title: The immunomodulatory and cytotoxic effects of different forms of recombinant adenylate cyclase toxin
Creators: name: Cheung, Yiu Chong Gordon
Publisher: name: University of Glasgow
Year: 2006
Subjects: subject: Immunology, adenylate cyclase, pertussis toxin.

description: This thesis describes the small- and large-scale purification of four different recombinant forms of CyaA, namely; fully functional enzymically-active, acylated and invasive CyaA; an acylated and invasive CyaA form lacking adenylate cyclase (AC) enzymic activity (CyaA*); and the non-acylated, poorly-invasive forms of these toxins, proCyaA and proCyaA*, respectively. Only proCyaA and CyaA showed AC enzymic activity. Only CyaA and CyaA* were haemolytic towards sheep erythrocytes and cytotoxic towards mouse J774.2 macrophage-like cells. Both haemolysis and cytotoxicity by CyaA and CyaA* only occurred in the presence of calcium. CyaA was cytotoxic towards J774.2 cells at low concentrations (50% killing at 0.1 ?g protein/ml) and this paralleled low levels of caspase 3/7 activity, a measure of apoptotic activity. At higher toxin concentrations of CyaA (&gt; 0.5 ?g protein/ml), caspase 3/7 activity declined despite high levels of cell killing measured by the cytotoxicity assay. This may have been due to the onset of necrotic killing at higher concentrations. CyaA* could only kill J774.2 cells at a toxin concentration greater than 0.5 ?g protein/ml and the effect was more calcium-dependent than that of CyaA. Cell killing by CyaA* also occurred in the absence of apoptosis. These data suggested that CyaA killed J774.2 cells by two different mechanisms, involving apoptosis at low concentrations (which was cAMP dependent) and necrosis by pore-formation at higher concentrations. ProCyaA and proCyaA* showed no apoptotic, cytotoxic or haemolytic activities. CyaA inhibited the zymosan stimulated oxidative burst of J774.2 cells. ProCyaA and CyaA* were ~500-fold less active at inhibiting the zymosan-stimulated oxidative burst of J774.2 cells compared with native CyaA. Thus, inhibition of the zymosan-stimulated oxidative burst by CyaA was a result of both invasive and AC enzymic activities. This also indicated that proCyaA was able to invade cells, albeit at a very low level, but that any increase in intracellular cAMP levels created by intracellular proCyaA was unable to induce apoptosis. Circular dichroism (CD) was used to assess any differences in the secondary structure of the four CyaA forms. There were similar changes in tertiary structure of all the CyaA forms as shown by intrinsic tryptophan fluorescence and near UV CD studies in the absence and presence of 1 mM CaCl2. Similar conformational changes in protein secondary structure of all four CyaA forms, at 0.5 mg protein/ml, were observed by far UV CD in the absence and presence of 1 mM CaCl2. However, at 0.05 mg protein/ml, the spectral amplitude of CyaA was decreased by 2-fold suggesting that CyaA was able to adopt two stochiometric forms. Preliminary analytical ultracentrifugation (AUC) studies indicated that CyaA did not exist as a single molecular species in solution. At 3 mg protein/ml, different oligomeric forms of CyaA (monomers, dimers and trimers) as well as the presence of degradation products (20 - 80 kDa) were present. Further optimisation of AUC studies is required to overcome the problem of non-ideality (caused by either a repulsion between charged molecules (proteins) that is not shielded by solvent counter-ions or by size-exclusion effects arising from extended/elongated conformations) which may have interfered with the analyses. (Abstract shortened by ProQuest.).