Masaya Takehara
Tokushima Bunri University
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Featured researches published by Masaya Takehara.
Biochimica et Biophysica Acta | 2015
Masahiro Nagahama; Soshi Seike; Hidenori Shirai; Teruhisa Takagishi; Keiko Kobayashi; Masaya Takehara; Jun Sakurai
BACKGROUND Clostridium perfringens beta-toxin is a pore-forming toxin (PFT) and an important agent of necrotic enteritis and enterotoxemia. We recently reported that beta-toxin strongly induced cell death in THP-1 cells via the formation of oligomers. We here describe that the P2X(7) receptor, which is an ATP receptor, interacts with beta-toxin. METHODS We tested the role of P2X(7) receptor in beta-toxin-induced toxicity using specific inhibitors, knockdown of receptor, expression of the receptor and interaction by dot-blot assay. The potency of P2X(7) receptor was further determined using an in vivo mouse model. RESULTS Selective P2X(7) receptor antagonists (oxidized ATP (o-ATP), oxidized ADP, and Brilliant Blue G (BBG)) inhibited beta-toxin-induced cytotoxicity in THP-1 cells. o-ATP also blocked the binding of beta-toxin to cells. The P2X(7) receptor and beta-toxin oligomer were localized in the lipid rafts of THP-1 cells. siRNA for the P2X(7) receptor inhibited toxin-induced cytotoxicity and binding of the toxin. In contrast, the siRNA knockdown of P2Y(2) or P2Y(6) had no effect on beta-toxin-induced cytotoxicity. The addition of beta-toxin to P2X(7)-transfected HEK-293 cells resulted in binding of beta-toxin oligomer. Moreover, beta-toxin specifically bound to immobilized P2X(7) receptors in vitro and colocalized with the P2X(7) receptor on the THP-1 cell surface. Furthermore, beta-toxin-induced lethality in mice was blocked by the preadministration of BBG. CONCLUSIONS The results of this study indicate that the P2X(7) receptor plays a role in beta-toxin-mediated cellular injury. GENERAL SIGNIFICANCE P2X(7) receptor is a potential target for the treatment of C. perfringens type C infection.
Scientific Reports | 2016
Masaya Takehara; Teruhisa Takagishi; Soshi Seike; Kaori Ohtani; Keiko Kobayashi; Kazuaki Miyamoto; Tohru Shimizu; Masahiro Nagahama
Although granulopoiesis is accelerated to suppress bacteria during infection, some bacteria can still cause life-threatening infections, but the mechanism behind this remains unclear. In this study, we found that mature neutrophils in bone marrow cells (BMCs) were decreased in C. perfringens-infected mice and also after injection of virulence factor α-toxin. C. perfringens infection interfered with the replenishment of mature neutrophils in the peripheral circulation and the accumulation of neutrophils at C. perfringens-infected sites in an α-toxin-dependent manner. Measurements of bacterial colony-forming units in C. perfringens-infected muscle revealed that α-toxin inhibited a reduction in the load of C. perfringens. In vitro treatment of isolated BMCs with α-toxin (phospholipase C) revealed that α-toxin directly decreased mature neutrophils. α-Toxin did not influence the viability of isolated mature neutrophils, while simultaneous treatment of BMCs with granulocyte colony-stimulating factor attenuated the reduction of mature neutrophils by α-toxin. Together, our results illustrate that impairment of the innate immune system by the inhibition of neutrophil differentiation is crucial for the pathogenesis of C. perfringens to promote disease to a life-threatening infection, which provides new insight to understand how pathogenic bacteria evade the host immune system.
PLOS ONE | 2016
Soshi Seike; Kazuaki Miyamoto; Keiko Kobayashi; Masaya Takehara; Masahiro Nagahama
Clostridium perfringens delta-toxin is a β-pore-forming toxin and a putative pathogenic agent of C. perfringens types B and C. However, the mechanism of cytotoxicity of delta-toxin remains unclear. Here, we investigated the mechanisms of cell death induced by delta-toxin in five cell lines (A549, A431, MDCK, Vero, and Caco-2). All cell lines were susceptible to delta-toxin. The toxin caused rapid ATP depletion and swelling of the cells. Delta-toxin bound and formed oligomers predominantly in plasma membrane lipid rafts. Destruction of the lipid rafts with methyl β-cyclodextrin inhibited delta-toxin-induced cytotoxicity and ATP depletion. Delta-toxin caused the release of carboxyfluorescein from sphingomyelin-cholesterol liposomes and formed oligomers; toxin binding to the liposomes declined with decreasing cholesterol content in the liposomes. Flow cytometric assays with annexin V and propidium iodide revealed that delta-toxin treatment induced an elevation in the population of annexin V-negative and propidium iodide-positive cells. Delta-toxin did not cause the fragmentation of DNA or caspase-3 activation. Furthermore, delta-toxin caused damage to mitochondrial membrane permeability and cytochrome c release. In the present study, we demonstrate that delta-toxin produces cytotoxic activity through necrosis.
Toxins | 2017
Masaya Takehara; Teruhisa Takagishi; Soshi Seike; Masataka Oda; Yoshihiko Sakaguchi; Junzo Hisatsune; Sadayuki Ochi; Keiko Kobayashi; Masahiro Nagahama
Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins.
Biochimica et Biophysica Acta | 2016
Teruhisa Takagishi; Masataka Oda; Masaya Takehara; Keiko Kobayashi; Masahiro Nagahama
BACKGROUND Clostridium perfringens epsilon-toxin is responsible for fatal enterotoxemia in ungulates. The toxin forms a heptamer in the lipid rafts of Madin-Darby Canine Kidney (MDCK) cells, leading to cell death. Here, we showed that epsilon-toxin requires neutral sphingomyelinase (nSMase) activity during oligomerization. METHODS We tested the role of nSMase in the oligomerization of epsilon-toxin using specific inhibitors, knockdown of nSMase, formation of ceramide, and localization of epsilon-toxin and ceramide by immunofluorescence staining. RESULTS Epsilon-toxin induced the production of ceramide is a dose- and time-dependent manner in ACHN cells. GW4869, an inhibitor of nSMase, inhibited ceramide production induced by the toxin. GW4869 and knockdown of nSMase blocked toxin-induced cell death and oligomer formation of epsilon-toxin. Confocal microscopy images showed that the toxin induced ceramide clustering and colocalized with ceramide. CONCLUSIONS These results demonstrated that oligomer formation of epsilon-toxin is facilitated by the production of ceramide through activation of nSMase caused by the toxin. GENERAL SIGNIFICANCE Inhibitors of nSMase may confer protection against infection.
Infection and Immunity | 2017
Masahiro Nagahama; Masaya Takehara; Teruhisa Takagishi; Soshi Seike; Kazuaki Miyamoto; Keiko Kobayashi
ABSTRACT Clostridium botulinum C2 toxin consists of an enzyme component (C2I) and a binding component (C2II). Activated C2II (C2IIa) binds to a cell receptor, giving rise to lipid raft-dependent oligomerization, and it then assembles with C2I. The whole toxin complex is then endocytosed into the cytosol, resulting in the destruction of the actin cytoskeleton and cell rounding. Here, we showed that C2 toxin requires acid sphingomyelinase (ASMase) activity during internalization. In this study, inhibitors of ASMase and lysosomal exocytosis blocked C2 toxin-induced cell rounding. C2IIa induced Ca2+ influx from the extracellular medium to cells. C2 toxin-induced cell rounding was enhanced in the presence of Ca2+. ASMase was released extracellularly when cells were incubated with C2IIa in the presence of Ca2+. Small interfering RNA (siRNA) knockdown of ASMase reduced C2 toxin-induced cell rounding. ASMase hydrolyzes sphingomyelin to ceramide on the outer leaflet of the membrane at acidic pH. Ceramide was detected in cytoplasmic vesicles containing C2IIa. These results indicated that ASMase activity is necessary for the efficient internalization of C2 toxin into cells. Inhibitors of ASMase may confer protection against infection.
Biochimica et Biophysica Acta | 2016
Soshi Seike; Masaya Takehara; Keiko Kobayashi; Masahiro Nagahama
BACKGROUND Beta-toxin produced by Clostridium perfringens is a key virulence factor of fatal hemorrhagic enterocolitis and enterotoxemia. This toxin belongs to a family of β-pore-forming toxins (PFTs). We reported recently that the ATP-gated P2X7 receptor interacts with beta-toxin. The ATP-release channel pannexin 1 (Panx1) is an important contributor to P2X7 receptor signaling. Hence, we investigated the involvement of Panx1 in beta-toxin-caused cell death. METHODS We examined the effect of Panx1 in beta-toxin-induced cell death utilizing selective antagonists, knockdown of Panx1, and binding using dot-blot analysis. Localization of Panx1 and the P2X7 receptor after toxin treatment was determined by immunofluorescence staining. RESULTS Selective Panx1 antagonists (carbenoxolone [CBX], probenecid, and Panx1 inhibitory peptide) prevented beta-toxin-caused cell death in THP-1 cells. CBX did not block the binding of the toxin to cells. Small interfering knockdown of Panx1 blocked beta-toxin-mediated cell death through inhibiting the oligomer formation of the toxin. Beta-toxin triggered a transient ATP release from THP-1 cells, but this early ATP release was blocked by CBX. ATP scavengers (apyrase and hexokinase) inhibited beta-toxin-induced cytotoxicity. Furthermore, co-administration of ATP with beta-toxin enhanced the binding and cytotoxicity of the toxin. CONCLUSIONS Based on our results, Panx1 activation is achieved through the interaction of beta-toxin with the P2X7 receptor. Then, ATP released by the Panx1 channel opening promotes oligomer formation of the toxin, leading to cell death. GENERAL SIGNIFICANCE Pannexin 1 is a novel candidate therapeutic target for beta-toxin-mediated disease.
Biochimica et Biophysica Acta | 2018
Soshi Seike; Masaya Takehara; Teruhisa Takagishi; Kazuaki Miyamoto; Keiko Kobayashi; Masahiro Nagahama
Clostridium perfringens delta-toxin is a β-barrel-pore-forming toxin (β-PFT) and a presumptive virulence factor of type B and C strains, which are causative organisms of fatal intestinal diseases in animals. We showed previously that delta-toxin causes cytotoxicity via necrosis in sensitive cells. Here, we examined the effect of delta-toxin on intestinal membrane integrity. Delta-toxin led to a reduction in transepithelial electrical resistance (TEER) and increased the permeability of fluorescence isothiocyanate-conjugated dextran in human intestinal epithelial Caco-2 cells without changing the tight junction proteins, such as zonula occludens-1 (ZO-1), occludin, and claudin-1. On the other hand, delta-toxin reduced the cellular levels of adherence junction protein E-cadherin before cell injury. A disintegrin and metalloprotease (ADAM) 10 facilitates E-cadherin cleavage and was identified as the cellular receptor for alpha-toxin, a β-PFT produced by Staphylococcus aureus. ADAM10 inhibitor (GI254023X) blocked the toxin-induced decrease in TEER and cleavage of E-cadherin. Delta-toxin enhanced ADAM10 activity in a dose- and time-dependent manner. Furthermore, delta-toxin colocalized with ADAM10. These results indicated that ADAM10 plays a key role in delta-toxin-induced intestinal injury.
Toxins | 2018
Masahiro Nagahama; Masaya Takehara; Kazuaki Miyamoto; Kazumi Ishidoh; Keiko Kobayashi
Clostridium perfringens iota-toxin is a binary actin-ADP-ribosylating toxin composed of the enzymatic component Ia and receptor binding component Ib. Ib binds to a cell surface receptor, forms Ib oligomer in lipid rafts, and associates with Ia. The Ia-Ib complex then internalizes by endocytosis. Here, we showed that acid sphingomyelinase (ASMase) facilitates the cellular uptake of iota-toxin. Inhibitions of ASMase and lysosomal exocytosis by respective blockers depressed cell rounding induced by iota-toxin. The cytotoxicity of the toxin increased in the presence of Ca2+ in extracellular fluids. Ib entered target cells in the presence but not the absence of Ca2+. Ib induced the extracellular release of ASMase in the presence of Ca2+. ASMase siRNA prevented the cell rounding induced by iota-toxin. Furthermore, treatment of the cells with Ib resulted in the production of ceramide in cytoplasmic vesicles. These observations showed that ASMase promotes the internalization of iota-toxin into target cells.
Toxins | 2018
Masahiro Nagahama; Masaya Takehara; Keiko Kobayashi
Iota toxin produced by Clostridium perfringens is a binary, actin ADP-ribosylating toxin that is organized into the enzymatically active component Ia and the binding component Ib. Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a cellular receptor of Ib. Here, we investigated the functional interaction between Ib and LSR, where siRNA for LSR blocked the toxin-mediated cytotoxicity and the binding of Ib. The addition of Ib to LSR-green fluorescence protein (GFP)-transfected cells at 4 °C resulted in colocalization with LSR and Ib on the cell surface. Upon transfer of the cells from 4 °C to 37 °C, LSR and Ib were internalized and observed in cytoplasmic vesicles. When the cells were incubated with Ib at 37 °C and fractionated using the Triton-insoluble membrane, Ib oligomer was localized in insoluble factions that fulfilled the criteria of lipid rafts, and LSR was clustered in lipid rafts. To examine the interaction between N-terminal extracellular region of LSR and Ib, we constructed a series of LSR N-terminal deletions. Ten amino acids residues can be deleted from this end without any reduction of Ib binding. However, deletion of 15 N-terminal residues drastically reduces its ability to bind Ib. These results demonstrate that Ib binds to the LSR N-terminal 10 to 15 residues and endocytoses into trafficking endosomes together with LSR.