Bordetella dermonecrotizing toxin causes assembly of actin stress fibers and focal adhesions in some cultured cells and induces mobility shifts of the small GTP-binding protein Rho on electrophoresis. We attempted to clarify the molecular basis of the toxin action on Rho. Analysis of the amino acid sequence of toxin-treated RhoA revealed the deamidation of Gln-63 to Glu. The substitution of Glu for Gln-63 of RhoA by site-directed mutagenesis caused a mobility shift on electrophoresis, which was indistinguishable from that of the toxintreated RhoA. Neither mutant RhoA-bearing Glu-63 nor toxintreated RhoA significantly differed from untreated wild type RhoA in guanosine 5-[␥-thio]triphosphate binding activity but both showed a 10-fold reduction in GTP hydrolysis activity relative to untreated RhoA. C3H10T1͞2 cells transfected with cDNA of the mutant RhoA bearing Glu-63 showed extensive formation of actin stress fibers similar to the toxin-treated cells. These results indicate that the toxin catalyzes deamidation of Gln-63 of Rho and renders it constitutively active, leading to formation of actin stress fibers.Dermonecrotizing toxin (DNT) produced by bacteria of the Bordetella species has lethal, dermonecrotic, and splenoatrophic activities (1-3). DNT is considered to be one of the virulence factors responsible for turbinate atrophy in swine atrophic rhinitis and has been suggested to damage bone tissues in turbinates (4, 5). Using osteoblast-like MC3T3-E1 cells, we demonstrated that DNT changes cell morphology and inhibits the differentiation into osteoblasts (6). The morphological changes in DNT-treated cells were accompanied by the assembly of actin stress fibers and focal adhesions, indicating anomalous activation of the small GTP-binding protein Rho (7). It was also found that Rho in the lysate from DNT-treated cells showed shifts in its electrophoretic mobility. Similar mobility shifts of Rho were observed by in vitro treatment of recombinant Rho with DNT. These results indicate that DNT covalently modifies and activates Rho, leading to the assembly of actin stress fibers and focal adhesions. However, the nature of the DNT-induced modification of Rho, which relates to its activation remains unknown.Here, we show that DNT catalyzes the deamidation of Gln-63 of RhoA and converts it into Glu. We conclude that the deamidated RhoA bearing Glu-63 becomes constitutively active and stimulates the formation of actin stress fibers in DNT-treated cells.

The small GTPase Rho, which regulates a variety of cell functions, also serves as a specific substrate for bacterial toxins. Here we demonstrate that Bordetella dermonecrotizing toxin (DNT) catalyzes cross‐linking of Rho with ubiquitous polyamines such as putrescine, spermidine and spermine. Mass spectrometric analyses revealed that the cross‐link occurred at Gln63, which had been reported to be deamidated by DNT in the absence of polyamines. Rac1 and Cdc42, other members of the Rho family GTPases, were also polyaminated by DNT. The polyamination, like the deamidation, markedly reduced the GTPase activity of Rho without affecting its GTP‐binding activity, indicating that polyaminated Rho behaves as a constitutively active analog. Moreover, polyamine‐linked Rho, even in the GDP‐bound form, associated more effectively with its effector ROCK than deamidated Rho in the GTP‐bound form and, when microinjected into cells, induced the anomalous formation of stress fibers indistinguishable from those seen in DNT‐treated cells. The results imply that the polyamine‐linked Rho, transducing signals to downstream ROCK in a novel GTP‐independent manner, plays an important role in DNT cell toxicity.

Summary Vibrio parahaemolyticus is a Gram-negative marine bacterium that causes acute gastroenteritis in humans. The virulence of V. parahaemolyticus is dependent upon a type III secretion system (T3SS2). One effector for T3SS2, VopC, is a homolog of the catalytic domain of cytotoxic necrotizing factor (CNF), and was recently reported to be a Rho family GTPase activator and to be linked to internalization of V. parahaemolyticus by nonphagocytic cultured cells. Here, we provide direct evidence that VopC deamidates Rac1 and CDC42, but not RhoA, in vivo. Our results also suggest that VopC, through its activation of Rac1, contributes to formation of actin stress fibers in infected cells. Invasion of host cells, which occurs at a low frequency, does not seem linked to Rac1 activation, but instead appears to require CDC42. Finally, using an infant rabbit model of V. parahaemolyticus infection, we show that the virulence of V. parahaemolyticus is not dependent upon VopC-mediated invasion. Genetic inactivation of VopC did not impair intestinal colonization nor reduce signs of disease, including fluid accumulation, diarrhea, and tissue destruction. Thus, although VopC can promote host cell invasion, such internalization is not a critical step of the disease process, consistent with the traditional view of V. parahaemolyticus as an extracellular pathogen.

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In this study, we compared the apoptotic activities of clinical and environmental isolates of Vibrio vulnificus toward macrophages in vitro and in vivo. The clinical isolates induced apoptosis in macrophage-like cells in vitro and in macrophages in vivo. This suggests that macrophage apoptosis may be important for the clinical virulence of V. vulnificus.Many bacterial pathogens that infect mammals have developed specific traits to avoid the innate and specific immune defenses of the host (3,6,12,17). A characteristic common to several invasive enteric pathogens (e.g., Shigella, Salmonella, and Yersinia species) is the ability to induce macrophage apoptosis via a type III protein secretion system (8,9,16,21). Macrophage apoptosis in response to Shigella and Salmonella infections triggers severe inflammation via the action of proinflammatory cytokines (22). Yersiniae induce apoptosis in macrophages by suppressing the signaling pathway that leads to the production of proinflammatory cytokines (1, 10). Macrophage cell death may lead to either the induction or the inhibition of an inflammatory response. In studying the interaction between phagocytes and Vibrio vulnificus, most efforts have focused on the capsule (7,11,13,18). Encapsulated isolates of V. vulnificus are more resistant to phagocytosis by human polymorphonuclear leukocytes and murine peritoneal macrophages than are unencapsulated isolates (4,5,15). However, the cytotoxic effects on phagocytes have not been clearly demonstrated. In this study, we examined the apoptotic effects of nine isolates of V. vulnificus on macrophages.First, we examined each isolate of V. vulnificus for apoptotic activity toward a macrophage-like cell line, J774, by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) analysis. Five clinical isolates of V. vulnificus, the K series of strains, were isolated from the blood of individual septicemic patients at Kurashiki Central Hospital in Japan between 1985 and 1999. Two environmental strains, E4 and E10, were isolated from seafood in Florida. Strain R41 was isolated from plankton in Okayama Prefecture in Japan. Strain G83 was isolated from seafood in the Republic of Korea. Bacteria in the logarithmic growth phase were obtained by cultivation with Luria-Bertani broth at 37°C. The desired bacterial concentration was checked by plating serial dilutions of the samples on agar and counting CFU after incubation. J774 cells were grown in Dulbecco's modified Eagle's medium (Gibco BRL Life Technologies, Rockville, Md.) supplemented with 2 mM glutamine, 2 mM sodium pyruvate, and 20% heattreated fetal calf serum. Cells were seeded in 24-well tissue culture plates at 10 5 cells/well. Each isolate of V. vulnificus was inoculated into the wells at a multiplicity of infection of approximately 1.0. After incubation at 37°C for 150 min, the Mebstain Apoptosis Kit (Immunotech, Marseilles, France) was used to label the free 3Ј-OH ends of DNA fragments with fluorescein as recommended by the manufacturer. The wellkno...

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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...

Dermonecrotic toxin (DNT), commonly produced by Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis, exerts lethal, dermonecrotic, and splenoatrophic activities in a variety of experimental animals (2,3,7,8,12,19). B. bronchiseptica DNT is considered to be responsible for turbinate atrophy in swine atrophic rhinitis (4, 6, 9). The turbinate atrophy caused by DNT likely results from a deficiency of osteoblastic differentiation in bone tissues (9, 11). DNT also alters cell morphology, stimulating the anomalous reorganization of actin stress fibers and focal adhesions, which is elaborately regulated by the small GTPase Rho (5, 10). The small GTPases function as a molecular switch regulating various cell functions besides the reorganization of actin cytoskeletons by changing between the GDP-bound inactive and the GTPbound active forms. The GDP-bound GTPases in resting cells exchange GDP for GTP in response to various stimulations, transduce the signals downstream by interacting with effector proteins, and thereafter revert to the GDP-bound inactive form by hydrolyzing the bound GTP. Our research group recently demonstrated that DNT was a transglutaminase catalyzing deamidation or polyamination at Gln63 of Rho and the corresponding Gln residues of the other members of the Rho family, Rac and Cdc42 (5, 18). The deamidation and polyamination result in a reduction of the GTP hydrolyzing activity. In addition, the polyaminated Rho comes to interact with a downstream effector, ROCK, in a GTP-independent manner (17). Thus, these modifications render the intracellular GTPases constitutively active, which probably mediates various effects of DNT on target cells.DNT is a single-chain polypeptide which consists of 1,464 amino acids with a calculated molecular mass of 160,602 (13). Previously, we localized the catalytic domain of DNT to the C-terminal region from Ile 1176 to the C-terminal end. We also found that the N-terminal fragment spanning Met 1 to Pro 531 of DNT competitively blocked the intoxication of cells by the full-length DNT (13), implying that this fragment retains the receptor-binding or internalizing property. In the present study, we attempted to define the N-terminal receptor-binding region of DNT by using a series of toxin mutants with various lengths and a monoclonal antibody (MAb) that neutralizes the toxin. The results presented here indicate that DNT binds to the cells through the N-terminal region consisting of 54 amino acids, in which the MAb recognized the region including Arg 44 . MATERIALS AND METHODSMaterials. DNT was purified from B. bronchiseptica S798 as described previously (8). The numbering of the amino acids of DNT was based on the sequence available from the DDBJ/EMBL/GenBank databases under accession no. AB020025. C3 exoenzyme was provided by S. Kozaki, University of Osaka Prefecture, Osaka, Japan. MC3T3-E1 cells were cultured at 37°C in ␣-minimum essential medium (␣-MEM; Gibco Laboratories, Grand Island, N.Y.) supplemented with 10% fetal calf serum under 5% CO 2 ...