Yasuhiko Horiguchi

Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein

Claudins (claudin‐1 to ‐18) with four transmembrane domains and two extracellular loops constitute tight junction strands. The peptide toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to claudin‐3 and ‐4, but not to claudin‐1 or ‐2. We constructed claudin‐1/claudin‐3 chimeric molecules and found that the second extracellular loop of claudin‐3 conferred CPE sensitivity on L fibroblasts. Furthermore, overlay analyses revealed that the second extracellular loop of claudin‐3 specifically bound to CPE at the K a value of 1.0×108 M−1. We concluded that the second extracellular loop is the site through which claudin‐3 interacts with CPE on the cell surface.

Clostridium perfringens Enterotoxin Utilizes Two Structurally Related Membrane Proteins as Functional Receptors in Vivo

Human and mouse cDNAs showing homology to theClostridium perfringens enterotoxin (CPE) receptor gene (CPE-R) from Vero cells (DDBJ/EMBL/GenBankTMaccession no. D88492) (Katahira, J., Inoue, N., Horiguchi, Y., Matsuda, M., and Sugimoto, N. (1997) J. Cell Biol. 136, 1239–1247) were cloned. They were classified into two groups, the Vero cell CPE receptor homologues and rat androgen withdrawal apoptosis protein (RVP1; accession no. M74067) homologues, based on the similarities of primary amino acid sequences. L929 cells that were originally insensitive to CPE became sensitive to CPE on their transfection with cDNAs encoding either the CPE receptor or RVP1 homologues, indicating that these gene products are not only structurally similar but also functionally active as receptors for CPE. By binding assay, the human RVP1 homologue showed differences in affinity and capacity of binding from those of the human CPE receptor. Northern blot analysis showed that mouse homologues of the CPE receptor and RVP1 are expressed abundantly in mouse small intestine. The expression ofCPE-R mRNA in the small intestine was restricted to cryptic enterocytes, indicating that the CPE receptor is expressed in intestinal epithelial cells. These results are consistent with reports that CPE binds to the small intestinal cells via two different kinds of receptors. High levels of expression of CPE-R and/orRVP1 mRNA were also detected in other organs, including the lungs, liver, and kidneys, but only low levels were expressed in heart and skeletal muscles. These results indicate that CPE uses structurally related cellular proteins as functional receptors in vivo and that organs that have not so far been recognized as CPE-sensitive have the potential to be targets of CPE.

Activation of Rho through a cross-link with polyamines catalyzed by Bordetella dermonecrotizing toxin

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.

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin

Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575–1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a “feet”-,“body”-, and “head”-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys–His–Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying Cys–His–Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.

A Novel Tumor-Targeted Therapy Using a Claudin-4-Targeting Molecule

Carcinogenesis is often accompanied by dysfunctional tight junction (TJs), resulting in the loss of cellular polarity. Claudin, a tetra-transmembrane protein, plays a pivotal role in the barrier and fence functions of TJs. Claudin-4 is deregulated in various cancers, including breast, prostate, ovarian, and gastric cancer. Claudin-4 may be a promising target molecule for tumor therapy, but the claudin-targeting strategy has never been fully developed. In the present study, we prepared a claudin-4-targeting molecule by fusion of the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) with the protein synthesis inhibitory factor (PSIF) derived from Pseudomonas aeruginosa exotoxin. PSIF was not cytotoxic to claudin-4-expressing cells, whereas C-CPE-PSIF was cytotoxic. Cells that express claudin-1, -2, and -5 were less sensitive to C-CPE-PSIF. Pretreatment of the cells with C-CPE attenuated C-CPE-PSIF-induced cytotoxicity, and mutation of C-CPE in the claudin-4-binding residues attenuated the cytotoxicity of C-CPE-PSIF. TJ-undeveloped cells were more sensitive to C-CPE-PSIF than TJ-developed cells. It is noteworthy that polarized epithelial cells are sensitive to C-CPE-PSIF applied to the basal side, whereas the cells were less sensitive to C-CPE-PSIF applied to the apical side. Intratumoral injection of C-CPE-PSIF reduced tumor growth. This is the first report to indicate that a claudin-4-targeting strategy may be a promising method to overcome the malignant tumors.

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.

Domain mapping of a claudin-4 modulator, the C-terminal region of C-terminal fragment of Clostridium perfringens enterotoxin, by site-directed mutagenesis.

A C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) is a modulator of claudin-4. We previously found that upon deletion of the C-terminal 16 amino acids, C-CPE lost its ability to modulate claudin-4. Tyrosine residues in the 16 amino acids were involved in the modulation of claudin-4. In the present study, we performed functional domain mapping of the 16-amino acid region of C-CPE by replacing individual amino acids with alanine. To evaluate the ability of the alanine-substituted mutants to interact with claudin-4, we carried out a competition analysis using claudin-4-targeting protein synthesis inhibitory factor. We found that Tyr306Ala, Tyr310Ala, Tyr312Ala, and Leu315Ala mutants had reduced binding to claudin-4 compared to C-CPE. Next, we investigated effects of each alanine-substituted mutant on the TJ-barrier function in Caco-2 monolayer cells. The TJ-disrupting activity of C-CPE was reduced by the Tyr306Ala and Leu315Ala substitutions. Enhancement of rat jejunal absorption was also decreased by each of these mutations. The double mutant Tyr306Ala/Leu315Ala lost the ability to interact with claudin-4, modulate TJ-barrier function, and enhance jejunal absorption. These data indicate that Tyr306 and Leu315 are key residues in the modulation of claudin-4 by C-CPE. This information should be useful for the development of a novel claudin modulator based on C-CPE.

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.

Human herpesvirus 6 ganciclovir-resistant strain with amino acid substitutions associated with the death of an allogeneic stem cell transplant recipient

BACKGROUND Viral resistance to antiviral drugs can cause serious complications in immunosuppressed patients. We isolated from an allogeneic stem cell transplant (SCT) recipient an antiviral-resistant human herpesvirus 6 (HHV-6) strain with mutations that caused amino acid substitutions. OBJECTIVE To study the impact of mutations in the U38 and U69 genes of the ganciclovir (GCV)-resistant HHV-6 strain associated with the death of the SCT recipient. STUDY DESIGN Viruses were obtained from blood taken during symptomatic disease. Mutations in the genes for U69 protein kinase and U38 DNA polymerase were analyzed and the effects of the U69 mutations on GCV resistance were assayed using a recombinant baculovirus system. RESULTS Increasing HHV-6 antigenemia occurred after 2-3 months of preemptive GCV therapy, followed by symptomatic HHV-6 disease that ended in fatal fungus-related septic shock. The HHV-6 strain isolated from the patient was 100-fold more resistant to GCV than was a wild-type strain. New mutations were found in HHV-6 genes U38 (P462S and A565V) and U69 (L202I and L213I). The mutation of U38 P462S corresponds to a mutation in the UL54 gene (P522S) of a GCV-resistant HCMV. The U69 mutations did not alter GCV sensitivity in baculovirus GCV-resistant assay system. CONCLUSIONS Drug-resistant mutations arising during preemptive therapy may complicate post-transplant HHV-6 disease in SCT recipients. The increased copy number during GCV treatment of this new GCV-resistant HHV-6 strain correlated with mutations in the U69 and U38 genes. Since the kinase mutation did not alter sensitivity to GCV when tested in the in vitro system, it is likely that the substitutions in the polymerase related to GCV resistance.

Purification and characterization of Bordetella bronchiseptica dermonecrotic toxin

Dermonecrotic toxin produced by Bordetella bronchiseptica was purified by chromatography on DEAE Toyopearl 650M and on Bio-Gel HTP, gel filtration on Sephadex G-200, and subsequent chromatography on Bio-Gel HTP and on SP Toyopearl 650M. The purified toxin was homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis and high performance liquid chromatography. There was a 90-fold increase in the dermonecrotic titer per mg protein in guinea pigs and the recovery of activity was 17.6% of that of the original cell extract. The purified toxin is a single-chain protein with a molecular weight of 145,000 and an isolelectric point of 6.3-6.7. Its minimal necrotizing dose is approximately 0.4 ng. It was completely inactivated by heating for 20 min at 56 degrees C. It contained no endotoxin, carbohydrates, nucleic acids, or hemagglutinins.