Bordetella Dermonecrotic Toxin Undergoes Proteolytic Processing to Be Translocated from a Dynamin-related Endosome into the Cytoplasm in an Acidification-independent Manner

Abstract

Bordetella pertussis dermonecrotic toxin (DNT), which activates intracellular Rho GTPases, is a single chain polypeptide composed of an N-terminal receptorbinding 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 (⌬B) 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. ⌬B, 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 ⌬B, dissociated from the binding domain, fully possesses the ability to enter the cytoplasm across the lipid bilayer membrane. The translocation ability of ⌬B 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. ⌬B, liberated from the binding domain after the processing, begins to translocate the enzymatic domain into the cytoplasm.Bacterial protein toxins that enzymatically modify cytosolic substances of eukaryotic cells consist of functionally distinct domains. The designation A-B toxin refers to toxins composed of an A domain conducting enzymatic action and a B domain binding to a surface receptor on target cells. In addition, these toxins are equipped with transmembrane domains carrying a delivery system to transport the A domain across lipid bilayer membranes after the B domain binds to the receptor. The A-B toxins are classified into at least three groups on the basis of structure (1). In the first group, which includes diphtheria toxin, botulinum neurotoxin, and Bordetella pertussis adenylate cyclase toxin, both the A and B domains originally reside on a single polypeptide chain. The toxins of the second group possess the A and B domains on different subunits that are noncovalently associated with each other. Cholera toxin, pertussis toxin, and Shiga toxin belong to this group. The third group is composed of binary toxins, in which components carrying the A and B domains are produced as distinct peptides and assembled on the target cells. The toxins of this type are exemplified by botulinum C2 toxin, anthrax toxin, and Clostridium perfringens -toxin. Regardless of molecular structure, many of the A-B toxins undergo prot...

Molecular Pathogenesis, Epidemiology, and Clinical Manifestations of Respiratory Infections Due toBordetella pertussisand OtherBordetellaSubspecies

“…Translocation is independent of acidification of endosomes and retrograde vesicular transport and requires the N-terminal region of the DNT enzymatic domain, which includes a putative transmembrane domain. On endocytosis, DNT undergoes proteolytic nicking by mammalian proteases such as furin, which is necessary for the cellular activity of DNT (502).…”

Inhibition of Furin/Proprotein Convertase-catalyzed Surface and Intracellular Processing by Small Molecules

“…Furin activity contributes to numerous chronic pathological conditions, including Alzheimer disease (8), other non-Alzheimer cerebral amyloidoses (9), osteoarthritis (10), atherosclerosis (11), and tumor progression and malignancy (12). Moreover, activation by host cells of bacterial toxins such as anthrax toxin, Pseudomonas exotoxin A, diphtheria toxin (13), Shiga toxin (14), and Bordetella dermonecrotic toxin (15), requires cleavage by furin or other PCs. Furin or furin-like cleavage of viral envelope glycoproteins is necessary for propagation of many lipid-enveloped viral pathogens including H5N1 avian influenza (16), human immunodeficiency virus-1 (17), ebola (18), measles (19), cytomegalovirus (20), and flaviviruses (21).…”

CNF and DNT

“…Proteolytic processing of DNT by furin seems to be necessary for translocation of the toxin across cellular membranes. After cleavage, the translocation seems to be independent of the DNT receptor (Matsuzawa et al 2003).…”