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Phosphoglycerate Kinase and Phosphoenolpyruvate Synthase of the Enteric Pathogen Helicobacter Pylori

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Phosphoglycerate Kinase and Phosphoenolpyruvate Synthase of the Enteric Pathogen Helicobacter Pylori Phosphoglycerate kinase and phosphoenolpyruvate synthase of the enteric pathogen Helicobacter pylori Thesis submitted for the degree of Doctor of Philosophy At the University of Leicester By Aziz Yasir Hasan Al-Ethari Department of Molecular and Cell Biology University of Leicester June 2018 Phosphoglycerate kinase and phosphoenolpyruvate synthase of the enteric pathogen Helicobacter pylori Aziz Yasir Hasan Al-Ethari Abstract Helicobacter pylori is a globally distributed enteric pathogen implicated in several serious diseases. Understanding the genetics and metabolism of the pathogen is of significant importance to developing new therapies for eradication. However, its metabolism is poorly characterised. The genome lacks coding sequences of some key glycolytic enzymes, however the gluconeogenic enzymes fructose-1,6-bisphosphatase and phosphoenolpyruvate synthase (hpPPSA) are present. This suggests H. pylori uses the glycolytic/gluconeogenic pathway for anabolic biosynthesis rather than for catabolic energy production. This study examines the structure and function of hpPPSA and phosphoglycerate kinase (hpPGK) and investigates the conditional essentiality of these genes, which were identified by in silico double deletion mutational studies of H. pylori. The ppsA and pgk mutants (with controls) were constructed using experimental knock out strategies, and their role in synthetic lethality was investigated. The ppsA-mutated allele alone showed evidence of essentiality. The Krebs cycle in H. pylori deviates from the text book examples such as in humans and E. coli, thus pgk may be essential alone or in combination with other enzymes. The X-ray crystal structure of apo hpPGK was determined and compared to human PGK. Structural superposition showed that both the substrate and the nucleotide binding residues are well conserved. Four sulphate ions were identified bound in the hpPGK dimer. The positions of these molecules allowed the path of phosphoryl transfer during catalysis to be modelled. The enzyme was further characterised using kinetic techniques and compared to homologous enzymes. Prediction of the hpPPSA structure by homology modelling analysis located the essential His and Cys catalytic conserved residues in their respective domains. Superimposition of the N-terminal ATP binding domain superposition showed that these residues forming this binding site are well conserved. Understanding H. pylori metabolism may provide directions for the development of therapeutics. I Acknowledgements It would not have been possible to write this thesis without the help and support of kind people, only some of whom it is possible to give particular mention here. I am grateful to my supervisor Prof. Peter Moody whose expertise, understanding, guidance and support made it possible for me to work on a topic that was of great interest to me. It was a pleasure working under his supervision and learning from him as a scientist and human. I would like to express the deepest appreciation to my committee members Prof. Julian Ketley (for his suggestions during and after meetings and for giving me the opportunity to do the genetics part of my research in his lab and under his supervision) and Dr. Mark Leyland (for his suggestions during and after meetings). I would like to acknowledge all the members of Prof. Peter Moody’s research group for assistance and advice and for providing me with a supportive and friendly environment to work in. In particular, I would like to thank Dr. Adnan Ayna, Dr. Samah Almehmadi and Dr Hanna Kwon. My thanks also go to Prof. Julian Ketley’s lab, in particular Dr. Mohamed Elamen Fadel and Dr Richard Haigh for their unrestrained help during my genetic experimental work of this research. I would also like to thank Prof. John Schwabe’s lab especially Dr. Louise Fairall (for her advices in protein crystallisation trials and helping in data collection from Diamond), Prof. Emma Raven’s lab, in particular Dr Sofia M. Kapetanaki (for her support and providing all the experimental material when needed), Dr Mohammed El-Mezgueldi and Dr Jaswir Basran (for helping on enzyme kinetics), Dr Ralf Schmid (for useful discussions on bioinformatics work), Sura M. Abbood (for the scientific discussions and support) and lastly Dr. Xiowen Yang of blessed memory (for advises during and after cloning in expression vectors). My acknowledgment would not be complete without giving special thanks to my beloved wife Hawraa Mahdi Hussein Al-Ethari and our beloved children Tabarek, Mohammed Sadeq, Baha Alddin and Amina. I could not make this work without your support. Thanks for always believing in me and for being there for me every single day of my life. For all that, thank you so very much. Finally, my gratitude is to the Ministry of Higher Education and Scientific Research in Iraq for providing the funding for this PhD. II To my beloved Father and mother, Wife and children, Brothers and sisters, I dedicate this thesis. III Table of Contents Abstract ............................................................................................................................. I Acknowledgements .......................................................................................................... II Table of Contents ........................................................................................................... IV Abbreviations................................................................................................................... XI Chapter 1 Introduction ..................................................................................................... 1 1.1 Introduction ............................................................................................................ 1 1.2 Helicobacter pylori .................................................................................................. 1 1.2.1 Taxonomy ......................................................................................................... 2 1.2.2 Morphology and growth requirements ........................................................... 4 1.2.3 Stomach colonisation and acid acclimation ..................................................... 5 1.3 H. pylori diseases and treatment ............................................................................ 8 1.3.1 Epidemiology .................................................................................................... 8 1.3.2 Transmission .................................................................................................. 10 1.3.3 Disease spectrum ........................................................................................... 11 1.3.3.1 Gastritis .................................................................................................... 11 1.3.3.2 Peptic ulcer disease ................................................................................. 12 1.3.3.3 Atrophic gastritis, intestinal metaplasia and gastric cancer ................... 13 1.3.3.4 MALT lymphoma ..................................................................................... 13 1.3.4 Therapy regimes ............................................................................................. 14 1.3.5 Emergence of antibiotic resistance ................................................................ 16 IV 1.3.5.1 Clarithromycin resistance ........................................................................ 17 1.3.5.2 Metronidazole ......................................................................................... 17 1.4 Genome and strain diversity of H. pylori .............................................................. 19 1.5 Metabolism within H. pylori ................................................................................. 20 1.5.1 Carbohydrate metabolism ............................................................................. 21 1.5.2 Pyruvate metabolism ..................................................................................... 25 1.5.3 The Krebs cycle ............................................................................................... 27 1.6 Phosphoglycerate kinase and Phosphoenolpyruvate synthase ......................... 30 1.6.1 Phosphoglycerate kinase ............................................................................... 30 1.6.2 PGK as drug target .......................................................................................... 32 1.6.3 Phosphoenolpyruvate synthase ..................................................................... 32 1.6.4 Gene essentiality of ppsA ............................................................................... 35 1.7 Aims of this study .................................................................................................. 37 Chapter 2. Materials and methods................................................................................. 39 2.1 Materials ............................................................................................................... 39 2.1.1 Chemicals and reagents ................................................................................. 39 2.1.2 Chromatographic media and membranes ..................................................... 39 2.1.3 Crystallisation accessories .............................................................................. 39 2.2 Bacterial culture conditions and storage .............................................................. 40 2.3 Chromosomal DNA extraction .............................................................................
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