Shorter, Susan Amanda (2017) Biochemical characterisation of the translocation of material through the protective antigen (PA) Pore. PhD thesis, University of Greenwich.

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Abstract

Bacillus anthracis secretes a tripartite protein complex, forming the toxin known as anthrax toxin. It is composed of the catalytically active proteins, lethal factor (LF) and oedema factor (EF), and protective antigen of 83 kDa (PA83). This toxin complex mediates cytosolic entry through the formation of an oligomeric PA pore, facilitating translocation of LF and EF, an event critical for EF and LF to exert their cytotoxic effects. The requirements for translocation are still poorly understood, and essential for understanding the limitations and possible utility to this pore. Once the dynamics of translocation are more clearly understood, therapeutics for inhibiting LF and EF toxicity could be developed for anthrax treatment, as well as the potential to develop this pore into a drug delivery platform. The project outlined herein was to evaluate the requirements for molten globular (MG) transition during translocation of LF and EF through the PA pore. This study documented the translocation of a variety of biochemically and thermodynamically distinct constructs (protein and protein:DNA), and their ability to pass through the PA pore. Pore translocation was detected by monitoring gene modulation, toxicity, fluorescent microscopy and neutron reflectometry. The ability of LFn-GAL4, antisense oligonucleotide (ASO) complex to translocate and mediate gene modulation was assayed in vitro. The components of this complex were found to be non-toxic individually and together (PA8 3, LFn-GAL4:ASO). This complex was found to be able to deliver ASOs and mediate cytosolic access as demonstrated by the decreased level of endogenous syntaxin 5 post-treatment (77 % ± 11.6). Then the ability of LFn-Ricin toxin A chain (RTAC) to translocate through the pore was evaluated by cytotoxicity. This demonstrated limited cytotoxicity in vitro, indicating that this cationic protein either couldn’t translocate or once translocated was no longer a functional enzyme. Further to this, LFn-GFPs ability to translocate, assayed in the presence and absence of the PA pore. LFn-GFP demonstrated translocation in the presence and absence of PA in HeLa cells over 4, 6 and 16 hours. In the presence of PA cytosolic signal increased over this time frame (from 0% of the input to 4%) whereas in the absence of PA cytosolic signal decreased from 4% to 0%). Binding and translocation of deuterated LFn-GFP with the both a lipid-nanodisc and a lipid-nancomplex (NC – a nanodisc containing the protective antigen pore) was followed in real time at pH7.4 and 37oC mimicking physiological conditions and at pH 5.5 mimicking the acidity of late endosomes. Measurements were performed in a variety of solvents, namely H2O, D2O, SiMW and 4MW. Neutron reflectometry (NR) measurements showed approximately 90% coverage of ND and approximately 10% coverage of NC at the silica/solvent interface. A significant change in spectra was observed following ddition of dLFn-GFP, alteration of pH from pH7.4 to pH5.5 resulted in translocation of LFn-GFP into the PA layer on the NC as determined by a decrease in hydration of the ND and an increase in hydration of the PA layer.

Item Type:	Thesis (PhD)
Uncontrolled Keywords:	Chemistry; PA pore; protein constructs; drug delivery systems;
Subjects:	Q Science > QD Chemistry
Faculty / School / Research Centre / Research Group:	Faculty of Engineering & Science Faculty of Engineering & Science > School of Science (SCI)
Last Modified:	08 Apr 2019 15:47
URI:	http://gala.gre.ac.uk/id/eprint/23492

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