title: Interaction of Bordetella pertussis adenylate cyclase toxin with target cells
Creators: name: Brotherston, Christopher
Publisher: name: University of Glasgow
Year: 1997
Subjects: subject: Microbiology.

description: Adenylate cyclase toxin (CyaA) is one of the many virulence factors produced by B.pertussis, the causative organism of whooping cough. The mechanisms governing the interaction of CyaA with target cells and the role toxin-activation plays in these are unclear. This study investigated these mechanisms. For the purposes of this study CyaA was purified from B.pertussis 348 (pRMB1) by a two step-process involving urea extraction of whole cells followed by calmodulin-agarose affinity chromatography. The purification of CyaA was confirmed by SDS-PAGE and Western blotting. The functional significance of toxin activation was studied by preparing activated and non-activated forms of CyaA from recombinant E.coli strains. Urea extraction from the recombinant strains yielded a 200kDa protein which was relatively free from contaminating host cell protein as determined by Coomassie blue staining. Enzymic assays showed that catalytically active forms of CyaA had been purified from the recombinant E.coli strains and from B.pertussis 348 (pRMB1). The activated form of CyaA possessed toxic activity which was absent from the non-activated form. Binding studies were employed to investigate the interaction of CyaA with various cell types. It was found that activated CyaA could bind to / associate with sheep erythrocytes in a time and dose-dependent manner. Studies revealed that binding increased even at very high doses of toxin, suggesting that the binding process is non-saturatable and therefore non-specific. Regarding the specificity of interaction of CyaA with cells it was found that pre- trypsinisation of sheep erythrocytes increased the susceptibility of the cells to the haemolytic activity of the toxin and this activity was directly dependent on the concentration of trypsin used to treat the erythrocytes. Trypsinisation of cells may have made membrane recognition sights more accessible for CyaA insertion and pore formation. The binding of CyaA to trypsinised cells was low compared to control cells suggesting that the processes of binding and pore-formation are separable events. In this study it was found that none of the sugars tested; D-galactose, D-glucose, D-mannose, D-maltose, D-fucose or N-acetyl neuraminic acid (NANA), inhibited the binding of CyaA to sheep erythrocytes suggesting that these sugars may not play essential roles in toxin-cell interaction. Interestingly, it was found that activated and non-activated derivatives of CyaA could both bind to / associate with sheep erythrocytes. However, binding of non-activated CyaA to cells from the murine macrophage cell line (J774.2) was much less when compared to the activated form. These results are the first evidence of differences in cell-affinity between activated and non-activated CyaA. No binding of activated or non-activated CyaA was detected with BHK or BL3 cells. It is possible that the solubilisation conditions of hypotonic lysis of these cell types may have resulted in the dissociation of bound toxin or that the assay was not sensitive enough to detect low levels of cell-bound toxin. Sheep erythrocyte membrane proteins were investigated as potential receptors for CyaA. No binding was detected between CyaA and the membrane proteins isolated from sheep erythrocytes. To detect if CyaA interacts directly with glycolipid a thin-layer chromatogram overlay assay was developed. CyaA bound to a range of gangliosides (GM1, GD1a, GD1b, and GT1b) and to some but not all of the components in a sheep erythrocyte lipid extract. These results suggest that glycolipids may be possible membrane recognition sites for CyaA. Activated CyaA extracts were found to be lytic towards sheep erythrocytes and liposomes in a time and dose dependent manner. Unlike haemolysis the lytic effect of CyaA on liposomes proceeded without a lag period. This may be because liposomes do not have the same constraints on membrane integrity that erythrocytes have and can leak contents without lysis. Toxin activation was required for haemolytic and liposome permeabilising activities. This is further evidence of the essential role activation plays in the productive interaction of CyaA with cell membranes. The activity of CyaA was much greater towards liposomes with a net negative charge than to liposomes with no net charge or a net positive charge. Gangliosides were not essential and other negatively charged lipids could fulfil the same function. These results suggest that the interaction of CyaA with membranes may be at the level of a charge-charge interaction whereby negatively charged moieties on the membrane surface act as the initial recognition sites.