Negus, David; (2011) An investigation of poly-gamma-D-glutamic acid depolymerases; a novel therapeutic for the treatment of systemic anthrax infections. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Naturally occurring anthrax is acquired following contact with animals or animal products contaminated with the spores of Bacillus anthracis. Internalisation of spores, typically by inhalation, results in a lethal infection due to the expression by the vegetative bacilli of a toxin complex and a protective capsule composed of poly-γ-D-glutamic acid (PDGA). As a result of the potential for the malicious dissemination of multidrug resistant strains of B. anthracis, novel approaches to the treatment of anthrax should be investigated. Experimental infections due to bacterial pathogens expressing a polysaccharide or polypeptide capsule can be resolved by administration of depolymerases that selectively hydrolyse the external protective layer. As all isolates of B. anthracis elaborate a PDGA capsule that is essential for the full expression of pathogenesis, anthrax represents an attractive target for this therapeutic approach. Therefore, three potential sources of PDGA depolymerases were investigated: the capD gene product produced by B. anthracis, putative depolymerases from anthrax bacteriophages and hydrolytic enzymes elaborated by bacteria with the ability to utilize PDGA as the sole source of carbon and energy. Characterisation of recombinant CapD, including an accelerated storage stability study, revealed that whilst CapD rapidly hydrolyses PDGA, it is markedly unstable. Owing to the enzymes labile nature and poor in vivo characteristics documented elsewhere, it was concluded that CapD does not support further investigation as a therapeutic agent. The majority of phages which infect capsule-bearing hosts carry capsule depolymerases that facilitate interaction with the cell surface. Therefore, B. anthracis phages were investigated for the identification of associated PDGA depolymerases. Examination of seven anthrax bacteriophages, in association with HPA Porton Down, established that anthrax bacteriophages do not carry depolymerases. Soil enrichment culture, using minimal salts medium containing 0.2% PDGA as the sole source of carbon and energy, led to the isolation of a mixed culture of two Gram-negative rods which together elaborated an enzyme that hydrolysed PDGA. Depolymerase was isolated from the bacterial cytoplasm by ion-exchange and electrophoretic separation combined with 2D gel zymography. Activity was attributable to a single protein of approximately 30 kDa which could not be identified by LC/MS-MS, suggesting the depolymerase is a novel enzyme with a sequence currently not deposited in any searchable database. Characterisation of partially purified depolymerase preparations revealed potent hydrolytic activity specific for PDGA, with no general proteolytic activity, in marked contrast to CapD, the depolymerase was stable in a preliminary accelerated stability study conducted over a 30-day period at 40°C. Its properties suggest that the enzyme warrants further in vitro and in vivo investigation as an anti-anthrax therapeutic agent. This study represents the first stage in the development of a novel therapeutic for the treatment of systemic anthrax infections.

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