Molecular and Biochemical Studies of a Mycobacterial

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Molecular and Biochemical Studies of a Mycobacterial Ana Branco Maranha Tiago Molecular and biochemical studies of a mycobacterial maltokinase and a unique octanoyltransferase: Towards reconstruction of the pathway for methylglucose lipopolysaccharides biosynthesis Tese de Doutoramento em Biociências, ramo de especialização em Microbiologia, realizada sob orientação científica do Doutor Nuno Miguel da Silva Empadinhas e apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra 2015 Cover image: Illustration of a Ziehl-Neelsen-stained photomicrograph sputum smear sample containing Mycobacterium tuberculosis. Courtesy of Centers for Disease Control Public Health Image Library (CDC PHIL). i ii Agradecimentos Gostaria de agradecer em primeiro lugar ao Doutor Nuno Empadinhas por me ter acolhido no grupo e pela confiança que depositou em mim. Por contrariar as minhas dúvidas com uma constante atitude positiva e, ao longo destes anos, sempre me ter encorajado, orientado, ensinado e incentivado a aprender. Especificamente, gostaria de agradecer as muitas horas dispendidas a rever este manuscrito. A todos os meus companheiros no laboratório de micobacteriologia, os que me acompanham no dia-a-dia e aos que já me acompanharam. Ao Vítor Mendes, com quem iniciei o trabalho de bancada, que me transmitiu o entusiasmo pela investigação e que tanto me ensinou. Um obrigada muito especial à Susana Alarico pela sua infindável paciência e carinho. À Mafalda Costa minha “consultora de imagem”, à Daniela Costa, à Rita Pereira, à Andreia Lamaroso e ao Diogo Reis, pela ajuda, boa disposição e brincadeiras. I am profoundly grateful to Prof. Anthony Clarke for opening his lab to me, for sharing his expertise and making me feel welcome. To everyone at the Clarke Lab (U of G, Canada) my deepest thanks for taking such good care of me during my stay. A special thanks to Patrick Moynihan for all I learned from him. À Doutora Sandra de Macedo-Ribeiro e ao Doutor Pedro Pereira e aos seus grupos de trabalho no IBMC no Porto, que sempre tão bem me receberam, que me incluiram no quotidiano do laboratório como se estivesse “em casa.” Um especial obrigada à Joana Fraga que alinha sempre nas minhas “loucuras laboratoriais”. Não poderia deixar de expressar a minha enorme gratidão à Doutora Rita Ventura e a toda a sua equipa no ITQB, Oeiras, que foram sempre incansáveis tanto a esclarecer qualquer dúvida que eu como bióloga, não-química, pudesse ter, como a tentar satisfazer a minha insaciável necessidade de substratos. À Fundação para a Ciência e a Tecnologia por me ter concedido o apoio financeiro através de uma Bolsa de Doutoramento (SFRH/BD/74845/2010) e pelo suporte financeiro para realização do trabalho experimental através dos projetos EU-FEDER Programa COMPETE FCOMP-01-0124- FEDER-014321 (PTDC/BIA-PRO/110523/2009), FCOMP-01-0124-FEDER-028359 (PTDC/BIA- MIC/2779/2012), FCOMP-01-0124-FEDER-037276 [PEst-C/SAU/LA0001/2013-2014] e UID/NEU/04539/2013. iii I would also like to acknowledge the support of the Mizutani Foundation for Glycoscience, Japan, through grant 120123 to Dr. Nuno Empadinhas and also the Strategic Programme InovC-2014 and DITS (Divisão de Inovação e Transferências do Saber), University of Coimbra. Uma especial nota de agradecimento à Prof. Doutora Paula Veríssimo do Departamento de Ciências da Vida da UC, pela sua constante disponibilidade e pelas profícuas discussões científicas sobre proteínas e enzimologia. Gostaria também de agradecer ao Doutor Tiago Faria, que tantas vezes me ajudou e ao Doutor Hugo Osório pelas análises por MALDI-TOF. Assinalo também a ajuda preciosa da equipa técnica do CNC. Não poderia deixar de agradecer a todos os colegas dos diversos laboratórios por onde passei. Aos colegas do laboratório de microbiologia no patronato, obrigada por me terem ensinado que a ciência não é uma actividade solitária. Aos colegas da microbiologia do 3º piso da FMUC, obrigada por terem tornado a nossa transição tão fácil. Um obrigada especial à Lisa, minha companheira de “infortúnio”. Ao “pessoal do almoço” pelas conversas estimulantes, as conversas tontas, a amizade e por tantas vezes me guiarem neste labirinto que é a vida científica. Aos meus amigos que têm uma paciência infinita para me aturarem, que me perguntam pelo meu trabalho, mesmo quando não percebem metade do que lhes estou a explicar, que me incitam sempre a continuar e que me ajudam a manter sã. À minha família, especialmente à minha mãe e aos meus irmãos, o meu grande sistema de apoio, que seguram as pontas do meu mundo. A todos os que de alguma forma contribuiram para a elaboração deste trabalho o meu muito obrigada. Ana Coimbra, Agosto 2015 iv Table of contents Abstract ................................................................................................................................ vii Resumo ................................................................................................................................ viii Abbreviation list .................................................................................................................... ix List of genes and enzymes ..................................................................................................... xi List of figures ....................................................................................................................... xiii List of tables ......................................................................................................................... xv Chapter 1 General Introduction ............................................................................................ 1 Chapter 2 Structural and biochemical characterization of Mycobacterium vanbaalenii maltokinase ........................................................................................................ 33 Chapter 3 Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides ............................................................................................ 51 Chapter 4 Concluding remarks and future directions ......................................................... 83 References ........................................................................................................................... 91 v vi Abstract Mycobacteria belong to an extremely diversified bacterial genus that contains both saprophytic organisms and dangerous pathogens such as Mycobacterium leprae and Mycobacterium tuberculosis, the etiological agents of leprosy and tuberculosis, respectively. Tuberculosis (TB) is one of the deadliest diseases caused by a single infectious agent and its eradication is considered a major global health priority. Nonetheless the lack of proper access to medical care in developing countries, the burden of chronic illnesses in developed countries and the emergence of multidrug-resistant strains has hindered the process towards that goal. Mycobacteria possess a distinctive lipid-rich cell envelope to which they owe much of their pathogenicity. Among the unique molecules synthesized by mycobacteria there are two intracellular polymethylated polysaccharides (PMPSs) that likely assume a helical conformation in the cytoplasm and are postulated to interact with fatty acid synthase I (FAS-I) to modulate fatty acid synthesis. PMPSs can be polysaccharides of 3-O-methylmannose (MMPs) or lipopolysaccharides of 6-O-methylglucose (MGLPs). These molecules were discovered in the 1960’s but only recently with the unveiling of the mycobacterial genome was it possible to uncover more aspects of their biogenesis. MGLPs in particular have been the target of renewed attention because they probably represent the only type of PMPSs present in the slowly-growing phenotype frequently associated with pathogenic mycobacteria such as M. tuberculosis, while the distribution of MMP appears to be almost exclusively restricted to rapidly-growing mycobacteria. In addition, some genes involved in the MGLP biosynthetic pathway have been considered essential for M. tuberculosis growth, making their products potential drug targets. Recently a novel metabolic pathway (GlgE pathway) has been uncovered, connecting trehalose and glycogen through the intermediate metabolite maltose-1-phosphate. This pathway may also represent an alternative route for polymerization of the seemingly vital mycobacterial MGLP that has a glycogen-like arrangement of glucose units in the main chain. We pursued the identification and characterization of enzymes with probable involvement in the MGLP pathway and simultaneously proposed to be essential for M. tuberculosis growth. As a consequence of functional and genomic context surveys two genes were selected for study. Gene Rv0127 encoding a maltokinase for the synthesis of maltose-1-phosphate and Rv2418c of unknown function, but located in an operon containing the atypical glucosyl-3- phosphoglycerate phosphatase responsible for the production of the MGLP primer, glucosylglycerate. The work presented in this thesis contributes for the elucidation of the maltokinase structure and the functional characterization of a novel sugar octanoyltransferase adding new insights into an intricate and vital pathway of mycobacterial glycobiology. vii Resumo As micobactérias pertencem a um género bacteriano extremamente diverso que contém bactérias saprófitas e patogénicas. Das patogénicas destacam-se Mycobacterium leprae e Mycobacterium tuberculosis, os agentes etiológicos da lepra e da tuberculose, respectivamente. A tuberculose encontra-se entre as doenças
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