Schubert, Wolf-Dieter2024-05-232024-05-232024-092024-05-16*S2024http://hdl.handle.net/2263/96188Thesis (PhD (Biochemistry))--University of Pretoria, 2024.Enterotoxigenic Escherichia coli (ETEC) is a diarrhoeal pathogen associated with severe morbidity and mortality among young children in developing countries. At present, there is no vaccine for ETEC. One candidate vaccine antigen, EtpA, is a conserved secreted adhesin that presumably binds to the tips of the E. coli flagellum to link ETEC to host intestinal glycans. EtpA is exported through a Gram-negative, two-partner secretion (TPS) system (type Vb) comprised of the secreted EtpA passenger (TpsA) protein and EtpB (TpsB) a transporter integrated into the outer bacterial membrane. TpsA proteins share a conserved, N-terminal domain followed by an extensive C-terminal domain with divergent sequence repeats. Three constructs of EtpA were prepared and analysed respectively including residues 67 to 447 (EtpA67-447), residues 1 to 606 (EtpA1-606) and residues 67-930 (EtpA67-930). The crystal structure of EtpA67-447 solved at 1.76 Å resolution revealed a right-handed parallel β-helix with two extra-helical hairpins and an N-terminal β-strand cap. Analyses by circular dichroism spectroscopy confirmed the β-helical fold and indicated high resistance to chemical and thermal denaturation as well as rapid refolding. A theoretical model of full-length EtpA by AlphaFold largely concurs with the crystal structure adding a long β-helical C-terminal domain after an interdomain kink. We propose that robust folding of the TPS domain upon secretion provides a template to extend the N-terminal β-helix into the C-terminal domains of TpsA proteins. Unexpectedly, size exclusion chromatography and molecular pulldown assays with the N-terminal domain of EtpA failed to show any interaction with flagellin implying that other factors may be involved.en© 2023 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.UCTDSustainable Development Goals (SDGs)Infectious diseaseEnterotoxigenic EscherichiaSDG-03: Good health and well-beingNatural and agricultural sciences theses SDG-03Thesisu14334012