title: Purification, formulation and characterisation of the adenylate cyclase toxin of Bordetella pertussis
Creators: name: Khosravani, Abdolmajid
Publisher: name: University of Glasgow
Year: 2006
Subjects: subject: Microbiology.

description: Adenylate cyclase toxin (CyaA) toxin is an important virulence factor of Bordetella pertussis, the causative agent of whooping cough, and a potential component of acellular pertussis vaccine. The work involved the production of three purified forms of CyaA with different enzymic and invasive properties. These were: the native enzymatically-active, invasive toxin (CyaA), an invasive derivative lacking AC enzymic activity (CyaA*) and a non-acylated, non-invasive form of CyaA (proCyaA). These were expressed in E. coli BL21/DE3 as recombinant proteins. After purification by a combination of chromatographic methods (Q-and Butyl-Sepharose) their properties were investigated by several assays. The LPS content of these preparations was very low. The AC enzymic activity was assayed by a conductimetric method. CyaA and proCyaA had a high level of enzymic activity but that of CyaA* was very low. The cytotoxic properties of CyaA* and pro-CyaA towards J774.2 cells were similar and were 100-fold less than that of CyaA, but these differences were shown to some, extent to be Ca2+ -dependent for CyaA*. Caspase 3/7 activities were measured over a range of toxin concentrations. At these concentrations, neither urea buffer alone nor CyaA* induced any significant increase in caspase 3/7 from different mammalian cells. The greatest effect of CyaA was observed on J774.2 and RBL-2H3 cells where increasing concentration of toxin gave increasing activity. Different concentrations of CyaA and CyaA* were used to investigate dose-dependent effects of the toxins on phagocytosis and the oxidative burst in U937 human monoblastic cells, J774.2 mouse macrophagelike cells and fresh human granulocyte cells (whole blood used). Significant effects were seen with CyaA on both phagocytosis and oxidative burst, but CyaA* did not have a significant effect on either. The results of this first part of the study showed that both enzymatic and invasive functions are required for the cytotoxic effects of adenylate cyclase toxin. In the second part of the work, CyaA was formulated as protein-coated microcrystals (PCMCs) on the surface of microcrystals of DL-valine. The aims of this formulation were to remove the urea, normally used to stabilize the protein, and to determine the stability of the enzymic and cytotoxic activities of the protein in this form, as a dry powder and when the PCMCs were redissolved in aqueous solution. The CyaA in the PCMCs was shown not to be readily soluble in aqueous buffers, but could be resolubilised in urea buffer and retained high AC and cytotoxic activity. Many different types of PCMC formulation were prepared in attempts to increase solubility of PCMCs in aqueous solution. PCMCs were made with CyaA coprecipitated with different combination of CaM, BSA, CaCl2 or ATP and crystals were dissolved in different buffers at various pHs. The most promising results were obtained with CyaA-CaM- BSA-PCMCs where the highest levels of both AC enzymic and cytotoxic activities were seen when the PCMCs were dissolved in l00mM Bicine (pH 8). CaM alone preserved only the AC enzymic activity of PCMCs when coprecipitated with CyaA and DL-valine. AC and cytotoxic activities of CyaA were stable in PCMCs for up to a week at 37°C. CyaA-CaM-BSA-PCMCs induced a strong serum IgG response to CyaA and BSA when injected subcutaneously into mice. The results indicated that CyaA-CaM-BSA- PCMCs offer a promising way to preserve the activity and antigenicity of CyaA in a non-aqueous formulation. Neutralisation tests proved that the antibody generated from mice immunised with CyaA-BSA-CaM-PCMCs was able to neutralise the AC enzymic and cytotoxic activities. Such PCMCs could have application for presentation of protein antigens that normally require cold storage for stability.