Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins.
Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400 amino acid 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 CD8+ T-cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted cytotoxic T-cell (CTL) responses in vivo. To explore the tolerance of CyaA to insertion of polypeptides of larger size, we constructed and characterized different recombinant CyaA toxins with protein inserts of 87 to 206 amino acids in length. Several of these recombinant CyaA toxins were found to be invasive. Furthermore, we showed that the unfolding of the passenger protein is a prerequisite for the translocation of the recombinant toxins into eukaryotic cells. Our results highlight the remarkable tolerance of the CyaA toxin and suggest that CyaA might be used to deliver proteins into eukaryotic cells.
Gmira
S
S
Unité de Biochimie Cellulaire, CNRS URA 2185, Institut Pasteur, Paris, France.
Karimova
G
G
Ladant
D
D
D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000485:Allergens; D052179:Antigens, Plant; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active; D001886:Bordetella pertussis / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D005057:Eukaryotic Cells / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D015696:Gene Products, tat / Q000378:metabolism; D005822:Genetic Vectors; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000235:genetics / Q000378:metabolism*
C081947:ASPF1 protein, Aspergillus fumigatus; D037361:Adenylate Cyclase Toxin; D000485:Allergens; D052179:Antigens, Plant; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005656:Fungal Proteins; D015696:Gene Products, tat; D011498:Protein Precursors; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; C036196:MITF protein, Aspergillus restrictus; D013762:Tetrahydrofolate Dehydrogenase; D012260:Ribonucleases; D000262:Adenylyl Cyclases