Regulation of host tyrosine kinases by the Escherichia coli and Salmonella ADP-ribosyltransferases EspJ, SeoC and SboC Ph.D. Thesis Dominic James Pollard Imperial College London Department of Life Sciences Centre for Molecular Bacteriology and Infection Supervisor – Prof. Gad Frankel This thesis is presented for the degree of Doctor of Philosophy of Imperial College London and Diploma of Imperial College London 2018 1 Candidate’s declaration I, Dominic Pollard, declare that this thesis constitutes my own work and that any external contributions are appropriately acknowledged or referenced from hereon in. 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. 2 Abstract The human pathogens enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC) and Salmonella enterica are responsible for a significant proportion of the global diarrhoeal disease burden. EPEC, EHEC and the mouse-restricted pathogen C. rodentium (CR) make up the attaching and effacing (A/E) pathogens. CR is used to model EPEC/EHEC pathogenesis in vivo, where it colonises the apical surface of mouse intestinal mucosae, destroying microvilli and inducing colonic crypt hyperplasia (CCH) and diarrhoeal disease symptoms. The type three secretion system (T3SS) is instrumental to the infectivity of the A/E pathogens, as well as Salmonella, through the injection of sets of effector proteins into the host cell cytosol for the manipulation of host cell signalling. Prior to this study the A/E pathogens T3SS effector EspJ was identified as a novel enzyme that can disrupt FcγR- and C3R-mediated phagocytosis, dependent upon a functional ADP-ribosyltransferase (ART) domain. In vitro EspJ was shown to inhibit Src kinase through simultaneous amidation and ADP-ribosylation of a conserved kinase domain residue, E310. This study shows that the inhibition of phagocytosis is conserved by the EspJ homologues Salmonella enterica subsp. salamae and arizonae SeoC, S. bongori SboC and Y. enterocolitica YspJ. SeoC is secreted by the Salmonella pathogenicity island (SPI)-1 T3SS but does not impact Salmonella entry into host cells. An in vitro proteomics assay showed that EspJEHEC could ADP-ribosylate a multitude of host non-receptor tyrosine kinases (NRTK), including Src and its inhibitor Csk which were also ADP-ribosylated by SeoC and SboC, but not YspJ. Finally, a proteomics comparison of IECs purified from mice infected with CR expressing functional or non-functional EspJ, suggested that EspJ modulates host inflammation and the immune system in vivo. 3 Acknowledgements Technical assistance Animal work was performed by Valerie Crepin, Izabela Glegola-Madejska, Agnes Sågfors and Rachael Barry. Julia Morales Sanfrutos and Kate Hadavizadeh (Imperial College London) performed mass spectrometry for the analysis of in vitro EspJ targets, and Lu Yu (The Institute of Cancer Research, London) for the analysis of mouse IEC proteomes. Colon histopathology was performed by Lorraine Lawrence (ICL), and colon immunofluorescence by Rachael Barry and Lourdes Osuna-Almagro. FACS analysis was run by Maryam Habibzay. Thanks are extended to Jyoti Choudhary (ICR), Ed Tate (ICL) and Pete DiMaggio (ICL) and lab members of their research teams for mass spectrometry machine time, consultation, and provision of reagents that made these aspects of the project possible. General acknowledgements Huge thanks go to Gadi for enabling this PhD, providing invaluable guidance and supporting my independence as a scientist throughout. As well as being a great mentor you have provided endless entertainment from tomato ‘stress balls’ to cycling in pyjamas. I would like to thank Cedric for your supervision and willingness to discuss experiments at length over coffee! It is testament to you that I felt comfortable directing my own project from an early stage. Ernie, thanks for teaching Captain Liability to do mass spectrometry and Joanna thanks for setting up such a great PhD project prior to my arrival in the lab. Gratitude is extended to Vladimir Pelicic and Stephen Curry for support as my PhD progress review panel. I would also like to thank the BBSRC for providing funding. I will savour many happy memories from the past four years thanks to some great people on level 1. Alex for sharing a flat, bank accounts, grand designs, nachos and generally too much; Ernie for karaoke, chicken, gym and mass spec banter (yes, it is possible); Dani for returning 4 as much abuse as you get, and for pouring beer down where it shouldn’t go; Chris for cycling, Pie-Day Friday and the original pub-team; Agnes, Josh, Eleni, Pippa, Becky, Sabrina, and Elli for general good times in and out of the lab; Olga thanks for being my biochem buddy and sharing many crazy laughs. Jayne I can’t thank you enough. Thanks for caring, understanding and entertaining, for being the only person to find me funny and for telling me when I need to exercise because I am grumpy! Finally, I am eternally grateful to my close and extended family for their support. Thanks to my parents Christine and Andrew for making this possible in every single dimension, I couldn’t have reached this point without your support. Tom and Hannah thanks for putting up with me for the last 9 months, I can’t have been much fun! 5 Table of contents Candidate’s declaration .............................................................................................................. 2 Copyright declaration................................................................................................................. 2 Acknowledgements .................................................................................................................... 4 Table of contents ........................................................................................................................ 6 Table of figures ........................................................................................................................ 10 Table of tables .......................................................................................................................... 12 Abbreviations ........................................................................................................................... 13 Chapter 1: Introduction ............................................................................................................ 18 1 Bacterial pathogenesis ...................................................................................................... 18 1.1 Diarrhoeagenic bacteria Escherichia coli and Salmonella ........................................ 18 1.1.1 Diarrhoeagenic Escherichia coli epidemiology ................................................. 19 1.1.2 Salmonella spp. epidemiology ........................................................................... 23 1.2 Gram-negative bacterial secretion systems ............................................................... 25 1.2.1 The type three secretion system ......................................................................... 27 1.3 T3SS-directed pathogenesis ...................................................................................... 30 1.3.1 Pathogenicity islands ......................................................................................... 30 1.3.2 Induction of diarrhoeal symptoms ..................................................................... 33 1.3.3 Cytoskeleton modulation ................................................................................... 35 1.4 The host response to infection ................................................................................... 43 1.4.1 Inflammatory and ROS signalling ..................................................................... 44 1.4.2 Pro-inflammatory and anti-inflammatory cell death .......................................... 47 1.4.3 Phagocytosis ...................................................................................................... 51 1.5 Tyrosine kinases ........................................................................................................ 57 1.5.1 Src family kinases and Csk ................................................................................ 60 1.5.2 NRTKs in immune cell signalling ..................................................................... 64 1.5.3 NRTKs and pathogen recognition receptors ...................................................... 65 1.5.4 Bacterial utilisation of NRTKs during infection ................................................ 65 1.6 ADP-ribosylation ...................................................................................................... 66 1.6.1 Mammalian ADP-ribosyltransferases and hydrolases ....................................... 68 1.6.2 Bacterial ARTs................................................................................................... 69 1.6.3 The T3SS-secreted ADP-ribosyltransferase EspJ .............................................. 70 1.7 Aims and objectives .................................................................................................. 73 2 Chapter 2: Materials