Identification of functional domains of Bordetella dermonecrotizing toxin

Bordetella dermonecrotizing toxin (DNT) stimulates the assembly of actin st ress fibers and focal adhesions by deamidating Gln63 of the small GTPase Rh o. To clarify the functional and structural organization of DNT, we cloned and sequenced the DNT gene and examined the functions of various DNT mutant s. Our analyses of the nucleotide and amino acid sequences revealed that th e start codon of the DNT gene is a GTG triplet located 39 bp upstream of th e reported putative initiation ATG codon; consequently, DNT contains an add itional 13 amino acids at its N-terminal end. All of the N-terminally trunc ated mutants were found to modify Rho. The shortest fragment of DNT possess ing the Rho modification activity consists of amino acids from Ile1176 to t he C-terminal end. This fragment overlaps the region homologous to Escheric hia coli cytotoxic necrotizing factors (CNFs), which show activity similar to that of DNT. The introduction of a mutation at Cys1305 located in the hi ghly conserved region between CNFs and DNT eliminated the activity, indicat ing that this domain is the catalytic center of DNT. The N-terminal fragmen t (1 to 531) of DNT failed to modify Rho but reduced the DNT-induced polynu cleation in MC3T3-E1 cells when simultaneously added with the holotoxin, su ggesting competitive inhibition in the receptor-binding or internalizing st ep. Our finding that DNT consists of an N-terminal receptor-binding and/or internalizing domain and a C-terminal catalytically active domain may facil itate analysis of the overall action of the toxin on the mammalian target c ells.