduate research supervision

      My teaching

      My publications

      Highlights

      Borah, Khushboo, Mendum, Tom A., Hawkins, Nathaniel, Ward, Jane, Beale, Michael, Larrouy-Maumus, Gerald, Bhatt, Apoorva, Pichler, Harald, Moulin, Martine, Haertlein, Michael, Forsyth, Trevor, Noack, Stephan, Goulding, Celia McFadden, Johnjoe, Beste, Dany J. V. Metabolic flux partitioning between the TCA cycle and glyoxylate shunt combined with a reversible methyl citrate cycle provide nutritional flexibility for Mycobacterium tuberculosis. bioRxiv 2021.01.29.428863; doi: https://doi.org/10.1101/2021.01.29.428863

      Burley K. H., Cuthbert B. J., Basu P., Newcombe J. , Irimpan E. M. , Quechol R. , Foik I. P. , Mobley D. L. , Beste D. J. V. , Goulding C. W. Structural and Molecular Dynamics of Mycobacterium tuberculosis Malic Enzyme, a Potential Anti-TB Drug Target. ACS Infect Dis. 2021 Jan 8;7(1):174-188. doi: 10.1021/acsinfecdis.0c00735. Epub 2020 Dec 23. PMID: 33356117.

      Mackenzie, Jared S., Lamprecht, Dirk A., Asmal, Rukaya, Adamson, John H. ,Borah, Khushboo, Beste, Dany J. V., Lee, Bei Shi, Pethe, Kevin, Rousseau, Simon, Krieger, Inna, Sacchettini, James C.,Glasgow, Joel N.,Steyn, Adrie J. C.Bedaquiline reprograms central metabolism to reveal glycolytic vulnerability in Mycobacterium tuberculosis. Nat Commun 11, 6092 (2020). https://doi.org/10.1038/s41467-020-19959-4.

      Publications

      Sacchettini James C, Glasgow Joel N, Steyn Adrie J C, Mackenzie Jared S, Lamprecht Dirk A, Asmal Rukaya, Adamson John H, Borah Khushboo, Beste Dany J V, Pethe Kevin, Rousseau Simon, Krieger Inna, Lee Bei Shi (2020)Bedaquiline reprograms central metabolism to reveal glycolytic vulnerability in Mycobacterium tuberculosis, In: Nature communications11(1)pp. 6092-6092
      DOI: 10.1038/s41467-020-19959-4
      The approval of bedaquiline (BDQ) for the treatment of tuberculosis has generated substantial interest in inhibiting energy metabolism as a therapeutic paradigm. However, it is not known precisely how BDQ triggers cell death in Mycobacterium tuberculosis (Mtb). Using C isotopomer analysis, we show that BDQ-treated Mtb redirects central carbon metabolism to induce a metabolically vulnerable state susceptible to genetic disruption of glycolysis and gluconeogenesis. Metabolic flux profiles indicate that BDQ-treated Mtb is dependent on glycolysis for ATP production, operates a bifurcated TCA cycle by increasing flux through the glyoxylate shunt, and requires enzymes of the anaplerotic node and methylcitrate cycle. Targeting oxidative phosphorylation (OXPHOS) with BDQ and simultaneously inhibiting substrate level phosphorylation via genetic disruption of glycolysis leads to rapid sterilization. Our findings provide insight into the metabolic mechanism of BDQ-induced cell death and establish a paradigm for the development of combination therapies that target OXPHOS and glycolysis.
      Saunders Elizabeth (2020)Metabolic analysis of solventogenic clostridium saccharoperbutylacetonicum N1-4 (HMT). University of Surrey
      DOI: 10.15126/thesis.00852363

      The market for solvent production is predicted to reach $43.4 billion in 2018, with n-butanol having over 20% market share value where n-Butanol is the chemical precursor of several industrially important products, such as butyl-acetate, butyl-acrylate, glycol-ethers, and plasticisers. Butanol is currently produced from crude oil, and therefore in light of dwindling fossil fuel reserves, and more importantly, the need for green and clean production processes, synthesis of bio-butanol from biomass using Clostridia represents a viable and desirable alternative method.

      This project focuses on the metabolic and physiologic characterisation of the acetone-butanol-ethanol (ABE) producing species Clostridium saccharoperbutylacetonicum (Csb). A minimal medium for Csb was defined based on literature data, modified by the addition of glutamate to support growth. Interestingly, batch cultures using this medium showed that Csb was able to grow and produce butanol under aerobic conditions, with titres of approximately 74% of those observed under anaerobic conditions. Steady state cultures in chemostats are essential to elucidate and characterise physiological features of microorganisms. Steady state cultures of Csb were used to determine the effect of acid production on solventogenesis, bacterial growth, and energy metabolism. Studies at different pH in the range 5.5 to 6.5 showed no correlation with the onset of solventogenesis. However, the pH and the growth rate seem to influence the productivity of butanol. In those experiments, significant increases in the production rate of butanol were observed when the dilution (growth) rate increased from 0.01 h-1 to 0.03 h-1 and the pH decreased from 6.5 to 5.5. Growth is potentially linked to production rate due to an increased demand for ATP and NADH recycling.

      The use of genome scale metabolic models allows for the interpretation of metabolic and physiological changes upon changes in the culture conditions. A metabolic model of Csb was constructed based on the genome sequence of the microorganism and incorporating biomass synthesis equations specific for Csb which were constructed based on the analysis of the composition of the cells grown in the chemostat experiments, as opposed to current models that use biomass composition from related species (e.g. B. subtilis). The metabolic model was used to perform flux balance analysis to identify and interpret the changes in the distribution of metabolic fluxes that would explain the metabolic changes observed in Csb cultured under different conditions.

      This work has demonstrated the basis for the presence of monophasic solventogenesis in C. saccharoperbutylacetonicum and provided important tools (defined media, GSMN equations) to improve industrial scale production of renewable sources of carbon-based feedstocks and thus reducing reliance on crude oil.

      Beste DJV, Laing E, Bonde B, Avignone-Rossa C, Bushell ME, McFadden JJ (2007)Transcriptomic analysis identifies growth rate modulation as a component of the adaptation of mycobacteria to survival inside the macrophage, In: J BACTERIOL189(11)pp. 3969-3976 AMER SOC MICROBIOLOGY
      DOI: 10.1128/JB.01787-06
      Beste DJV, McFadden J (2012)System-level Strategies for Studying the Metabolism of Mycobacterium tuberculosis, In: Robertson BD, Wren BW (eds.), Systems Microbiology: Current Topics and Applications(7)
      Brian D. Robertson and Brendan W. Wren
      Lawrence ER, Arias CA, Duke B, Beste D, Broughton K, Efstratiou A, George RC, Hall LMC (2000)Evaluation of serotype prediction by cpsA-cpsB gene polymorphism in Streptococcus pneumoniae, In: JOURNAL OF CLINICAL MICROBIOLOGY38(4)pp. 1319-1323 AMER SOC MICROBIOLOGY
      Beste DJV, McFadden J (2013)Metabolism of Mycobacterium tuberculosis, In: Systems biology of tuberculosis(4)
      Bonde BK, Beste DJV, Laing E, Kierzek AM, McFadden J (2011)Differential Producibility Analysis (DPA) of Transcriptomic Data with Metabolic Networks: Deconstructing the Metabolic Response of M. tuberculosis, In: PLOS COMPUTATIONAL BIOLOGY7(6)ARTN epp. ?-? PUBLIC LIBRARY SCIENCE
      DOI: 10.1371/journal.pcbi.1002060
      Beste DJV, McFadden J (2010)System-level strategies for studying the metabolism of Mycobacterium tuberculosis, In: Molecular Biosystems6(12)pp. 2363-2372 Royal Society of Chemistry
      DOI: 10.1039/C003757P
      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 M. tuberculosis’s metabolism and pathogenesis have 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 allow metabolism to be studied on a genome scale. Here we discuss the progress being made in applying system level approaches to studying the metabolism of this important pathogen. The information from these studies will fundamentally change our approach to tuberculosis research and lead to new targets for therapeutic drugs and vaccines.
      Bakatselou C, Beste D, Kadri AO, Somanath S, Clark CG (2003)Analysis of genes of mitochondrial origin in the genus Entamoeba, In: JOURNAL OF EUKARYOTIC MICROBIOLOGY50(3)pp. 210-214 SOC PROTOZOOLOGISTS
      DOI: 10.1111/j.1550-7408.2003.tb00119.x
      Beste DJ, Espasa M, Bonde B, Kierzek AM, Stewart GR, McFadden J (2009)The genetic requirements for fast and slow growth in mycobacteria., In: PLoS One4(4)e5349 PLoS
      DOI: 10.1371/journal.pone.0005349
      Mycobacterium tuberculosis infects a third of the world's population. Primary tuberculosis involving active fast bacterial replication is often followed by asymptomatic latent tuberculosis, which is characterised by slow or non-replicating bacteria. Reactivation of the latent infection involving a switch back to active bacterial replication can lead to post-primary transmissible tuberculosis. Mycobacterial mechanisms involved in slow growth or switching growth rate provide rational targets for the development of new drugs against persistent mycobacterial infection. Using chemostat culture to control growth rate, we screened a transposon mutant library by Transposon site hybridization (TraSH) selection to define the genetic requirements for slow and fast growth of Mycobacterium bovis (BCG) and for the requirements of switching growth rate. We identified 84 genes that are exclusively required for slow growth (69 hours doubling time) and 256 genes required for switching from slow to fast growth. To validate these findings we performed experiments using individual M. tuberculosis and M. bovis BCG knock out mutants. We have demonstrated that growth rate control is a carefully orchestrated process which requires a distinct set of genes encoding several virulence determinants, gene regulators, and metabolic enzymes. The mce1 locus appears to be a component of the switch to slow growth rate, which is consistent with the proposed role in virulence of M. tuberculosis. These results suggest novel perspectives for unravelling the mechanisms involved in the switch between acute and persistent TB infections and provide a means to study aspects of this important phenomenon in vitro.
      Beste DJV, McFadden J (2010)Systems biology of the metabolism of Mycobacterium tuberculosis, In: Biochemical Society Transactions38(5)pp. 1286-1289 PORTLAND PRESS LTD
      DOI: 10.1042/BST0381286
      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.
      Beste DJV, Peters J, Hooper T, Avignone Rossa CA, Bushell ME, McFadden JJ (2005)Compiling a molecular inventory for Mycobacterium bovis BCG at two growth rates: Evidence for growth rate-mediated regulation of ribosome biosynthesis and lipid metabolism, In: Journal of Bacteriology187(5)pp. 1677-1684 American Society of Microbiology
      DOI: 10.1128/JB.157.5.1677-1684.2005
      An experimental system of Mycobacterium tuberculosis growth in a carbon-limited chemostat has been established by the use of Mycobacterium bovis BCG as a model organism. For this model, carbon-limited chemostats with low concentrations of glycerol were used to simulate possible growth rates during different stages of tuberculosis. A doubling time of 23 h (D 0.03 h 1) was adopted to represent cells during the acute phase of infection, whereas a lower dilution rate equivalent to a doubling time of 69 h (D 0.01 h 1) was used to model mycobacterial persistence. This chemostat model allowed the specific response of the mycobacterial cell to carbon limitation at different growth rates to be elucidated. The macromolecular (RNA, DNA, carbohydrate, and lipid) and elemental (C, H, and N) compositions of the biomass were determined for steady-state cultures, revealing that carbohydrates and lipids comprised more than half of the dry mass of the BCG cell, with only a quarter of the dry weight consisting of protein and RNA. Consistent with studies of other bacteria, the specific growth rate impacts on the macromolecular content of BCG and the proportions of lipid, RNA, and protein increased significantly with the growth rate. The correlation of RNA content with the growth rate indicates that ribosome production in carbon-limited M. bovis BCG cells is subject to growth rate-dependent control. The results also clearly show that the proportion of lipids in the mycobacterial cell is very sensitive to changes in the growth rate, probably reflecting changes in the amounts of storage lipids. Finally, this study demonstrates the utility of the chemostat model of mycobacterial growth for functional genomic, physiology, and systems biology studies.
      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, In: GENOME BIOLOGY8(5)ARTN rpp. ?-? BIOMED CENTRAL LTD
      DOI: 10.1186/gb-2007-8-5-r89
      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, In: PLOS ONE8(9)ARTN e7591 PUBLIC LIBRARY SCIENCE
      DOI: 10.1371/journal.pone.0075913
      Wallqvist Anders, López-Agudelo Víctor A., Mendum Tom A., Laing Emma, Wu Huihai, Baena Andres, Barrera Luis F., Beste Dany J. V., Rios-Estepa Rigoberto (2020)A systematic evaluation of Mycobacterium tuberculosis Genome-Scale Metabolic Networks, In: PLOS Computational Biology16(6)e1007533 Public Library of Science
      DOI: 10.1371/journal.pcbi.1007533
      Metabolism underpins the pathogenic strategy of the causative agent of TB, Mycobacterium tuberculosis (Mtb), and therefore metabolic pathways have recently re-emerged as attractive drug targets. A powerful approach to study Mtb metabolism as a whole, rather than just individual enzymatic components, is to use a systems biology framework, such as a Genome-Scale Metabolic Network (GSMN) that allows the dynamic interactions of all the components of metabolism to be interrogated together. Several GSMNs networks have been constructed for Mtb and used to study the complex relationship between the Mtb genotype and its phenotype. However, the utility of this approach is hampered by the existence of multiple models, each with varying properties and performances. Here we systematically evaluate eight recently published metabolic models of Mtb-H37Rv to facilitate model choice. The best performing models, sMtb2018 and iEK1011, were refined and improved for use in future studies by the TB research community.
      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., In: Chem Biol20(8)pp. 1012-1021 Elsevier
      DOI: 10.1016/j.chembiol.2013.06.012
      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.
      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.
      Fotopoulou N, Tassios PT, Beste DV, Ioannidou S, Efstratiou A, Lawrence ER, Papaparaskevas J, George RC, Legakis NJ (2003)A common clone of erythromycin-resistant Streptococcus pneumoniae in Greece and the UK, In: CLINICAL MICROBIOLOGY AND INFECTION9(9)pp. 924-929
      DOI: 10.1046/j.1469-0691.2003.00689.x
      Borah Khushboo, Beyß Martin, Theorell Axel, Wu Huihai, Basu Piyali, Mendum Tom A., Nӧh Katharina, Beste Dany J.V., McFadden Johnjoe (2019)Intracellular Mycobacterium tuberculosis Exploits Multiple Host Nitrogen Sources during Growth in Human Macrophages, In: Cell Reports29(11)pp. 3580-3591.e4 Elsevier
      DOI: 10.1016/j.celrep.2019.11.037
      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.
      Borah Khushboo, Girardi Karina do Carmo de Vasconcelos, Mendum Thomas A., Lery Leticia Miranda Santos, Beste Dany J.V., Lara Flavio Alves, Pessolani Maria Cristina Vidal, McFadden Johnjoe, Boyle Jon P. Intracellular Mycobacterium leprae Utilizes Host Glucose as a Carbon Source in Schwann Cells, In: Boyle Jon P. (eds.), mBio10(6) American Society for Microbiology
      DOI: 10.1128/mBio.02351-19
      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.
      Ward JL, Beale MH, Noack S, Nöh K, Kruger NJ, Ratcliffe RG, McFadden J, Beste DJ, Bonde B, Hawkins N (2011)¹³C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation., In: PLoS Pathog7(7)pp. e1002091-? PLoS
      DOI: 10.1371/journal.ppat.1002091
      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 CO₂ into biomass. As the human host is abundant in CO₂ this finding requires further investigation in vivo as CO₂ 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.
      Beste DJV, Laing E, Bonde B, Avignone-Rossa C, Bushell ME, McFadden JJ (2007)Transcriptomic analysis identifies growth rate modulation as a component of the adaptation of mycobacteria to survival inside the macrophage, In: JOURNAL OF BACTERIOLOGY189(11)pp. 3969-3976 AMER SOC MICROBIOLOGY
      DOI: 10.1128/JB.01787-06
      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, In: Journal of Biological Chemistry293(15)pp. 5695-5704 American Society for Biochemistry and Molecular Biology
      DOI: 10.1074/jbc.RA118.001839
      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.
      Al Hashmi Salim M. (2016)Studies on the stress responses of M. tuberculosis : tmRNA and α-crystallins.
      Mycobacterium tuberculosis remains a major human health problem killing millions of people around the world. Therefore, the need for an in depth understanding of its pathogenicity is very important to enable rational development of new control strategies.  Not all M. tuberculosis infected individuals progress to active disease at the time of primary infection as some carry an asymptomatic persistent infection that may reactivate later in life to cause disease. Despite a great deal of M. tuberculosis research, the ability of M. tuberculosis to cause long-term persistent infection in immune-competent hosts is poorly understood. When M. tuberculosis is inside the body it faces many environmental stress conditions including hypoxia, starvation and oxidative stress that damage proteins, DNA and other molecules. Gene expression studies have identified many bacterial genes that are differentially regulated when M. tuberculosis is subjected to such environmental stresses. This thesis focusses on three of these genes, the ssrA gene, which encodes the tmRNA molecule involved in ribosome recycling and degradation of denatured proteins, and two genes encoding -crystallin molecular chaperones (acr1 and acr2). This study was designed to achieve two aims: (1) to examine the role of tmRNA in translational control and protein homeostasis in stressed mycobacteria; and (2) to understand the roles of Acr1 and Acr2 in the stress response of M. tuberculosis and to reveal the degree of redundancy between them. In this thesis it was shown that ssrA/tmRNA is essential for bacterial viability as it was not possible to delete the gene unless a second fully functional copy was introduced elsewhere in the genome. The results suggested that the protease tagging function of tmRNA is essential alongside its role in ribosome recycling. A recombinant His-tagged tmRNA was expressed in the mycobacteria in an attempt to identify if tmRNA is directly involved in the translation of stress proteins. Expression of the His-tagged tmRNA was detrimental to the cell and appeared to preclude successful tagging of tmRNA substrate polypeptides. Thus there was insufficient evidence to support the hypothesis. Ultrastructural localisation of Acr1 and Acr2 by immuno-electron microscopy and Western blotting of subcellular fractions of mycobacteria showed that Acr1 and Acr2 were localised in different parts of the cell. Assay of the phenotypes of single and double deletion mutants of Acr1 and Acr2 in different in vitro conditions failed to show any evidence that the two chaperones are functionally redundant. Indeed, experiments on intracellular infection of macrophages showed no phenotypic consequences resulted from loss of Acr1 but deletion of Acr2 resulted in an altered cytotoxic effect on the host cell.
      Alfagih Nargs (2017)Understanding the regulation of host mRNA translation initiation during Mycobacterium bovis BCG infection.
      Tuberculosis (TB) is a major health problem worldwide resulting in 1.4 million deaths, caused by Mycobacterium tuberculosis. Despite all the efforts to target and eliminate this chronic disease, the control of tuberculosis has been severely thwarted by the emergence of multidrug and extensively resistant strains. The long treatment duration and its association with various side effects result in noncompliance of the patients. To improve treatment outcomes and reduce duration of therapy, host-directed TB therapies could provide a solution for the resolution of the disease. The development of host directed therapies will be expedited by further understanding of host-mycobacterium interaction and how the pathogen hijacks host cell processes to facilitate survival. Key to this process is the regulation of host gene expression. However, very little is known about translational control by bacterial pathogens, including Mycobacterium tuberculosis and how this contributes to pathogenesis. By using Mycobacterium bovis Bacillus Calmette-Guerin (BCG) as a surrogate of Mycobacterium.tuberculosis, we aimed to dissect how Mycobacterium bovis BCG alters translation in the infected macrophages, and how the regulation of eIF4E activity participates in this response to infection. Our results suggest that mycobacterial infection induces eIF4E phosphorylation in murine macrophages. Furthermore, the kinases ERK and MNK are responsible for eIF4E phosphorylation and their activation contributes to changes in the translational state of host mRNAs, as identified by polysome profiling. These changes alter the macrophage response to mycobacteria, affecting intracellular bacterial survival and macrophage viability. As it is believed that in up to 50 % of TB exposed individuals, the infection is cleared without the involvement of the adaptive immune system, indicating that the innate immune system may be able to control or clear the infection if activated appropriately. Further testing of the mechanisms used by macrophages to keep the infection under control has been done by measuring TNFα and IL-10 production, phagosomal acidity and cellular autophagy in the presence of ERK and MNK inhibitors. We found that the activation of ERK-MNK-eIF4E pathway regulates the cytokines production, but only ERK plays a regulatory role on macrophage phagosomal acidification as well as cell autophagy. Our finding suggests that Mycobacterium bovis BCG benefits from the activated ERK-MNK- eIF4E signalling to survive inside the cell. We conclude that regulating eIF4E phosphorylation is a key component of the hostpathogen interaction during mycobacterium infection and therefore, we suggest the possibility of using selective MNK and/or ERK inhibitors as host-directed immuno-therapeutics for tuberculosis.

      Additional publications