Investigation into the Glycosylation Mechanism of Polyglycerolphosphate Lipoteichoic Acid in Firmicutes Matthew G. Percy Department of Medicine Imperial College London December 2014 A thesis submitted for the degree of Doctor of Philosophy Abstract Lipoteichoic acid (LTA) constitutes a major component of the cell wall in Gram- positive bacteria belonging to the phylum Firmicutes. The chemical structure of LTA varies between different bacteria. Type I LTA, a polyglycerolphosphate polymer often further decorated with D-alanine and glycosyl moieties, is linked to the outside of the membrane via a glycolipid anchor, and is one of the most common and best characterized structures. While the D-alanyl modification is known to protect bacteria against the action of cationic antimicrobial peptides, the function of the glycosyl modification has never been determined, and the genes involved in this process are unknown. In this work, a bioinformatics approach was used to identify candidate genes encoding glycosyltransferases involved in the LTA glycosylation process in Listeria monocytogenes and Bacillus subtilis. Mutant strains with deletions in these genes were constructed and the absence of glycosyl modifications on LTA confirmed by nuclear magnetic resonance experiments. Using strains lacking glycosyl modification on their LTA, a possible role of this modification in the stress response to NaCl, H2O2, and ethanol was examined. Previous studies on L. monocytogenes suggested that LTA acts as an anchor for cell wall proteins containing GW domains, so named because they possess a glycine - tryptophan dipeptide. In this work, a role of the glycosyl modification on LTA in the retention of the GW domain proteins was explored. However, not only was it found that the glycosyl groups on LTA were dispensable for the binding of GW domain proteins, but also that these proteins were still retained in the cell wall of a L. monocytogenes strain completely lacking LTA. Further experiments using purified GW domain proteins and purified peptidoglycan in binding assays, suggested that GW domain containing proteins are retained in the cell wall by direct binding to the peptidoglycan structure. In summary, this thesis explores possible roles of the glycosyl modification to LTA. 2 Declaration of Authorship I certify that this thesis entitled "Investigation into the Glycosylation Mechanism of Polyglycerolphosphate Lipoteichoic Acid in Firmicutes" is written entirely by myself and that the research to which it refers to is my own. I confirm that all main sources of help have been acknowledged and that any ideas or quotations from the work of others have always clearly been referenced. Matthew G. Percy 3 Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work’ 4 Acknowledgements First and foremost, I would like to thank Angelika for being my supervisor. I am grateful for all the support and guidance she has given me throughout my PhD, and for her help in taking the next step towards my future career. I have learned an immense amount during my time as her student and I would not be where I am without her help and perseverance. It has been a privilege to work in her lab. I also thank CIPM for funding me during my PhD. I would also like to thank my clinical mentor Shiranee, for her support throughout my time at Imperial. It has not always been an easy path, and your advice and guidance has been invaluable. In particular, thank you for giving me opportunities at Imperial and supporting me in my future career. Next, I would like to thank the people that made the lab such a fun place to work. I especially want to thank Becca and Alex as, without your advice through my time in the lab and afterwards during writing, I would never have finished this PhD. Thanks also to the other members of the Gründling lab past and present, in particular Nathalie, Tharsh, Ivan, Lisa, and Lauren, who have endured my unique sense of humour and exhaustive discussions about how to find a phenotype. I would also like to thank the rest of CMMI 3 and 4, in particular Ellen, Dan, Jamie, Sophie and everyone who was up for a drink at the end of a bad day. Finally, I would like to thank my family and friends. Most of all I want to thank my wonderful girlfriend Gemma, for her support and for keeping up my morale when the going got tough. I could not have reached the end without you. 5 Table of Contents Abstract Declaration of Authorship .................................................................................................. 3 Copyright Declaration ......................................................................................................... 4 Acknowledgements ............................................................................................................ 5 List of figures .................................................................................................................... 14 Chapter 1: Introduction .................................................................................................... 18 1.1 Staphylococcus aureus .............................................................................................. 19 1.2 Listeria monocytogenes ............................................................................................. 19 1.3 Bacillus subtilis .......................................................................................................... 20 1.4 The Gram-positive cell wall ........................................................................................ 21 1.4.1 Peptidoglycan structure and synthesis in Gram-positive bacteria ........................ 21 1.4.2 Cell wall anchoring mechanisms of proteins in L. monocytogenes ...................... 24 1.4.2.1 GW domain containing proteins in L. monocytogenes .................................. 25 1.5 The teichoic acids ...................................................................................................... 27 1.6 Wall teichoic acid ....................................................................................................... 28 1.7 Lipoteichoic acid ........................................................................................................ 30 1.7.1 Type I LTA structure and synthesis ..................................................................... 30 1.7.2 Complex LTAs in Gram-positive bacteria ............................................................ 35 1.7.3 Pneumococcal LTA structure and synthesis ........................................................ 36 1.8 LTA modifications ...................................................................................................... 39 1.8.1 D-alanylation of lipoteichoic acid ......................................................................... 39 1.8.2 Glycosyl modification of Type I LTA .................................................................... 42 1.8.3 Choline modification to type IV lipoteichoic acid .................................................. 48 1.9 Function of LTA and its clinical applications ............................................................... 51 1.10 Aims and objectives of this study ............................................................................. 54 Chapter 2: Materials and methods ................................................................................... 55 2.1 Bacterial strains, growth and storage conditions ........................................................ 56 2.2 DNA manipulation techniques .................................................................................... 57 2.2.1 Plasmid purification from E. coli .......................................................................... 57 2.2.2 Preparation of chromosomal DNA ....................................................................... 57 2.2.2.1 Isolation of chromosomal DNA from S. aureus ............................................. 57 6 2.2.2.2 Isolation of chromosomal DNA from Listeria monocytogenes ....................... 57 2.2.2.3 Isolation of chromosomal DNA from Bacillus subtilis ..................................... 58 2.2.3 Estimation of DNA concentrations ....................................................................... 58 2.2.4 Separation of DNA by agarose gel electrophoresis ............................................. 58 2.2.5 Standard PCR reaction conditions ...................................................................... 58 2.2.6 Restriction enzyme digestion of DNA .................................................................. 59 2.2.7 Ligation of DNA fragments .................................................................................. 59 2.2.8 Recombination with pKOR1 ................................................................................ 60 2.2.9 Sequencing ......................................................................................................... 60 2.2.10 Transformation of chemically competent E. coli cells ........................................ 60 2.2.11 Transformation of S. aureus strains ..................................................................