Studies of the cholesterol-dependent cytolysins

Cowan, Graeme James Macfarlane (2006) Studies of the cholesterol-dependent cytolysins. PhD thesis, University of Glasgow.

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Abstract

The cholesterol-dependent cytolyslns (CDCs) are a group of toxins produced by several genera of Gram-positive bacteria, that bind to and form large oligomeric pores in target cell membranes that contain cholesterol. In addition to cell lysis, a great number of other biological functions have been described for many members of the group, including induction of cytokine release and complement activation. Some of the CDCs have been shown to be critical virulence factors of their producing organisms and immunisation with a number of the CDCs has been shown to be protective against disease caused by those producing organisms. The CDCs are also being used in a diverse array of applications that utilise their pore-forming and toxic properties, including anti-cancer therapy, anti-viral therapy and cell biology. Pneumolysin (PLY) is a member of the CDCs produced by S. pneumoniae and is an important virulence factor and vaccine candidate. A deletion mutant of PLY, Delta6PLY, has been described (Kirkham et al., 2006b), and this mutant is capable of cell binding but incapable of pore formation. Enhanced green fluorescent protein (eGFP) tagged forms of PLY and Delta6PLY were produced and the tagged toxins had similar haemolytic and cytotoxic effects to the parent toxins. Toxin binding to host cells was visualised by epifluorescence microscopy and laser scanning confocal fluorescence microscopy. Binding of the tagged toxins to erythrocytes could also be measured by flow cytometry and an increase in the quantity of bound toxin could be detected in a dose-dependent manner over a large range of toxin concentrations. Rod-like structures were observed on membranes treated with Delta6eGFPPLY and these were further studied by SEM. These rod-like structures may be responsible for the strong aggregation effect observed upon Delta6PLY treatment of erythrocytes. Intermedilysin (ILY) is a member of the CDCs produced by S. intermedius. It is unique within the group in that it exhibits human-specific cytolysis and initial studies suggested that this is due to binding of a different cellular receptor to other toxins of the family and that this receptor was a protein (Nagamune et al, 1996). eGFP-tagged forms of ILY and Delta6ILY were produced to allow tracking of the localisation of ILY and for use in development of a quantitative binding assay. It was confirmed that these proteins could be easily visualised by fluorescence microscopy and that binding could be detected by flow cytometry. A two-hybrid screen was also used to screen for proteins from a human brain cDNA library that were capable of interaction with ILY in order to identify candidate protein receptors for ILY, however no likely receptor candidates were identified. In order to determine which region is responsible for the human specificity of intermedilysin, a bank of chimeras between ILY and PLY was created. The chimeric toxins were expressed and purified and the specificity of the mutants was determined by haemolytic assay on human and rabbit erythrocytes. The specificity of the chimeric toxin was determined by the origin of the C-terminal 53/56 residues, indicating that the latter part of domain 4 is responsible for the human specificity of intermedilysin. To further resolve the region involved in human specificity or cholesterol-binding, a series of small substitution mutants was created. These revealed that the promiscuous cell binding activity of the other CDCs was conferred by residues in the undecapeptide loop as this property could be transferred to ILY by introduction of the typical undecapeptide sequence. Surface-plasmon resonance analysis of substitutions of PLY was used to detect any mutants possessing reduced binding affinity. However, problems with aggregation of purified proteins prevented quantitative data from being collected. Anthrolysin O (ALO) is a toxin produced by Bacillus anthracis, the causative agent of anthrax. It is a member of the cholesterol-dependent cytolysin (CDC) group of toxins, many of which are potential vaccine candidates that protect against their producing organisms. Pore formation by ALO was studied by transmission electron microscopy and pores were found to be consistent with those formed by other members of this toxin family. A novel genetic toxoid of anthrolysin O, Delta6mAL0, was constructed and characterised and was able to bind to cells but was incapable of pore- formation or haemolysis. The capacity of the haemolytic and non-haemolytic forms of ALO to protect against challenge with the toxin or B. anthracis was determined. Immunisation with both active and non-haemolytic forms of ALO elicited protection against lethal i.v. challenge with ALO but neither was protective against B. anthracis in a murine i.p. challenge model. Immunisation with another CDC, pneumolysin, did not confer cross-protection against challenge with ALO. Histopathological investigation following lethal i.v. challenge with ALO revealed acute pathology in the lungs with occlusion of alveolar vessels by fibrin deposits.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Tim Mitchell
Keywords: Molecular biology
Date of Award: 2006
Depositing User: Enlighten Team
Unique ID: glathesis:2006-71047
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71047

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