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Title: Elucidating Legionella pneumophila effector function using proteomic approaches
Author: So, Ernest
ISNI:       0000 0004 6496 1871
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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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.
Supervisor: Frankel, Gad ; Tate, Ed Sponsor: Engineering and Physcial Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral