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

Abstract

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).

Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo

“…Earlier studies by Haverstick and Glaser on rabbit erythrocytes labeled with NBD-phospholipid (20, 21) using fluorescence digital imaging microscopy showed uneven distribution of the phospholipids in the membrane and attributed this heterogeneity to the presence of membrane domains of 1-2 μm in size. Fluorescent antibodies for CyaA (adenylate cyclase toxin) from Bordetella pertussis were used to identify rafts (regions rich in sphyingomyline and cholesterol easily disrupted by methyl-betacyclodextrin) as binding sites on sheep erythrocytes (22). Using Laurdan GP, imaging authors agreed on the observation that the plasma membrane of erythrocytes has similar fluidity than nucleated cells but does not show macrodomains separation in vivo.…”

Role of CD11b/CD18 in the Process of Intoxication by the Adenylate Cyclase Toxin of Bordetella pertussis

“…With the greater receptor expression in K562 CR3 ϩ cells, receptor saturation would be expected to occur at higher concentrations of ACT than in CHO CR3 ϩ cells. In addition to expressing different levels of transfected receptor, the parent CHO and K562 cells are very different cell lines, probably with different cell surface lipid organization and protein components which may interact with CR3 to affect ACT translocation and oligomerization (7,39,57).…”

Identification of a Region That Assists Membrane Insertion and Translocation of the Catalytic Domain of Bordetella pertussis CyaA Toxin

“…In these conditions, AC489 bound to the resin and was eluted by 20 Protein batches were more than 95% pure, as determined from SDS-PAGE analysis and N-terminal sequencing. The integrity and identity of the samples were confirmed by measurement of the absolute molecular mass by surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS model PCS 4000, Ciphergen).…”

“…Once in the cytosol, AC interacts with endogenous calmodulin, which is a potent activator of its catalytic activity, and produces supraphysiologic levels of cAMP. Besides, CyaA is also able to form cation-selective pores in the membrane of target cells, leading eventually to cell lysis, although the contribution of this activity in the pathophysiological effects of CyaA remains less clear (17)(18)(19)(20)(21).…”