Investigation into Glycosylation of the Aeromonas caviae Polar Flagellum Rebecca Catherine Lowry A thesis submitted for the degree of Doctor of Philosophy Department of Infection and Immunity The University of Sheffield February 2015 i Acknowledgements I have found the past three and a half years both incredibly enjoyable, and immensely challenging. There are so many people I would like to thank who have helped and encouraged me along the way. First of all I would like to start by thanking my supervisors, Dr Jonathan Shaw and Dr Graham Stafford, for their constant help and support during my PhD; and Dr Narciso Couto for all his patience and wisdom during the mass spectrometry work, whom without, Chapter 4 of this thesis would not have been possible. I would also like to thank Dr Jennifer Parker for being an amazing teacher and friend from day one of this project (and her husband Dr Matthew Hicks). A big thank you also to Dr Sabela Balboa- Mendez for all her help and support, and for the amazing Spanish feasts that have kept me going through all of this. I would also like to thank the other members of Infection and Immunity and the School of Clinical Dentistry, in particular: Jamie Hall, Helena Spiewak, Ben Harvey, Sayali Haldipurkar, Yu Hang Zhao, Hannah McMellon, Sarah Oates and Sam Harding, for so many useful discussions and coffee breaks that have really spurred me on. Also, thank you to the Department of Biological Engineering for all the support, particularly Tom Minshull and Andrew Landells for being computer and mass spec geniuses. A big thank you also to the ‘world food club’ (ie. Beth and Megan) and the Spooner girls for being wonderful people, and getting me through the demanding times. A huge thank you also to my lovely undergraduate friends, Daniel Cozens, Liz Court, Kate Naylor and Charlotte Green, who have plied me with Fizzy Fangs and dry shampoo during the thesis writing period; I hope to return the favour when you guys are going through the same thing in a few months. I would also like to thank the lovely Palmer family who are always amazing and supportive, and have made regular trips to Sheffield to cheer my day. I would especially like to thank my mum, dad and Jason, for their continuous support with everything I do, and for their constant kindness – always making sure the larders are stocked and we have wood for the fire! And finally, I would like to thank JP for everyday being incredibly supportive and understanding, I can’t put into words how grateful I am. And you still want to marry me after all of my PhD craziness! ii Abstract The bacterial flagellum is an important appendage at the bacterial cell surface, not only for motility, but for adherence to host cells, and therefore has an extensive role in bacterial colonisation and virulence. A number of pathogenic bacteria modify their flagellins with nonulosonic acids, such as Aeromonas, Campylobacter and Helicobacter species, via an O- linked glycosylation process. This modification is essential for their ability to form flagella, thus having implications in pathogen virulence. However, the role of flagellin glycosylation is currently undetermined. The mesophilic aeromonad, Aeromonas caviae, forms a constitutively expressed polar flagellum necessary for motility in liquid environments. It is thought to be a good model for glycosylation as it decorates its flagellins solely with pseudaminic acid, and contains a genetically simple system for this process to occur. The work in this thesis has explored the pathway, and role of flagellin glycosylation in this microorganism. The function of a putative deglycosylation enzyme (AHA0618) and its possible role in the fine tuning of flagellin glycosylation was examined, where it was concluded that this protein is likely to be involved in peptidoglycan crosslinking at the cell wall, and not the flagellin glycosylation pathway. This demonstrates that subtle changes to bacterial cellular morphology are able to affect bacterial behaviour. Additionally, investigations into the sites of flagellin glycosylation via mass spectrometric methods concluded that the sites of modification on A. caviae flagellins can vary. However, certain residues were found to be predominantly glycosylated, suggesting partial selectivity to the glycosylation process via the putative glycosyltransferase, Maf1. Finally, protein interaction studies have provided evidence that glycosylation is likely to occur in the cytoplasm before binding of the flagellin-specific chaperone and flagellin export. Moreover, Maf specificity investigations, together with these interaction studies have suggested that the Maf proteins may be recognising and docking to the N-terminal region of the flagellins. iii Abbreviations ACN Acetonitrile Ambic Ammonium bicarbonate Amp Ampicillin ATP Adenosine triphosphate BHIB Brain heart infusion broth Bp Base pairs cAMP Cyclic adenosine monophosphate CAP Catabolite gene activator protein CBD Chaperone binding domain CID Collision induced dissociation Cm Chloramphenicol C-terminal Carboxy terminal DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid dNTPs Deoxynucleotides DTT Dithiothreitol ETD Electron transfer dissociation GlcNAc N-acetylglucosamine Gm Gentamycin HPLC High performance liquid chromatography Hrs Hours Hz Hurtz IPTG Isopropyl-β-thiogalactoside Kb kilobase Km Kanamycin LPS Lipopolysaccharide mAU milliabsorabance units Min Minutes MS Mass spectrometry MS/MS Tandem mass spectrometry Nal Nalidixic acid N-terminal Amino terminal NTR N-terminal region OD600 Optical density (measured at 600 nm) PBS Phosphate buffered saline PCR Polymerase chain reaction Pse5Ac7Ac Pseudaminic acid Q-TOF Quadrupole Time of Flight (Mass spectrometer) RNA Ribonucleic acid SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis Strep Streptomycin T3SS Type three section system T6SS Type six secretion system Tm Melting temperature (primers) UV Ultraviolet iv Table of Contents Chapter 1: Introduction ............................................................................................................ 1 1.1 – The genus Aeromonas ..................................................................................................... 1 1.1.1 – Host interactions ....................................................................................................... 1 1.1.2 – Colonisation factors .................................................................................................. 3 1.1.2.1 – Secreted factors ................................................................................................. 3 1.1.2.2 – Adherence factors .............................................................................................. 4 1.2 – Aeromonas motility ......................................................................................................... 8 1.2.1 – Polar Flagellum .......................................................................................................... 8 1.2.2 – Lateral Flagella ........................................................................................................ 13 1.2.3 – Export and assembly ............................................................................................... 14 1.2.4 – Gene expression and regulation ............................................................................. 16 1.3 – Protein Glycosylation .................................................................................................... 19 1.3.1 – Eukaryotic protein glycosylation ............................................................................. 19 1.3.2 – Bacterial glycosylation of surface proteins ............................................................. 22 1.3.2.1 – Campylobacter ................................................................................................. 22 1.3.2.2 – Helicobacter ..................................................................................................... 25 1.3.2.3 – Pseudomonas ................................................................................................... 26 1.3.2.4 – Neisseria ........................................................................................................... 27 1.3.2.5 – Gram-positive bacterial glycosylation .............................................................. 28 1.4 – Glycosylation in Aeromonas species............................................................................. 29 1.4.1 – The O-linked flagellin glycosylation pathway in A. caviae Sch3 .............................. 31 1.4.1.1 – Pseudaminic acid biosynthesis ......................................................................... 31 1.4.1.2 – Flagellin glycosylation ...................................................................................... 33 1.5 – Project aims ................................................................................................................... 35 Chapter 2 – Materials and methods ..................................................................................... 36 2.1 – Strains and Plasmids ..................................................................................................... 36 2.2 – Media and Antibiotics ................................................................................................... 42 2.3 – Solutions and Buffers .................................................................................................... 43 2.4 – Growth Conditions and Genetic Manipulations ..........................................................