Bordetella Dermonecrotic Toxin Research Articles

Hierarchical determinants in cytotoxic necrotizing factor (CNF) toxins driving Rho G-protein deamidation versus transglutamination.

The cytotoxic necrotizing factor (CNF) family of AB-type bacterial protein toxins catalyze two types of modification on their Rho GTPase substrates: deamidation and transglutamination. It has been established that E. coli CNF1 and its close homolog proteins catalyze primarily deamidation and Bordetella dermonecrotic toxin (DNT) catalyzes primarily transglutamination. The rapidly expanding microbial genome sequencing data have revealed that there are at least 13 full-length variants of CNF1 homologs. CNFx from E. coli strain GN02091 is the most distant from all other members of the CNF family with 50%-55% sequence identity at the protein level and 0.45-0.52 nucleotide substitutions per site at the DNA level. CNFx modifies RhoA, Rac1, and Cdc42, and like CNF1, activates downstream SRE-dependent mitogenic signaling pathways in human HEK293T cells, but at a 1,000-fold higher EC50 value. Unlike other previously characterized CNF toxins, CNFx modifies Rho proteins primarily through transglutamination, as evidenced by gel-shift assay and confirmed by MALDI mass spectral analysis, when coexpressed with Rho-protein substrates in E. coli BL21 cells or through direct treatment of HEK293T cells. A comparison of CNF1 and CNFx sequences identified two critical active-site residues corresponding to positions 832 and 862 in CNF1. Reciprocal site-specific mutations at these residues in each toxin revealed hierarchical rules that define the preference for deamidase versus a transglutaminase activity in CNFs. An additional unique Cys residue at the C-terminus of CNFx was also discovered to be critical for retarding cargo delivery.IMPORTANCECytotoxic necrotizing factor (CNF) toxins not only play important virulence roles in pathogenic E. coli and other bacterial pathogens, but CNF-like genes have also been found in an expanding number of genomes from clinical isolates. Harnessing the power of evolutionary relationships among the CNF toxins enabled the deciphering of the hierarchical active-site determinants that define whether they modify their Rho GTPase substrates through deamidation or transglutamination. With our finding that a distant CNF variant (CNFx) unlike other known CNFs predominantly transglutaminates its Rho GTPase substrates, the paradigm of "CNFs deamidate and DNTs transglutaminate" could finally be attributed to two critical amino acid residues within the active site other than the previously identified catalytic Cys-His dyad residues. The significance of our approach and research findings is that they can be applied to deciphering enzyme reaction determinants and substrate specificities for other bacterial proteins in the development of precision therapeutic strategies.

Bordetella Dermonecrotic Toxin Is a Neurotropic Virulence Factor That Uses CaV3.1 as the Cell Surface Receptor.

Dermonecrotic toxin (DNT) is one of the representative toxins produced by Bordetella pertussis, but its role in pertussis, B. pertussis infection, remains unknown. In this study, we identified the T-type voltage-gated Ca2+ channel CaV3.1 as the DNT receptor by CRISPR-Cas9-based genome-wide screening. As CaV3.1 is highly expressed in the nervous system, the neurotoxicity of DNT was examined. DNT affected cultured neural cells and caused flaccid paralysis in mice after intracerebral injection. No neurological symptoms were observed by intracerebral injection with the other major virulence factors of the organisms, pertussis toxin and adenylate cyclase toxin. These results indicate that DNT has aspects of the neurotropic virulence factor of B. pertussis The possibility of the involvement of DNT in encephalopathy, which is a complication of pertussis, is also discussed.IMPORTANCEBordetella pertussis, which causes pertussis, a contagious respiratory disease, produces three major protein toxins, pertussis toxin, adenylate cyclase toxin, and dermonecrotic toxin (DNT), for which molecular actions have been elucidated. The former two toxins are known to be involved in the emergence of some clinical symptoms and/or contribute to the establishment of bacterial infection. In contrast, the role of DNT in pertussis remains unclear. Our study shows that DNT affects neural cells through specific binding to the T-type voltage-gated Ca2+ channel that is highly expressed in the central nervous system and leads to neurological disorders in mice after intracerebral injection. These data raise the possibility of DNT as an etiological agent for pertussis encephalopathy, a severe complication of B. pertussis infection.

Type III Effector VopC Mediates Invasion for Vibrio Species

Vibrio spp. are associated with infections caused by contaminated food and water. A type III secretion system (T3SS2) is a shared feature of all clinical isolates of V. parahaemolyticus and some V. cholerae strains. Despite its being responsible for enterotoxicity, no molecular mechanism has been determined for the T3SS2-dependent pathogenicity. Here, we show that although Vibrio spp. are typically thought of as extracellular pathogens, the T3SS2 of Vibrio mediates host cell invasion, vacuole formation, and replication of intracellular bacteria. The catalytically active effector VopC is critical for Vibrio T3SS2-mediated invasion. There are other marine bacteria encoding VopC homologs associated with a T3SS; therefore, we predict that these bacteria are also likely to use T3SS-mediated invasion as part of their pathogenesis mechanisms. These findings suggest a new molecular paradigm for Vibrio pathogenicity and modify our view of the roles of T3SS effectors that are translocated during infection.

Bordetella dermonecrotic toxin binds to target cells via the N-terminal 30 amino acids

Bordetella dermonecrotic toxin (DNT) affects the biological function of host cells by activating intracellular Rho GTPases. The toxin binds to unidentified receptor(s) via 54 N-terminal amino acids, undergoes intramolecular cleavage on the C-terminal side of Arg(44) by furin or furin-like protease, and eventually enters the cytoplasm where the Rho GTPases reside. The binding to the receptor(s) and intramolecular cleavage are essential for DNT to intoxicate cells, and the 54 amino-acid binding domain encompasses the cleavage site, however, it is unclear whether these two events are related. In this study, we could narrow down the cell-binding domain to the N-terminal amino acids 2-30. The region does not contain the furin-recognition site, indicating that the cell binding and the intramolecular cleavage are independent events.

Pleiotropic role of Rac in mast cell activation revealed by a cell permeable Bordetella dermonecrotic fusion toxin

To activate the GTPase Rac in rat basophilic leukemia (RBL) cells and mouse bone marrow-derived mast cells (BMMC) a TAT fusion toxin of Bordetella dermonecrotic toxin (DNT-TAT) was constructed. The fusion toxin activated Rac1 and RhoA in vitro but only Rac in RBL cells and BMMC. DNT-TAT caused an increase in inositol phosphate formation, calcium mobilization, ERK activation and degranulation of mast cells. All these effects were inhibited by the Rho GTPase-inactivating Clostridium difficile toxin B and Clostridium sordellii lethal toxin. Also the calcium ionophore A23187 caused mast cell activation, including ERK phosphorylation, by processes involving an activation of Rac. The data indicate pleiotropic functions of Rac in mast cell activation.

Association of Bordetella dermonecrotic toxin with the extracellular matrix

BackgroundBordetella dermonecrotic toxin (DNT) causes the turbinate atrophy in swine atrophic rhinitis, caused by a Bordetella bronchiseptica infection of pigs, by inhibiting osteoblastic differentiation. The toxin is not actively secreted from the bacteria, and is presumed to be present in only small amounts in infected areas. How such small amounts can affect target tissues is unknown.ResultsFluorescence microscopy revealed that DNT associated with a fibrillar structure developed on cultured cells. A cellular component cross-linked with DNT conjugated with a cross-linker was identified as fibronectin by mass spectrometry. Colocalization of the fibronectin network on the cells with DNT was also observed by fluorescence microscope. Several lines of evidence suggested that DNT interacts with fibronectin not directly, but through another cellular component that remains to be identified. The colocalization was observed in not only DNT-sensitive cells but also insensitive cells, indicating that the fibronectin network neither serves as a receptor for the toxin nor is involved in the intoxicating procedures. The fibronectin network-associated toxin was easily liberated when the concentration of toxin in the local environment decreased, and was still active.ConclusionsComponents in the extracellular matrix are known to regulate activities of various growth factors by binding and liberating them in response to alterations in the extracellular environment. Similarly, the fibronectin-based extracellular matrix may function as a temporary storage system for DNT, enabling small amounts of the toxin to efficiently affect target tissues or cells.

DERMONECROTIC TOXIN: THE OLD BUT NEW VIRULENCE FACTOR PRODUCED BY BORDETELLA SPP.

Bordetella dermonecrotic toxin (DNT) is a virulence factor produced by bacteria belonging to the genus Bordetella including B. pertussis, B. parapertussis, and B. bronchiseptica. Intensive studies have clarified the structure and function of DNT in the last decade long after its discovery by Bordet and Gengou (). DNT is a single-chain polypeptide composed of 1464 amino acids and possesses novel transglutaminase activity that catalyzes polyamination or deamidation of the small GTPases of the Rho family. The modified GTPases lose their GTP hydrolyzing activity, function as a constitutive active molecule, and continuously transduce signals to downstream effectors, which mediate various abnormal phenotypes in intoxicated cells.

Bordetella Dermonecrotic Toxin Exerting Toxicity through Activation of the Small GTPase Rho

Bordetella dermonecrotic toxin (DNT) is a virulence factor produced by bacteria belonging to the genus Bordetella. The toxin possesses novel transglutaminase activity that catalyzes polyamination or deamidation of the small GTPases of the Rho family. The modified GTPases loose their GTP hydrolyzing activity, function as a constitutive active molecule, and continuously transduce signals to downstream effectors, which mediate the consequent phenotypes of cells intoxicated by DNT. A dynamin-dependent endocytosis is required for the toxin to be internalized into cells although it is unlikely transported to deep organelles such as the Golgi apparatus or the ER. Several lines of evidence show that the toxin undergoes proteolytic cleavage by furin or furin-like protease probably in the early endosome, and then escapes into the cytoplasm to reach the GTPase.

Isotype-specific Degradation of Rac Activated by the Cytotoxic Necrotizing Factor 1

The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli activates members of the Rho family by deamidation of glutamine 61/63. Because this amino acid is crucial for GTP hydrolysis, deamidation of glutamine 61/63 results in constitutively active Rho proteins. Recently, it was shown that the level of CNF1-activated Rac is rapidly diminished in CNF1-treated cells by proteolytic degradation. Here, we studied the requirements for CNF1-induced Rac degradation. By overexpressing His-tagged activated Rac mutants we show that constitutive activation is necessary for degradation of Rac. However, permanent activation is not sufficient for degradation, because Rac that is constitutively activated by transamidation at glutamine 61 by the Bordetella dermonecrotic toxin is not degraded. Overexpression of His-tagged Rac mutants deficient in interaction with GTPase-activating protein (Rac(N92D) and Rac(Y64H)) and guanosine nucleotide dissociation inhibitor (Rac(H103E)) were degraded after activation by CNF1, whereas Rac(Y40C), which is not able to interact with CRIB domain effectors or plenty of SH3, was not degraded. Isoprenylation and the presence of a putative mitotic destruction box are essential for CNF-induced degradation. In contrast to Rac1, Rac2, and Rac3 were not degraded following constitutive activation by CNF1. Using site-directed mutagenesis, we defined the polybasic region and amino acids 90, 107, 147, and 151 as responsible for isotype-specific degradation.

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.

Escherichia coli cytotoxic necrotizing factors and Bordetella dermonecrotic toxin: the dermonecrosis-inducing toxins activating Rho small GTPases

Escherichia coli cytotoxic necrotizing factors (CNFs) and Bordetella dermonecrotic toxin (DNT) have been recently found to comprise a novel family of dermonecrosis-inducing toxins which activate the small GTPases of the Rho family. They are single chain polypeptides consisting of an N-terminal domain responsible for binding to target cells and a C-terminal catalytic domain. CNFs (CNF1 and 2) and DNT share in the catalytic domain about 30% identical residues and a consensus sequence where the catalytically active center Cys resides. Both toxins deamidate Rho and other members of the Rho family, Rac and Cdc42, at Gln in the switch II region, which plays an important role in their GTPase activity. DNT, in addition, catalyzes a cross-link of the Gln of the GTPases with ubiquitous polyamines such as putrescine, spermidine, and spermine. The deamidation and the polyamination result in abrogation of the GTPase activity, and in addition, the polyamination endows Rho with the ability to interact with a downstream effector, ROCK, in a GTP-independent manner. These effects render the GTPases constitutively active, which underlies the toxicities of CNFs and DNT.

The pathological effect of the Bordetella dermonecrotic toxin in mice.

The effect of dermonecrotic toxin (DNT) expression of Bordetella bronchiseptica was studied in mice by comparing the pathology induced by a wild type strain with that induced by an isogenic DNT- strain in which part of the structural gene has been replaced by an antibiotic resistance cassette. While extracts of strain B58 proved toxic in intravenously inoculated mice, similar extracts from strain B58GP had lost toxic activity. The parent (B58) and the mutant (B58GP) strains of B. bronchiseptica each possessed comparable virulence for mice. These findings confirmed that DNT production was successfully abolished in strain B58GP while other virulence characteristics required for pathogenicity in mice remained intact, at a comparable level to the parent strain. Turbinate atrophy was observed in mice infected with the DNT+ strain, but not in those infected with the DNT- strain. This indicates that DNT is the cause of turbinate atrophy in the mice and not other factors produced by phase I strains of B. bronchiseptica. B. bronchiseptica DNT showed a lienotoxic effect (lymphocyte depletion and a reduction in the intensity of extramedullar haemocytopoieis) that is considered to adversely alter the immune function of the host animal. In mice infected with strain B58GP, catarrhal pneumonia with characteristic lympho-histiocytic peribronchial and perivascular infiltration was noticed. In mice infected with strain B58, large necrotic areas were seen surrounded by an inflammatory reaction. The DNT appears to directly damage lung tissues, at least in mice. DNT production seems to enhance the establishment of B. bronchiseptica in the lungs, presumably by reducing the local resistance and causing severe local damage to the lung tissues.

Identification of the C-terminal Part of BordetellaDermonecrotic Toxin as a Transglutaminase for Rho GTPases

Bordetella dermonecrotic toxin (DNT) causes the deamidation of glutamine 63 of Rho. Here we identified the region of DNT harboring the enzyme activity and compared the toxin with the cytotoxic necrotizing factor 1, which also deamidates Rho. The DNT fragment (DeltaDNT) covering amino acid residues 1136-1451 caused deamidation of RhoA at glutamine 63 as determined by mass spectrometric analysis and by the release of ammonia. In the presence of dansylcadaverine or ethylenediamine, DeltaDNT caused transglutamination of Rho. Deamidase and transglutaminase activities were blocked in the mutant proteins Cys(1292) --> Ala, His(1307) --> Ala, and Lys(1310) --> Ala of DeltaDNT. Deamidation and transglutamination induced by DeltaDNT blocked intrinsic and Rho- GTPase-activating protein-stimulated GTPase activity of RhoA. DeltaDNT deamidated and transglutaminated Rac and Cdc42 in the absence and presence of ethylenediamine, respectively. Modification of Rho proteins by DeltaDNT was nucleotide-dependent and did not occur with GTPgammaS-loaded GTPases. In contrast to cytotoxic necrotizing factor, which caused the same kinetics of ammonia release in the absence and presence of ethylenediamine, ammonia release by DeltaDNT was largely increased in the presence of ethylenediamine, indicating that DeltaDNT acts primarily as a transglutaminase.

Identification of the Region of Rho Involved in Substrate Recognition by Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) and the Bordetella dermonecrotic toxin (DNT) activate Rho GTPases by deamidation of Gln(63) of RhoA (Gln(61) of Cdc42 and Rac). In addition, both toxins possess in vitro transglutaminase activity in the presence of primary amines. Here we characterized the region of Rho essential for substrate recognition by the toxins using Rho/Ras chimeras as protein substrates. The chimeric protein Ras55Rho was deamidated or transglutaminated by CNF1. Rat pheochromocytoma PC12 cells microinjected with Ras55Rho developed formation of neurite-like structures after treatment with the CNF1 holotoxin indicating activation of the Ha-Ras chimera and Ras-like effects in intact cells. The Ras59Rho78Ras chimera protein contained the minimal Rho sequence allowing deamidation or transglutamination by CNF1. A peptide covering mainly the switch II region and consisting of amino acid residues Asp(59) through Asp(78) of RhoA was substrate for CNF1. Changes of amino acid residues Arg(68) or Leu(72) of RhoA into the corresponding residues of Ras (R68ARhoA and L72QRhoA) inhibited deamidation and transglutamination of the mutants by CNF1. In contrast to CNF1, DNT did not modify Rho/Ras chimeras or the switch II peptide (Asp(59) through Asp(78)). Glucosylation of RhoA at Thr(37) blocked deamidation by DNT but not by CNF. The data indicate that CNF1 recognizes Rho GTPases exclusively in the switch II region, whereas the substrate recognition by DNT is characterized by additional structural requirements.