RESUMO
The Bordetella adenylate cyclase-hemolysin (CyaA, ACT, or AC-Hly) is a multifunctional toxin. Simultaneously with promoting calcium ion entry, CyaA delivers into host cells an adenylate cyclase enzyme (AC) and permeabilizes cell membrane by forming small cation-selective pores. Indirect evidence suggested that these two activities were accomplished by different membrane-inserted CyaA conformers, one acting as an AC-delivering monomer and the other as an uncharacterized pore-forming oligomer. We tested this model by directly detecting toxin oligomers in cell membrane and by assessing oligomerization of specific mutants with altered pore-forming properties. CyaA oligomers were revealed in sheep erythrocyte membranes by immunogold labeling and directly demonstrated by pulldown of membrane-inserted CyaA together with biotinylated CyaA-AC(-) toxoid. Membrane oligomers of CyaA could also be resolved by nondenaturing electrophoresis of mild detergent extracts of erythrocytes. Furthermore, CyaA mutants exhibiting enhanced (E581K) or reduced (E570K+E581P) specific hemolytic and pore-forming activity were found to exhibit also a correspondingly enhanced or reduced propensity to form oligomers in erythrocyte membranes. On the other hand, processed CyaA, with the AC domain cleaved off by erythrocyte proteases, was detected only in a monomeric form excluded from the oligomers of unprocessed CyaA. These results provide the first direct evidence that oligomerization is involved in formation of CyaA pores in target membranes and that translocation of the AC domain across cell membrane may be accomplished by monomeric CyaA.
Assuntos
Toxina Adenilato Ciclase/metabolismo , Bordetella/enzimologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Toxina Adenilato Ciclase/farmacocinética , Animais , Endocitose , Eritrócitos , Hemólise/efeitos dos fármacos , Mutação de Sentido Incorreto , Multimerização Proteica , OvinosRESUMO
Recombinant adenylate cyclase toxoids are shown to deliver inserted foreign CD4(+)-T-cell epitopes into the major histocompatibility complex class II presentation pathway, inducing a specific CD4(+)-T-cell response in vivo and yielding in vitro stimulation of specific CD4(+) T cells at a 100-times-higher molar efficiency than the free peptide containing the epitope.