Bordetella adenylate cyclase toxin induces a cascade of morphological changes of sheep erythrocytes and localizes into clusters in erythrocyte membranes

Adenylate cyclase toxin (CyaA) of Bordetella pertussis penetrates the membrane of eukaryotic cells, producing high levels of intracellular cAMP, as well as hemolysis that results from the formation of cation-selective toxin channels in the membrane. Using several microscopical approaches we studied the effects of CyaA action on the morphology of sheep erythrocytes during early phases preceding lysis and examined localization of CyaA molecules within the erythrocyte membrane. CyaA induced a cascade of morphological changes of erythrocytes, such as shrinkage, formation of membrane projections, and blebs and swelling. The use of an enzymatically inactive CyaA-AC(-) toxoid that is unable to produce cAMP and of a CyaA-E581K mutant exhibiting higher hemolytic activity than with CyaA showed that the hemolytic activity is responsible for the induction of morphological changes of erythrocytes. Further, immunolabeling of inserted CyaA-232/ FLAG molecules with specific anti-FLAG antibodies and IgG-gold particles indicated a clustered distribution of CyaA molecules in erythrocyte membrane. This was confirmed by immunofluorescence and confocal microscopy, which revealed uniform stoichiometry of CyaA clusters, suggesting CyaA binding into specific domains in erythrocyte membrane. Indeed, a decrease of CyaA binding after cholesterol depletion of erythrocytes suggests toxin targeting and binding to membrane microdomains (rafts).