Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.707073
Title: Structural and functional studies of the AHL tripartite pore forming toxin proteins from Aeromonas hydrophila
Author: Wilson, Jason
ISNI:       0000 0004 6060 5394
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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Abstract:
The Cytolysin A (ClyA) family of pore forming toxins include mono-, bi-, and tripartite members. Haemolysin BL (HBL-B,L1,L2) and non-haemolytic enterotoxin (NHE-A,B,C) are tripartite toxins where each subunit has a specific role in pore formation, and where pore formation is more complex than that of the monopartite ClyA. Homology searches have identified a large number of NHE homologues in a diverse group of bacterial species, including Aeromonas hydrophila. Each component of the Aeromonas hydrophila tripartite toxin system, AhlA, AhlB, and AhlC, was successfully overexpressed and purified, and haemolysis assays confirmed that these NHE homologues are haemolytic, and require all three components for maximum lysis. AhlC was successfully crystallised, and the structure was solved of two crystal forms to ~2.5 Å. The crystal structure of AhlC shows that the protein is a structural homologue to NheA, HblB, and ClyA, and consists of a bundle of four helices, and a long helical stalk and a hydrophobic hairpin. AhlC forms a tetramer to protect the hydrophobic hairpin from the solvent, unlike other ClyA family toxins that shield their membrane-spanning regions with conformational rearrangements. Electron microscopy studies have shown that AhlB is able to form pre-pores of ~12 nm in diameter, and that AhlB and AhlC together form pores, although more readily than AhlB in isolation. AhlBC pores also localise to certain liposomes, leading to saturation of pores in these vesicles. AhlB was successfully crystallised in a number of conditions, and analysis showed that many crystallisation conditions contained MPD. In MPD-containing crystallisation conditions, large unit cells were present, with a conserved 115 Å cell dimension, and a 10-fold non-crystallographic symmetry axis was identified in analysis of self rotations. These crystals are predicted, therefore, to contain the pre-pore observed by EM, and attempts are on-going to solve the structure.
Supervisor: Patrick, Baker Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.707073  DOI: Not available
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