The Eukaryotic Host Factor 14-3-3 Inactivates Adenylate Cyclase Toxins of Bordetella bronchiseptica and B. parapertussis, but Not B. pertussis.

Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis share highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles in Bordetella infections. We herein demonstrate that CyaAs function as virulence factors differently between B. bronchiseptica/B. parapertussis and B. pertussisBbronchiseptica CyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly to Bpertussis CyaA. The hemolytic activity of Bbronchiseptica CyaA on sheep erythrocytes was also preserved. However, in nucleated target cells, Bbronchiseptica CyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects of Bbronchiseptica CyaA based on its enzyme activity were markedly attenuated. Bparapertussis CyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly to Bbronchiseptica CyaA; however, Bpertussis CyaA has Phe375 instead of Ser, and thus, was not affected by 14-3-3. In addition, Bpertussis CyaA impaired the barrier function of epithelial cells, whereas Bbronchiseptica CyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity between B. bronchiseptica/Bparapertussis and B. pertussisIMPORTANCEBordetella pertussis, B. bronchiseptica, and B. parapertussis are bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by each Bordetella species. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor of Bordetella The toxins of B. bronchiseptica and presumably B. parapertussis were inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas the B. pertussis toxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences in Bordetella are attributed to the different activities of adenylate cyclase toxins.

Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated α subunit of the heterotrimeric GTPase Gq.

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 (G(q), G(12/13) and G(i))-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 Gα(q), to detect the actions of PMT by immunological techniques such as western blotting. Using the monoclonal antibodies, we found that the toxin deamidated Gα(q) 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 Gα(11), as well as Gα(q), could be a substrate for PMT.

Crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins.

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 ß-pore-forming toxins such as aerolysin, C. perfringens ε-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of ß-strands, two of which span its entire length. Domain II and domain III have three short ß-strands each, by which they are distinguished. In addition, domain II has an α-helix lying on the ß-strands. The sequence of amino acids composing the α-helix and preceding ß-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming ß-barrel-made pores. These structural features imply that CPE is a ß-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long ß-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins.

Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G(q)- and G(12/13)-dependent pathways through the deamidation of a glutamine residue in the alpha-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575-1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590-670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT DeltaC1(4H)) neither localized to the plasma membrane nor stimulated the G(q/12/13)-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT DeltaC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.

Clostridium perfringens enterotoxin interacts with claudins via electrostatic attraction.

Clostridium perfringens enterotoxin (CPE), a causative agent of food poisoning, is a pore-forming toxin disrupting the selective permeability of the plasma membrane of target cells, resulting in cell death. We previously identified claudin as the cell surface receptor for CPE. Claudin, a component of tight junctions, is a tetratransmembrane protein and constitutes a large family of more than 20 members, not all of which serve as the receptor for CPE. The mechanism by which the toxin distinguishes the sensitive claudins is unknown. In this study, we localized the region of claudin responsible for interaction with CPE to the C-terminal part of the second extracellular loop and found that the isoelectric point of this region in sensitive claudins was higher than insensitive claudins. Amino acid substitutions to lower the pI resulted in reduced sensitivity to CPE among sensitive claudins, whereas substitutions to raise the pI endowed CPE-insensitive claudins with sensitivity. The steric structure of the claudin-binding domain of CPE reveals an acidic cleft surrounded by Tyr(306), Tyr(310), Tyr(312), and Leu(315), which were reported to be essential for interaction with the sensitive claudins. These results imply that an electrostatic attraction between the basic claudin region and the acidic CPE cleft is involved in their interaction.

Enhancement of Shiga toxin production in enterohemorrhagic Escherichia coli serotype O157:H7 by DNase colicins.

Colicins are proteins produced by and active against several strains of Escherichia coli. Previously we reported that colicinogenic bacteria seemed beneficial in preventing the clinical manifestations of infectious disease caused by enterohemorrhagic E. coli O157 in humans. The inhibitory effects could be due to a decrease in O157 levels and/or pathogenicity. This study investigated the effects of colicinogenic E. coli on the production of Shiga toxin (Stx) by O157. Standard strains of colicinogenic bacteria carrying plasmids for each type of colicin (E3/5/8/9) were used for the study. The O157 strains were cultured in the presence of colicinogenic bacteria or extracted colicins. Compared with results for controls, DNase colicins (E8/9) facilitated an 8- to 64-fold increase in production of Stx2, while RNase colicins (E3/5) suppressed Stx production in only two strains. Stx prophages were induced in synchrony with Stx production. Semiquantitative real-time reverse transcription-PCR (RT-PCR) was then performed to examine SOS gene expression. The RT-PCR results clearly indicated a marked increase in mRNA levels of SOS reaction-associated genes after the addition of DNase colicins. We believe that Stx prophages are induced by the SOS response to DNA damage caused by DNase colicins, thus leading to higher Stx production. These findings suggest that while colicinogenic bacteria can be antagonistic to O157 infection, DNase colicins may enhance Stx production. Thus, colicinogenic flora is likely to be involved in the complex pathogenic pathways of O157 infection, and further investigation should be performed before the use of colicinogenic bacteria as an intervention method.

Detection and isolation of Escherichia coli with a coding gene for enteroaggregative Escherichia coli heat-stable enterotoxin 1 from food and comparison with fecal isolates.

Enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) was originally regarded as a putative enterotoxin of enteroaggregative Escherichia coli. Although its etiological role has not yet been elucidated, it has been epidemiologically suggested that some strains of E. coli possessing EAST1-coding gene (astA) but no other identifiable pathogenic properties comprise a new group of diarrhea-associated E. coli (EAST1EC). However, the source of the organisms and their prevalence in foods are still obscure. In this study, methods for detection of the organisms in foods heavily contaminated with coliforms were evaluated and properties of the isolated strains were compared with those of fecal strains. Four enrichment methods (brilliant green lactose bile broth, E. coli, lauryl tryptose broth, and a combination of brain heart infusion broth and tryptone phosphate broth) were evaluated through inspection of 115 samples. PCR showed positive results in 26 samples after enrichment with a combination of brain heart infusion broth and tryptone phosphate broth, and EAST1EC was successfully isolated from 18 samples. Fifteen samples showed a positive reaction in the PCR test after enrichment by the other methods, and the organisms were isolated from only 10 specimens. The highest prevalence of EAST1EC was found in animal products (16 of 54, 29.6%); the organism was rarely found in foods of plant origin (2 of 45, 4.4%) or fishery products (1 of 16, 6.3%). Although EAST1EC is unexpectedly common in animal products, its potential as a human pathogen remains uncertain because the possession of some virulence properties differs significantly between strains from fecal specimens and those from foods. Some food isolates, however, possess the same characteristics as diarrheal isolates do. It is necessary to clarify the pathogenicity of EAST1EC and the significance of food as a source of infection.