Yoichi Kamata

The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a.

125I‐labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high‐ (0.4 nM) and low‐affinity (4.1 nM) binding sites in synaptosomes. The high‐affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino‐terminal region of synaptotagmin II. These results suggest that this region of synaptotagmin II participates in the formation of the high‐affinity toxin binding site by associating with specific gangliosides.

Identification of Kudoa septempunctata as the Causative Agent of Novel Food Poisoning Outbreaks in Japan by Consumption of Paralichthys olivaceus in Raw Fish

BACKGROUND Outbreaks of an unidentified food-borne illness associated with the consumption of raw fish have increased in Japan since 2003. Those affected with this illness develop diarrhea and emesis within 2-20 hours after a meal including raw fish. No known causative agents such as bacteria, viruses, bacterial toxins, or toxic chemicals have been detected in the foods that were ingested. Fortunately, this illness is self-limiting with good prognosis in all cases. METHODS We conducted an epidemiological analysis of outbreaks that occurred during 2008 and 2010 and analysed a fish sample from one outbreak by metagenomic DNA sequencing, real-time polymerase chain reaction, and direct microscopic observations. The pathogenicity of a putative risk factor identified by these techniques was assessed using the suckling-mouse test and a house musk shrew emetic assay. RESULTS The epidemiological analysis of outbreaks in 24 municipalities involving >1300 subjects implicated an olive flounder (Paralichthys olivaceus) as the causative food source. The presence of Kudoa septempunctata, a recently-described myxosporean species in P. olivaceus, was prevalent in the causative foods. K. septempunctata induced watery stools and an elevated fluid accumulation ratio in suckling mice, as well as vomiting in house musk shrews. CONCLUSIONS These results identify K. septempunctata as the etiological agent of this novel food-borne illness outbreak associated with consumption of raw P. olivaceus. This is the first report, to our knowledge, demonstrating the human pathogenicity of Kudoa spores.

Kudoa septempunctata n. sp. (Myxosporea: Multivalvulida) from an aquacultured olive flounder (Paralichthys olivaceus) imported from Korea

A new myxosporean species, Kudoa septempunctata n. sp. (Myxosporea: Multivalvulida), is described from the trunk muscles of an aquacultured olive flounder (Paralichthys olivaceus) imported from Korea. This species formed pseudocysts in the myofiber without inflammatory reactions, and the infection was not evident macroscopically. Spores of the new species were irregularly stellate in apical view, with the majority having seven unequal valves, each with a polar capsule of variable size (the remaining spores had six valves and polar capsules). The spores had dimensions of: width 11.8 (11.1–13.1); thickness 9.4 (8.9–10.0); length 8.5 (7.9–8.9); polar capsule length 4.6 (3.7–5.3); and polar capsule width 2.4 (2.2–2.8; mean with range in parentheses; n = 10; all measurements in micrometers). Scanning electron microscopy of the spores revealed unequal positioning of the seven valves without a definite center, rounded posterior ends of valves, and tiny projections at the apex of each valve. The small subunit ribosomal RNA gene (SSU rDNA) sequence of the new species was closely related to Kudoa spp. with five or more valves, particularly Kudoa thalassomi (97.6% identity) recorded from the trunk muscles of a moon wrasse (Thalassoma lunare) around the Australian continent. However, the latter species has six valves with a pointed edge and six polar capsules of a uniform size. The new species was also distinct from all presently known Kudoa spp. with seven valves and polar capsules, i.e. Kudoa yasunagai and Kudoa lethrini, regarding tissue tropism (trunk muscles versus brain), spore shape or external appendages, and SSU rDNA sequence.

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

Rapid detection of nivalenol and deoxynivalenol in wheat using surface plasmon resonance immunoassay.

A surface plasmon resonance (SPR) immunoassay using a monoclonal antibody was developed to measure nivalenol (NIV) and deoxynivalenol (DON) contamination in wheat. A highly sensitive and stable DON-immobilized sensor chip was prepared, and an SPR detection procedure was developed. The competitive inhibition assay used a monoclonal antibody that cross-reacts with NIV and DON. The half maximal inhibitory concentration (IC(50)) values of the SPR assay were 28.8 and 14.9 ng mL(-1) for NIV and DON, respectively. The combined responses of NIV and DON in wheat were obtained using a simultaneous detection assay in a one-step cleanup procedure. NIV and DON were separated using a commercial DON-specific immunoaffinity column (IAC) and their responses were obtained using an independent detection assay. Spiked tests using these toxins revealed that recoveries were in the range 91.5-107% with good relative standard deviations (RSDs) (0.40-4.1%) and that detection limits were 0.1 and 0.05 mg kg(-1) for NIV and DON, respectively. The independent detection using IAC showed detection limits of 0.2 and 0.1 mg kg(-1) for NIV and DON, respectively. SPR analysis results were correlated with those obtained using a conventional LC/MS/MS method for wheat co-contaminated with NIV and DON. These results suggested that the developed SPR assay is a practical method to rapidly screen the NIV and DON co-contamination of wheat and one of a very few immunoassays to detect NIV directly.

Distinct characters of Clostridium botulinum type A strains and their toxin associated with infant botulism in Japan.

Four strains of Clostridium botulinum type A having been associated with infant botulism in Japan, and another strain isolated from honey not associated with infant botulism, were found to be hemagglutinin (HA) negative. These strains do not produce L (Mr 500 kDa) nor LL toxin (Mr 900 kDa) but M toxin (Mr 300 kDa) only. No marked difference was found between the HA-positive and HA-negative strains in other biochemical properties, but the HA-negative strains tended to colonize more easily in the intestines of infant mice than did HA-positive strains. The toxin of HA-positive strains and that of HA-negative strains differed in the antigenicity of part of the toxic component and that of the nontoxic component, and in the molecular size of the toxic component.

Kudoa iwatai and two novel Kudoa spp., K. trachuri n. sp. and K. thunni n. sp. (Myxosporea: Multivalvulida), from daily consumed marine fish in western Japan

Infection of marine fish by certain myxosporean species of the genus Kudoa results in unsightly cyst formation in the trunk muscle or post-mortem myoliquefaction, causing a great economic loss to aquaculture industries, capture fisheries, and fish dealers. In addition, consumers encountering unsightly Kudoa cysts in fish fillets believe them to be unknown foreign materials acquired during processing. To identify prevalent Kudoa spp. encountered in daily life by the Japanese population, fresh fish slices (sashimi) or fish fillets with whitish spots were collected during a 7-month period (May to December 2008) at local markets in the city of Yamaguchi, western Japan. Kudoa cysts were found in three Japanese seaperches (Lateolabrax japonicus), two black sea bream (Acanthopagrus schlegelii), two Japanese jack mackerel (Trachurus japonicus), and one albacore (Thunnus alalunga). Kudoa iwatai was identified in all the examined Japanese seaperch and black sea bream from Japan’s Inland Sea, as assessed by morphology and genetic analysis of the 18S and 28S ribosomal RNA gene (rDNA). Kudoa trachuri n. sp. from two Japanese jack mackerel fished in the Japanese Sea off Nagasaki and Kudoa thunni n. sp. from one albacore fished in the Pacific Ocean had a spore, which was semiquadrate in shape in apical views and ovoid in lateral views, with four equal shell valves and drop-like polar capsules. Scanning electron microscopy revealed that these three Kudoa species had different types of small projections at the apex of each valve. The 18S and 28S rDNA sequences of K. trachuri n. sp. and K. thunni n. sp. were found to be closely related to those of Kudoa crumena; however, these sequences were distinct in each of the species, which additionally exhibited different morphological features.

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 Tyr306, Tyr310, Tyr312, and Leu315, 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.

Immunological Characterization of the Neurotoxin Produced by Clostridium botulinum Type A Associated with Infant Botulism in Japan

The neurotoxin associated with type A infant botulism in Japan shows different antigenic properties from those produced by authentic strains. The monoclonal antibodies recognizing the light chain reacted to both neurotoxins, whereas half the antibodies recognizing the heavy chain reacted specifically to the respective neurotoxin. Each neurotoxin showed its own manner of binding to brain synaptosomes. These results indicate that the distinguishable characteristics are ascribable to the heavy chain but not to the light chain. In both neurotoxins, an epitope recognized by the monoclonal antibody that reacts to the light chain and neutralizes the toxin was found to be very close to the amino‐terminal half (H‐1 fragment) of the heavy chain. This may support the hypothesis that the H‐1 fragment functions in the transport of the light chain in the target cell.

Kudoa septempunctata Invasion Increases the Permeability of Human Intestinal Epithelial Monolayer

Kudoa septempunctata is a myxosporean parasite of Paralichthys olivaceus (olive flounder) and causes a foodborne illness that affects more than 100 cases in Japan each year. We previously reported that the consumption of raw olive flounder meat containing a high concentration of K. septempunctata spores induces transient but severe diarrhea and emesis through an unknown mechanism. Here, we demonstrate that K. septempunctata sporoplasm plays an important role in mediating the toxicity of K. septempunctata. When K. septempunctata spores were inoculated in Caco-2 human intestinal cells, K. septempunctata sporoplasms were released from spores, and they invaded the cells. Electron microscopic observations revealed that the sporoplasm invasion severely damaged the Caco-2 cells. The inoculation of K. septempunctata spores eliminated the transepithelial electrical resistance (TER) across the cell monolayer. Inhibiting the invasion of the sporoplasms prevented the observed loss in cell layer integrity, as illustrated by the rapid elimination of the TER. These results suggest that the invasion by sporoplasms severely damaged individual intestinal cells, resulting in a loss of cell monolayer integrity.