RESUMO
Bordetella pertussis adenylate cyclase (CyaA) is an invasive bacterial toxin that delivers its N-terminal catalytic domain into the cytosol of eukaryotic cells bearing the alpha(M)beta(2) integrin (CD11b/CD18), such as myeloid dendritic cells. This allows use of engineered CyaA for targeted delivery of CD8(+) T cell epitopes into the MHC class I pathway of APC and induction of robust and protective cytotoxic responses. In this study, we demonstrate that CyaA can efficiently codeliver both a CD8(+) T cell epitope (OVA(257-264)) and a CD4(+) T cell epitope (MalE(100-114)) into, respectively, the conventional cytosolic or endocytic routes of processing of murine bone marrow-derived dendritic cells. Upon CyaA delivery, a strong potentiation of the MalE(100-114) CD4(+) T cell epitope presentation is observed as compared with the MalE protein, which depends on CyaA interaction with its CD11b receptor and its subsequent clathrin-mediated endocytosis. In vivo, CyaA induces strong and specific Th1 CD4(+) and CD8(+) T cell responses against, respectively, the MalE(100-114) and OVA(257-264) epitopes. These results underscore the potency of CyaA for design of new vaccines.
Assuntos
Apresentação de Antígeno , Antígeno CD11b/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Sistemas de Liberação de Medicamentos/métodos , Epitopos de Linfócito T/metabolismo , Células Th1/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Actinas/metabolismo , Toxina Adenilato Ciclase/administração & dosagem , Toxina Adenilato Ciclase/genética , Toxina Adenilato Ciclase/imunologia , Toxina Adenilato Ciclase/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Células da Medula Óssea/enzimologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/metabolismo , Vesículas Revestidas por Clatrina/fisiologia , Citotoxicidade Imunológica/genética , Células Dendríticas/enzimologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endossomos/enzimologia , Endossomos/imunologia , Endossomos/metabolismo , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Proteínas de Escherichia coli/administração & dosagem , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/imunologia , Feminino , Genes Reporter , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Hibridomas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Ovalbumina/administração & dosagem , Ovalbumina/genética , Ovalbumina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Peptídeo Hidrolases/fisiologia , Proteínas Periplásmicas de Ligação/administração & dosagem , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismoRESUMO
Adenylate cyclase toxin (CyaA) is one of the major virulence factors produced by Bordetella pertussis, the whooping cough agent. CyaA belongs to the repeat in toxin protein family and requires a post-translational fatty acylation to form cation-selective channels in target cell membranes and to penetrate into cytosol. We have demonstrated recently that CyaA uses the alphaMbeta2 integrin (CD11b/CD18) as a specific cellular receptor. Here we show that the acylation of CyaA is required for a productive and tight interaction of the toxin with cells expressing CD11b. In addition, we demonstrate that the catalytic domain is not required for binding of CyaA to CD11b and that the main integrin interacting domain of CyaA is located in its glycine/aspartate-rich repeat region. These data decipher, for the first time, the interaction of CyaA with CD11b-positive cells and open new prospects for understanding the interaction of Bordetella pertussis with innate and adaptive immune systems.