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Inhibition of NF-Kappab Signalling by a Family of Type III Secretion System

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Inhibition of NF-Kappab Signalling by a Family of Type III Secretion System Inhibition of NF-B signalling by a family of type III secretion system effector proteins during Salmonella infection Elliott James Jennings Medical Research Council Centre for Molecular Bacteriology and Infection Department of Medicine Imperial College London Submitted for the degree of Doctor of Philosophy Supervised by Dr. Teresa L. M. Thurston and Prof. David W. Holden September 2018 ABSTRACT Abstract During Salmonella infection, bacterial proteins called ‘effectors’ are translocated into host cells by two type III secretion system apparatuses encoded by Salmonella-pathogenicity island 1 and 2. These effectors manipulate host cell processes to facilitate the formation of an intracellular replicative niche, to prevent bacterial clearance, and ultimately promote bacterial transmission to another susceptible host. A subset of SPI-2 T3SS effector proteins manipulate innate immune signalling pathways thereby preventing formation of an appropriate immune response. In this thesis, I identify three related effector proteins - GtgA, GogA, and PipA, as sufficient to inhibit NF-B signalling when expressed ectopically. Furthermore, I demonstrate that GtgA, GogA, and PipA are zinc metalloproteases that inhibit NF-B signalling by cleaving the NF-B transcription factor subunits p65, cRel, and RelB, but not NF-B1 (p105/p50) or NF-B2 (p100/p52). Accordingly, in Salmonella-infected cells, p65 was cleaved dependent on gtgA, gogA, and pipA leading to inhibition of NF-B signalling. To investigate the molecular basis for substrate recognition, mutational analysis of residues in close proximity to the p65 cleavage site (G40/R41) was done and showed that the P1’ residue (R41 in p65) is a critical determinant of substrate specificity. In NF-B1 and NF- B2, a proline residue is present at the corresponding site and this residue prevents cleavage by GtgA, GogA, and PipA. I also present the crystal structure of GtgA alone and in complex with the N-terminal domain of p65. The crystal structure demonstrates the importance of the P1’ residue in substrate specificity and supports a model of DNA mimicry as the mechanism of substrate recognition. This thesis therefore provides detailed insight into the functions and mechanism of substrate recognition, for a family of previously uncharacterised Salmonella virulence proteins. 2 ACKNOWLEDGEMENTS Acknowledgements First, I would like to thank my two supervisors - Teresa and David, for their advice, guidance, and mentorship, as well as for affording me opportunities to grow as a person and a scientist. I would also like to thank you both for your patience and understanding when I developed RSI in my first year. I know of others who were in a similar position with less understanding supervisors, so your support was very much appreciated. I would also like to thank Diego for taking me under his wing at the Crick and for teaching me all things crystallographic. Working alongside you has definitely been a highlight! Thanks also to Katrin without whom the collaboration would not have been possible. To all members of the Holden and Thurston lab, past and present – Regina, GG, Eric, Luciano, Charlotte, Camilla, Sophie M., Ondrej, Xin, Mei, Xiujun, Ethel, Alan, Hai Xia, and Julian, I say thank you! Thank you for all the help you all gave me, as well as for being a fantastic bunch of colleagues to work with. I’d also like to thank Megan, for keeping the lab running smoothly, as well as all other members of CMBI2. To all my friends outside the lab, thanks for your willingness to grab a beer - spending time with you guys was always a welcome escape from the lab. A huge thanks also to Mairi, who shared much of this adventure with me. I am glad that you were a part of it and am incredibly appreciative of the support you gave. The last few years would not have been the same without you. Lastly to my parents and to my sister Elise. Thanks for your continued support and encouragement, even though I know that most of the time it probably seemed as though I was speaking gibberish! At least you all now know what a Western blot is! 3 DECLARATION OF ORIGINALITY Declaration of originality I hereby declare that the work presented in this thesis is my own original work. Any contribution to this thesis by others, published or otherwise, is acknowledged throughout the text with all references listed in the bibliography. This thesis contains material, including figures and intellectual content, which is published in: JENNINGS, E., THURSTON, T. L. M. & HOLDEN, D. W. 2017. Salmonella SPI-2 Type III Secretion System Effectors: Molecular Mechanisms And Physiological Consequences. Cell Host Microbe, 22, 217-231. JENNINGS, E., ESPOSITO, D., RITTINGER, K. & THURSTON, T. L. M. 2018. Structure– function analyses of the bacterial zinc metalloprotease effector protein GtgA uncovers key residues required for deactivating NF-κB. Journal of Biological Chemistry. 4 COPYRIGHT DECLARATION Copyright declaration The copyright of this thesis rests with the author. Unless otherwise indicated, its contents are licensed under a Creative Commons Attribution-Non Commercial 4.0 International Licence (CC BY-NC). Under this licence, you may copy and redistribute the material in any medium or format. You may also create and distribute modified versions of the work. This is on the condition that: you credit the author and do not use it, or any derivative works, for a commercial purpose. When reusing or sharing this work, ensure you make the licence terms clear to others by naming the licence and linking to the licence text. Where a work has been adapted, you should indicate that the work has been changed and describe those changes. Please seek permission from the copyright holder for uses of this work that are not included in this licence or permitted under UK Copyright Law. 5 TABLE OF CONTENTS Table of Contents Abstract 2 Acknowledgements 3 Declaration of originality 4 Copyright declaration 5 List of Figures 10 List of Tables 12 Abbreviations 13 1 Introduction 16 1.1 Salmonella pathogenesis 16 1.1.1 Human diseases caused by Salmonella 16 1.1.1.1 Non-typhoidal Salmonella: gastroenteritis 17 1.1.1.2 Invasive non-typhoidal Salmonella 18 1.1.1.3 Typhoid and paratyphoid fever 18 1.1.2 Experimental models of Salmonellosis 19 1.1.2.1 Animal models of typhoidal Salmonella 19 1.1.2.2 Animal models of human gastroenteritis 20 1.1.2.3 In vitro tissue culture models 20 1.1.3 Molecular determinants of Salmonella virulence 21 1.2 Cell autonomous immunity 22 1.2.1 Autophagy 24 1.2.2 Innate immune signalling 24 1.2.2.1 The NF-B signalling pathway 25 1.2.2.2 The MAPK signalling pathway 27 1.2.3 Host cell death 28 1.2.3.1 Apoptosis 28 1.2.3.2 Necroptosis 29 1.2.3.3 Pyroptosis 30 1.3 Salmonella T3SS effector proteins 30 1.3.1 SPI-1 T3SS effector proteins 32 1.3.1.1 Epithelial cell invasion 32 1.3.1.2 Induction of intestinal inflammation 33 1.3.2 SPI-2 T3SS effector proteins 35 1.3.2.1 SCV membrane dynamics 35 6 TABLE OF CONTENTS 1.3.2.2 SCV intracellular positioning 39 1.3.2.3 Lipid droplets and cytoplasmic aggregates 39 1.3.2.4 Cytoskeletal remodelling 40 1.3.2.5 Modulation of innate immune signalling pathways 40 1.3.2.6 Manipulation of the adaptive immune system 43 1.3.2.7 Uncharacterised SPI-2 T3SS effectors 43 1.4 Zinc metalloprotease type III secretion system effector proteins 44 1.4.1 GtgA, GogA, and PipA 44 1.4.2 NleC – a bacterial zinc metalloprotease T3SS effector 46 1.4.3 Zinc metalloproteases 48 1.5 Aims of the project 49 2 Materials and methods 52 2.1 Materials 52 2.1.1 Bacterial strains 52 2.1.2 Plasmids 52 2.1.3 Antibodies 52 2.1.4 Mammalian cell lines 52 2.2 Methods 58 2.2.1 Bacterial growth conditions 58 2.2.2 Plasmid DNA purification 58 2.2.3 Isolation of bacterial genomic DNA 58 2.2.4 RNA extraction and complementary DNA synthesis 58 2.2.5 Construction of expression vectors 59 2.2.6 Preparation and transformation of electrocompetent bacteria 60 2.2.7 One-step PCR mutagenesis 60 2.2.8 P22 phage transduction 61 2.2.9 Mammalian cell culture 61 2.2.10 DNA transfections 61 2.2.11 Mammalian cell transduction 61 2.2.12 Salmonella Typhimurium infection of HeLa cells 62 2.2.13 Salmonella Typhimurium infection of RAW264.7 macrophages 62 2.2.14 Immunoblot analysis 63 2.2.15 Coomassie Blue staining 63 2.2.16 Flow cytometry 64 2.2.17 Luciferase assays 64 7 TABLE OF CONTENTS 2.2.18 Immunofluorescence microscopy 65 2.2.19 Protein purification 65 2.2.20 Cleavage of expression tags 66 2.2.21 Size-exclusion chromatography 67 2.2.22 Measurement of protein concentration 67 2.2.23 In vitro cleavage assays 67 2.2.24 LUMIER binding assay 68 2.2.25 Protein crystallisation 68 2.2.26 Structural determination 69 2.2.27 Statistical analysis 70 3 NF-B inhibition by Salmonella SPI-2 T3SS effector proteins 71 3.1 Results 72 3.1.1 SPI-2 T3SS-dependent NF- B inhibition in Salmonella-infected macrophages 72 3.1.2 GtgA is sufficient to inhibit TNF- -induced NF- B activation in 293ET cells 76 3.1.3 GtgA, GogA, and PipA inhibit NF- B signalling dependent on a functional zinc metalloprotease motif 79 3.1.4 NleC residues 281-330 are required for inhibition of IFN--induced ISRE signalling 85 3.1.5 GtgA, GogA, and PipA cleave the N-terminal domain of p65 85 3.1.6 GtgA, GogA, and PipA cleave p65, cRel, and RelB, but not NF-B1 (p50/p105), NF-B2 (p52/p100) or NFAT transcription factor subunits 89 3.1.7 GtgA, GogA, and PipA do not cleave NFAT transcription factor subunits 91 4 NF-B inhibition by GtgA, GogA, and PipA in Salmonella-infected cells 93 4.1 Results 94 4.1.1 Construction of Salmonella Typhimurium
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