Karimova G, Fayolle C, Gmira S, Ullmann A, Leclerc C, Ladant D
Unité de Biochimie Cellulaire (Centre National de la Recherche Scientifique, Unité de Recherche Associée 1129), Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Proc Natl Acad Sci U S A. 1998 Oct 13;95(21):12532-7. doi: 10.1073/pnas.95.21.12532.
Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400-aa residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver T cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted CD8(+) cytotoxic T cell responses in vivo. Here, we constructed a series of recombinant toxins harboring at the same insertion site various peptide sequences of 11-25 amino acids, corresponding to defined CD8(+) T cell epitopes and differing in the charge of the inserted sequence. We show that inserted peptide sequences containing net negative charges (-1 or -2) decreased or completely blocked (charge of -4) the internalization of the toxin into target cells in vitro and abolished the induction of cytotoxic T cell responses in vivo. The blocking of translocation due to the inserted acidic sequences can be relieved by appropriate mutations in the flanking region of CyaA that counterbalance the inserted charges. Our data indicate that (i) the electrostatic charge of the peptides inserted within the catalytic domain of CyaA is critical for its translocation into eukaryotic cells and (ii) the delivery of T cell epitopes into the cytosol of antigen-presenting cells by recombinant CyaA toxins is essential for the in vivo stimulation of specific cytotoxic T cells. These findings will help to engineer improved recombinant CyaA vectors able to stimulate more efficiently cellular immunity.
百日咳博德特氏菌分泌一种钙调蛋白激活的腺苷酸环化酶毒素CyaA，它能够将其N端催化结构域（400个氨基酸残基）直接通过细胞质膜递送到真核靶细胞的细胞质中。我们先前已经表明，CyaA可以用作载体，将插入毒素催化结构域内的T细胞表位递送到抗原呈递细胞中，并能在体内触发特定的I类限制性CD8(+)细胞毒性T细胞反应。在此，我们构建了一系列重组毒素，它们在相同的插入位点含有各种11至25个氨基酸的肽序列，这些序列对应于确定的CD8(+) T细胞表位，并且插入序列的电荷不同。我们发现，含有净负电荷（-1或-2）的插入肽序列在体外降低或完全阻断（-4电荷）了毒素进入靶细胞的内化，并在体内消除了细胞毒性T细胞反应的诱导。由于插入酸性序列导致的转位阻断可以通过CyaA侧翼区域的适当突变来缓解，这些突变可以抵消插入的电荷。我们的数据表明：（i）插入CyaA催化结构域内的肽的静电荷对其转运到真核细胞中至关重要；（ii）通过重组CyaA毒素将T细胞表位递送到抗原呈递细胞的细胞质中对于体内刺激特异性细胞毒性T细胞至关重要。这些发现将有助于设计出能够更有效地刺激细胞免疫的改良重组CyaA载体。
