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Title: Further investigation of the roles of fibronectin-binding proteins CadF and FlpA during Campylobacter jejuni interactions with intestinal epithelial cells
Author: Hussein, M.
ISNI:       0000 0004 7229 0692
Awarding Body: London School of Hygiene & Tropical Medicine
Current Institution: London School of Hygiene and Tropical Medicine (University of London)
Date of Award: 2018
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Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans. The ability of C. jejuni to invade human intestinal epithelial cells (IECs) is pivotal for pathogenicity. Two highly conserved fibronectin-binding proteins CadF & FlpA have been shown to play an important role in C. jejuni adhesion to IECs. CadF & FlpA are also associated with outer membrane vesicles (OMVs) and may play a role in the binding of OMVs to IECs. Mutation of cadF & flpA in 11168H & 81-176 reduced binding to fibronectin in vitro and bacterial adhesion to and invasion of both Caco-2 & T84 IECs, however intracellular bacterial numbers increased over time between 3 and 24 hours. Both cadF & flpA mutants were able to translocate as efficiently as the wild-type strain across a T84 IEC monolayer, an event not associated with any change in membrane permeability as indicated by TEER values. Mutation of cadF reduced C. jejuni cytotoxicity in the Galleria mellonella larvae model of infection to a greater extent than mutation of flpA. OMVs isolated from cadF or flpA mutants were less immunogenic and cytotoxic than OMVs isolated from wild-type strains. Results from inhibitors studies showed that cytochalasin D, methyl-beta-cyclodextrin, colchicine and wortmannin all reduced 11168H invasion of T84 IECs. However the invasion mediated by CadF and FlpA was shown to initiate different invasion pathways as wortmannin showed no effect on the ability of the 11168H cadF mutant to invade T84 IECs whilst colchicine showed no effect on the ability of the 11168H flpA mutant to invade T84 IECs. Using a 11168H strain expressing GFP at high levels, C. jejuni was shown to invade IECs, either residing within the Campylobacter containing vacuole (CCV), free within the cytoplasm and also in close proximity to parts of the trans-golgi network. A LAMP-1 stain showed co-localisation with late endosomal compartments in parts of the trans-golgi network. C. jejuni infection of IECs leads to actin cytoskeleton rearrangement as seen by the formation of filopodia, lamellapodia, membrane ruffles and F-actin stress fibres after 24 hours infection as a result of activation of the small GTPase Rac1. Mutation of cadF or flpA reduces Rac1 activation compared to wild-type strain. The significant finding of this study is that CadF and FlpA appear to initiate different invasion pathways to allow C. jejuni invasion of IECs. The results of this study support the view that both CadF and FlpA play equally important roles in C. jejuni pathogenesis.
Supervisor: Dorrell, N. ; Elmi, A. Sponsor: MARA, Malaysia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral