Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å . The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of ␤-pore-forming toxins such as aerolysin, C. perfringens ⑀-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of ␤-strands, two of which span its entire length. Domain II and domain III have three short ␤-strands each, by which they are distinguished. In addition, domain II has an ␣-helix lying on the ␤-strands. The sequence of amino acids composing the ␣-helix and preceding ␤-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming ␤-barrel-made pores. These structural features imply that CPE is a ␤-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long ␤-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins. Clostridium perfringens enterotoxin (CPE),2 which damages intestinal epithelia, is a causative agent of food poisoning. The toxin consists of a single chain polypeptide of 319 amino acids. The C-terminal domain of CPE (C-CPE, residues 184 -319) recognizes and binds to certain members of the claudin family, components of tight junctions, as a receptor on target cells (1-4), and the N-terminal region is believed to be involved in forming physiological pores to disrupt the selective permeability of the plasma membrane, resulting in cell death (5-7). It was also reported that the physiological pores are composed of a large complex comprising CPE and cellular components such as claudins (2,8).The binding between C-CPE and claudins has been well characterized. The 16 -17 C-terminal amino acids of C-CPE were reportedly important for the interaction (9, 10), especially, Tyr 306 , Tyr 310 , Tyr 312, and Leu 315 (11-13). According to the crystal structure of C-CPE (14), these residues organize a cleft space that is considered to interact directly with claudins. Claudins are tetratransmembrane proteins. The region of claudins responsible for binding to CPE was located on the C-terminal side of the second extracellular loop and recently designated CPE-SR for CPE sensitivity-related region (15). The bottom of the cleft space of CPE is negatively charged, whereas the CPESRs of CPE-sensitive claudins are positively charged. Therefore, it was proposed that electrostatic attraction at...

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through the activation of heterotrimeric GTPase (Gq, G12/13 and Gi)‐dependent pathways, by deamidating a glutamine residue in the α subunit of these GTPases. However, the enzymatic characteristics of PMT are yet to be analyzed in detail because the deamidation has only been observed in cell‐based assays. In the present study, we developed rat monoclonal antibodies, specifically recognizing the deamidated Gαq, to detect the actions of PMT by immunological techniques such as western blotting. Using the monoclonal antibodies, we found that the toxin deamidated Gαq only under reducing conditions. The C‐terminal region of PMT, C‐PMT, was more active than the full‐length PMT. The C3 domain possessing the enzyme core catalyzed the deamidation in vitro without any other domains. These results not only support previous observations on toxicity, but also provide insights into the enzymatic nature of PMT. In addition, we present several lines of evidence that Gα11, as well as Gαq, could be a substrate for PMT.

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins’ gene(s) among the Genus Clostridium.

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. IMPORTANCE Bordetella 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.

Bordetella pertussis causes whooping cough, a severe and prolonged respiratory disease that results inhas high morbidity and mortality rates, particularly in developing countries. The number incidence of whooping cough cases is increasing in many countries despite high vaccine coverage. Causes for the re-emergence of the disease include the limited duration of protection conferred by the acellular pertussis vaccines (aP)s and pathogenic adaptations that involve antigenic divergence from vaccine strains. Therefore, current vaccines therefore need to be improved. In the present study, we focused on five autotransporters: namely SphB1, BatB, SphB2, Phg, and Vag8, which were previously found to be expressed by B. bronchiseptica during the course of infection in rats and examined their protective efficiencies as vaccine antigens. The passenger domains of these proteins were produced in recombinant forms and used as antigens. An intranasal murine challenge assay showed that immunization with a mixture of SphB1 and Vag8 (SV) significantly reduced bacterial load in the lower respiratory tract and a combination of aP and SV acts synergistically in effects of conferring protection against B. pertussis infection, implying that these antigens have potential as components to for improvinge th the currently available acellular pertussis vaccine.