Bordetella dermonecrotic toxin undergoes proteolytic processing to be translocated from a dynamin-related endosome into the cytoplasm in an acidification-independent manner.
Bordetella pertussis dermonecrotic toxin (DNT), which activates intracellular Rho GTPases, is a single chain polypeptide composed of an N-terminal receptor-binding domain and a C-terminal enzymatic domain. We found that DNT was cleaved by furin, a mammalian endoprotease, on the C-terminal side of Arg(44), which generates an N-terminal fragment almost corresponding to the receptor-binding domain and a C-terminal remainder (deltaB) containing the enzymatic domain. These two fragments remained associated even after the cleavage and made a nicked form. DNT mutants insensitive to furin had no cellular effect, whereas the nicked toxin was much more potent than the intact form, indicating that the nicking by furin was a prerequisite for action. DeltaB, but not the nicked toxin, associated with artificial liposomes and activated Rho in cells resistant to DNT because of a lack of surface receptor. These results imply that deltaB, dissociated from the binding domain, fully possesses the ability to enter the cytoplasm across the lipid bilayer membrane. The translocation ability of deltaB was found to be attributable to the N-terminal region encompassing amino acids 45-166, including a putative transmembrane domain. Pharmacological analyses with various reagents disturbing vesicular trafficking revealed that the translocation requires neither the acidification of the endosomes nor retrograde vesicular transport to deeper organelles, although DNT appeared to be internalized via a dynamin-dependent endocytosis. We conclude that DNT binds to its receptor and is internalized into endosomes where the proteolytic processing occurs. DeltaB, liberated from the binding domain after the processing, begins to translocate the enzymatic domain into the cytoplasm.
Department of Bacterial Toxinology, Research Institute for Microbial Diseases, Osaka University, Yamada-oka 3-1, Suita, Osaka 565-0871, Japan.
Takeshi
T
Matsuzawa
Aya
A
Fukui
Takashige
T
Kashimoto
Kaori
K
Nagao
Kiyomasa
K
Oka
Masami
M
Miyake
Yasuhiko
Y
Horiguchi
D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D001886:Bordetella pertussis / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D034281:Dynamins / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D006868:Hydrolysis; D021381:Protein Transport; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000737:chemistry / Q000378:metabolism*
D001427:Bacterial Toxins; D010566:Virulence Factors, Bordetella; C060969:dermonecrotic toxin, Bordetella; D011503:Transglutaminases; D034281:Dynamins