Elucidating Legionella pneumophila effector function using proteomic approaches Ernest Cheng So Imperial College London Institute of Chemical Biology Department of Chemistry This thesis is presented for the degree of Doctor of Philosophy of Imperial College London and Diploma of Imperial College London 2016 Abstract Legionella pneumophila is the causative agent of Legionnaires’ disease, a severe and potentially fatal pneumonia. This intracellular pathogen proliferates by creating a replicative niche, the Legionella containing vacuole (LCV), inside the host and subverting host signalling pathways. Critical to L. pneumophila’s virulence strategy is its defect in organelle trafficking/intracellular multiplication (Dot/Icm) type IVB secretion system. Using the Dot/Icm, L. pneumophila translocates over 300 effector proteins into the host cell to manipulate signalling pathways. The novel effector LtpG localises to the nucleus upon Dot/Icm-dependent translocation. Genomic deletion of ltpG did not exhibit a L. pneumophila intracellular growth defect in all infection models tested. Although LtpG expression did not cause toxicity in mammalian cells, its filamentation induced by cAMP (Fic) domain caused cytotoxicity in yeast and has auto-AMPylation activity. However, small molecule substrate binding assays suggest a guanosine-containing metabolite is preferred. Determination of LtpG host targets using in vitro protein-protein interaction assays did not yield satisfactory results and consequently a more physiologically relevant infection-based mass spectrometric method was developed. Using the biotin ligase BirA, tagged-effectors were biotinylated in a translocation dependent manner. Effector-host protein complexes formed during infection were subsequently isolated and their composition deciphered using quantitative mass spectrometry. The method was downscaled by over 100-fold from the proof-of-concept study and critical parameters such as number of purifications, lysis conditions and crosslinker reactivity tested. This revealed the infection dependent Rab GTPase binding profiles of the promiscuous Rab binding effectors SidM and LidA. Additionally, HSP90, EEF2 and NACA were identified as high confidence physiological binding partners of LtpG, suggesting a role in manipulation of host translation and autophagic pathways as part of L. pneumophila’s virulence strategy. 1 Declaration of Originality I, Ernest So, declare that this thesis constitutes my own work and that any external contributions are appropriately acknowledged or referenced. 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 Acknowledgements “This is not science, this is bullshit…” Gadi Frankel 2016 (completely out of context) Firstly, I would like to thank my supersivors Gadi and Ed for welcoming me into their research groups and giving me the opportunity to pursue a PhD. Your insight and guidance have been essential for not only the progression of the project but also my development as a scientist. An insurmountable thanks to Gunnar who taught me everything I know in the lab. Thank you for your support and wisdom over the many years and always finding time to help me out. You have the patience of a saint for putting up with me for so long. A big thank you to Team Legionella (Corinna and Dani) for lending me a helping hand whenever I needed it and also providing much needed moral support. Thank you to everyone in the Frankel and Tate groups for general scientific discussions and especially for listening to my rants. Also thanks to everyone on CMBI1 for putting up with me for so many years and potentially a few more yet to come… In particular, I would like to thank Goska and Julia for all their help and discussions with the MS work and especially for looking after the rather temperamental Q-Exactive. I would also like to thank Jyoti Choudhary for invaluable help and expertise with mass spectrometry. Thanks to David Charles and Andreas Förster for all their help with the crystallography work and Eleni for her constant help with protein purifications. Also, thanks to Dan Brown with helping me with the radioactivity work. Thanks to the ICB and EPSRC for funding my PhD and in particular for allowing me to be in the same cohort as Jenny and Ben. Our frequent lunches kept me vaguely sane throughout the years. Finally, I would like to thank my wife-to-be Joanna. Thank you for your love and support throughout my PhD and in particular during the thesis writing period. I know it wasn’t easy watching me write in the most chaotic way possible and so I would like to dedicate this thesis to you. 3 Abbreviations AA amino acid ABC ATP-binding cassette ACES N-(2-Acetamido)-2-aminoethanesulfonic acid AD GAL4 activation domain ADP adenosine diphosphate AMBIC ammonium bicarbonate Amp ampicillin AMP adenosine monophosphate ARF1 ADP-ribosylation factor 1 ATP adenosine triphosphate AYE ACES buffered yeast extract AzTB Azide-TAMRA-biotin capture reagent BCA bicinchoninic acid BD GAL4 DNA binding domain BSA bovine serum albumin cAMP cyclic adenosine monophosphate CDK7 cyclin dependent kinase 7 CDP cytidine diphosphate CFU colony forming unit CHAPS 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate Cm chloramphenicol CMP cytidine monophosphate COPI coat protein complex I CTP cytidine triphosphate CYE buffered charcoal yeast extract DAPI 4',6-diamidino-2-phenylindole DDO double dropout SD (-Leu, -Trp) DMEM Dulbecco's Modified Eagle Medium DNA deoxyribonucleic acid dNTP deoxynucleoside triphosphate Dot/Icm defect in organelle trafficking/intracellular multiplication DSF differential scanning fluorimetry DSP dithiobis(succinimidyl propionate) DTME dithiobismaleimidoethane DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EEF2 elongation factor 2 EM electron microscopy EPF exponential phase form ER endoplasmic reticulum FACS fluorescence activated cell sorting FCS fetal calf serum FDR false discovery rate FF filamentous form 4 Fic filamentation induced by cAMP FL full length GAP GTPase activating protein GDI guanosine nucleotide dissociation inhibitor GDP guanosine diphosphate GEF guanine nucleotide exchange factor GFP green fluorescent protein GMP guanosine monophosphate GnCl guanidinium chloride GST glutathione S-transferase GTP guanosine triphosphate h hour HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HRP Horseradish peroxidase HSP90 heat shock protein 90 HYPE Huntingtin-interacting protein E ID identification IDA iminodiacetic acid IMPA1 inositol(myo)-1(or 4)-monophosphatase 1 IPTG isopropyl β-D-1-thiogalactopyranoside iTRAQ isobaric tagging for relative and absolute quantification Kn kanamycin LB Luria-Bertani LC-MS/MS liquid chromatography tandem mass spectrometry LCV Legionella-containing vacuole LPS lipopolysaccharide Lsp Legionella secretion pathway MIF mature infectious form min minute MOI multiplicity of infection MS mass spectrometry MVB multivesicular body NAC nascent polypeptide-associated complex NAD nicotinamide adenine dinucleotide NAPPA nucleic acid programmable protein array NDP nucleoside diphosphate NMP nucleoside monophosphate NTA nitrilotriacetic acid NTP nucleoside trisphosphate OD optical density 600 PBS phosphate buffered saline PBST PBS-Tween buffer PCR polymerase chain reaction PEG polyethylene glycol PFA paraformaldehyde PI phosphatidylinositol 5 PI(3)P phosphatidylinositol 3-phosphate PI(3,4)P2 phosphatidylinositol 3, 4-diphosphate PI(3,4,5)P3 phosphatidylinositol 3, 4, 5-triphosphate PI(3,5)P2 phosphatidylinositol 3, 5-diphosphate PI(4)P phosphatidylinositol 4-phosphate PI(4,5)P2 phosphatidylinositol 4, 5-diphosphate PI4K phosphatidylinositol 4-kinase PIP phosphatidylinositol phosphate PLA1 patatin-like phospholipase A1 PMA phorbol 12-myristate 13-acetate PPi pyrophosphate PVDF Polyvinylidene fluoride QDO quadruple dropout SD (-Leu, -Trp, -Ade, -His) RNA ribonucleic acid RNAi RNA interference RPF replicative phase form RPMI Roswell Park Memorial Institute medium RT room temperature SAP single Neutravidin affinity purification SCF SKP1-Cullin-F-box SD synthetic defined media SDB-XC poly(styrenedivinylbenzene) copolymer SDS sodium dodecyl sulphate SDS-PAGE SDS polyacrylamide gel electrophoresis Sec general secretory SILAC stable isotope labelling in cell culture SMCC 4-(N-maleimidomethyl)cyclohexane-1-carboxylate SNARE soluble N-ethylmaleimide-sensitive factor activating protein receptor SNX sorting nexin SOC super optimal broth with catabolite repression SPF stationary phase form SRP signal recognition particle T1SS type I secretion system T2SS type II secretion system T3SS type III secretion system T4SS type IV secretion system T5SS type V secretion system T6SS type VI secretion system TAE Tris-acetate-EDTA TAMRA 5,6-Carboxytetramethylrhodamine TAP tandem-affinity purification Tat twin arginine translocation TBTA Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine TCEP tris(2-carboxyethyl)phosphine TEM β-lactamase 6 TGS Tris-glycine-SDS Tm melting temperature TMT tandem mass tag TRITC tetramethylrhodamine UAT urinary antigen test UBE2T ubiquitin-conjugating enzyme E2 T UMP uridine monophosphate UTP uridine triphosphate