Oligomer formation of Clostridium perfringens epsilon-toxin is induced by activation of neutral sphingomyelinase

    Oligomer formation of Clostridium perfringens epsilon-toxin is induced by activation of neutral sphingomyelinase Teruhisa Takagishi, Masataka Oda, Masaya Takehara, Keiko Kobayashi, Masahiro Nagahama PII: DOI: Reference:

S0005-2736(16)30252-8 doi: 10.1016/j.bbamem.2016.07.009 BBAMEM 82270

To appear in:

BBA - Biomembranes

Received date: Revised date: Accepted date:

10 April 2016 14 July 2016 19 July 2016

Please cite this article as: Teruhisa Takagishi, Masataka Oda, Masaya Takehara, Keiko Kobayashi, Masahiro Nagahama, Oligomer formation of Clostridium perfringens epsilontoxin is induced by activation of neutral sphingomyelinase, BBA - Biomembranes (2016), doi: 10.1016/j.bbamem.2016.07.009

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ACCEPTED MANUSCRIPT Revised version 2

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Oligomer formation of Clostridium perfringens epsilon-toxin is induced by activation of neutral

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sphingomyelinase

Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University,

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Teruhisa Takagishi1, Masataka Oda2, Masaya Takehara1, Keiko Kobayashi1, Masahiro Nagahama1*

Yamashiro-cho 180, Tokushima 770-8514, Japan.

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Division of Microbiology and Infectious Diseases, Niigata University Graduate School of Medical

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and Dental Sciences, 2-5274, Gakkocho-dori, Chuo-ku, Niigata 951-8514, Japan.

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Running title: Oligomer formation of C. perfringens epsilon-toxin

*To whom correspondence should be addressed: Phone: +81-088-622-9611. Fax: +81-088-655-3051 E-mail: [email protected] Tokushima Bunri University

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ACCEPTED MANUSCRIPT Abstract

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Background Clostridium perfringens epsilon-toxin is responsible for fatal enterotoxemia in

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ungulates. The toxin forms a heptamer in the lipid rafts of Madin-Darby Canine Kidney (MDCK)

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cells, leading to cell death. Here, we showed that epsilon-toxin requires neutral sphingomyelinase (nSMase) activity during oligomerization.

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Methods We tested the role of nSMase in the oligomerization of epsilon-toxin using specific

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inhibitors, knockdown of nSMase, formation of ceramide, and localization of epsilon-toxin and ceramide by immunofluorescence staining.

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Results Epsilon-toxin induced the production of ceramide is a dose- and time-dependent manner in

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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

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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.

Key words: C. perfringens epsilon-toxin, oligomer, ceramide, neutral sphingomyelinase

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ACCEPTED MANUSCRIPT 1. Introduction

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Epsilon-toxin is produced by Clostridium perfringens types B and D, and it induces enteritis and

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enterotoxemia in sheep and other animals, which can have a serious financial impact for farmers

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[1-4]. Epsilon-toxin is produced as an inactive 32.9 kDa prototoxin, which is converted into active mature epsilon-toxin by proteolytic cleavage [2-4]. Epsilon-toxin possesses lethal, dermonecrotic

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activities and can cause blood pressure elevation. The toxin is one of the most potent clostridial

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toxins after botulinum and tetanus neurotoxins [5], and it is also considered as a potent, poisonous category B agent by the National Institutes of Health and the Centers of Disease Control [4].

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Although very few natural infections have been observed in humans [6], epsilon-toxin has been

sclerosis [7].

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reported to be a possible pathogenic factor in inducing demyelinating disorders such as multiple

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A comparative study of the deduced amino acid sequences of epsilon-toxin [8] and other protein toxins, e.g., Clostridium septicum alpha-toxin, Aeromonas hydrophila aerolysin, and Pseudomonas aeruginosa cytotoxin, revealed that epsilon-toxin is a member of the family of bacterial β-pore-forming toxins [9]. Structural analysis of epsilon-toxin indicated that it is composed of three domains, principally β-sheets, and is very similar to aerolysin. Putative roles for the toxin domains consist of receptor interaction (domain I), channel formation (domain II), and monomer-monomer interaction sites (domain III) [10]. Relatively few cell lines are sensitive to epsilon-toxin, and in vitro studies on the toxin have been carried out using the Madin-Darby Canine Kidney (MDCK) cells, the

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ACCEPTED MANUSCRIPT mouse kidney cell line mpkCCDcl4, and the human renal adenocarcinoma cell line ACHN [6, 11, 12].

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The cytotoxic activity epsilon-toxin of is correlated with a quick loss of intracellular K+ and ATP [13].

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The toxin induces rapid necrosis of sensitive cells in association with a remarkable reduction in

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nucleus size without DNA fragmentation. In addition, we reported that epsilon-toxin is endocytosed into MDCK cells and causes vacuole formation from late endosomes and lysosomes [14]. Hepatitis A

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virus cellular receptor 1 (HAVCR1) has been reported to play a role in the cytotoxicity of

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epsilon-toxin in MDCK and ACHN cells [11, 15]. However, it is remains unclear whether HAVCR1 is a cellular receptor.

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We showed that the fluidity of the membrane in artificial liposomes plays a key role in the binding

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and oligomer formation of epsilon-toxin [16]. It is important to determine the composition of the lipids and perform physical characterization of the effects of the membrane on toxin monomer

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assembly and insertion of the toxin pore into the membrane. In a recent study, epsilon-toxin bound to the lipid fraction present in lipid rafts in synaptosomal preparations and MDCK cells [17], and the authors suggested that sulfatide can interact with epsilon-toxin. Lipid rafts are microdomains within a membrane that are rich in cholesterol, sphingolipids, and specific proteins. Lipid rafts contribute to enriching platforms to accelerate oligomerization of a number of β-pore-forming toxins on the cell surface [18-21]. Epsilon-toxin seems to target lipid rafts in the plasma membrane [19]. It has been reported that receptors present in lipid rafts serve to concentrate toxins, allowing for oligomerization [4, 13]. Fennessey et al. [22] reported that

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ACCEPTED MANUSCRIPT caveolin-1 and caveolin-2 in lipid rafts augmented epsilon-toxin-induced cytotoxicity by promoting

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toxin oligomerization. Moreover, it was shown recently that epsilon-toxin binds to the myelin and

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lymphocyte protein (MAL) in membrane lipid rafts [23, 24].

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Sphingolipids may have key roles in membrane lipid raft structure and they are known to serve as intracellular messengers in signaling cascades relating to apoptosis, inflammation, cell proliferation,

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and differentiation [25, 26]. Ceramide can be produced by cleavage of sphingomyelin by either

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neutral sphingomyelinase (nSMase) or acid sphingomyelinase (aSMase) or by de novo synthesis [27]. Sphingomyelinase (SMase)-mediated ceramide production on the plasma membrane results in the

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formation of ceramide-rich platforms (CRPs) [27]. CRPs seem to be local sites of protein

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oligomerization [27]. Although oligomerization of epsilon-toxin is required for the cytotoxic effects, its detailed mechanism of oligomerization remains unknown. To investigate the mode of assembly of

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epsilon-toxin, we examined the relationship between the formation of ceramide in the plasma membrane and epsilon-toxin oligomerization.

2. Materials and Methods 2.1. Materials Purification of epsilon-prototoxin and toxin were carried out as described previously [14]. An antibody against epsilon-toxin was prepared as described previously [14]. The nSMase inhibitor GW4869 and purified Escherichia coli 1,2-diacylglycerol kinase (DGK) were purchased from

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ACCEPTED MANUSCRIPT Calbiochem (San Diego, CA). Rabbit anti-nSMase antibody (ab131330) was obtained from Abcam

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(Tokyo, Japan). Cardiolipin, n-octyl-β-glucopyranoside, DETAPAC, monoclonal anti-ceramide

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(mouse IgM) antibody and bovine serum albumin were purchased from Sigma-Aldrich (Tokyo,

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Japan). Alexa Fluor 488-conjugated anti-rabbit IgG, Alexa Fluor 546-conjugated anti-mouse IgM, and Hoechst 33342 were obtained from Life Technologies (Tokyo, Japan). An Amplex Red

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Sphingomyelinase Assay Kit was purchased from Invitrogen (Tokyo, Japan). Horseradish

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peroxidase-labeled anti-rabbit IgG and enhanced chemiluminescence kits were purchased from GE Healthcare (Tokyo, Japan). Dulbecco’s modified Eagle's medium (DMEM) and minimum essential

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media (MEM) were obtained from Gibco BRL (New York, NY, USA).

2.2. Cell culture and cell viability

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MDCK cells were obtained from Riken Cell Bank (Tsukuba, Japan). They were grown in DMEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 2 mM glutamine. ACHN cells were obtained from ATCC (USA). They were grown in MEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1% non-essential amino acids. Cells were cultivated at 37°C in a 5% CO2 incubator. ACHN cells (5.0 × 105 cells) were seeded in 24-well plates. Cell viability was measured by using the MTS (Promega, Tokyo, Japan) inner salt conversion assay (MTS assay). The optical density was determined at 490 nm using a microtiter plate reader. Cell viability was calculated as follows: mean absorbance of the toxin group/mean

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absorbance of the control [20].

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2.3. Determination of ceramide

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ACHN cells (5.0 × 106 cells) were incubated with or without epsilon-toxin (5 g/ml) at 37°C for the indicated times. Cellular lipids were extracted using chloroform-methanol-acetic acid-water

by

sonication

for

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min

in

a

water

bath

in

cardiolipin

(5

mM),

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solubilized

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(25:15:4:2, v/v/v/v), and the final organic phase was desiccated under nitrogen. Samples were

n-octyl-β-glucopyranoside (7.5%, w/v) and DETAPAC (1 mM). DGK in reaction buffer including 60

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mM PIPES-HCl buffer (pH 6.8), 20 mM MgCl2, 1 mM phospho(enol)pyruvate (PEP), 1 mM ATP, 1

for 30 min at

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mM NADH, and 2 mM EGTA plus [-32P]-ATP (Perkin Elmer, Japan). The samples were incubated 25°C, after which the

kinase reaction was

terminated by adding

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chloroform-methanol-1% perchloric acid (25:15:4, v/v/v). The organic phase was dried, resuspended in chloroform-methanol (1:1, v/v), and subjected to thin-layer chromatography on a silica 60 gel plate (Merck, Tokyo, Japan) using chloroform-methanol-acetic acid-formic acid (10:85:5:0.5, v/v) as a solvent. Labeled lipids on the plate were visualized using a Bio-Imaging Analyzer FLA-2000 (Fujifilm, Tokyo, Japan). In some experiments, heat-inactivated epsilon-toxin was prepared by heating at 95°C for 10 min.

2.4. Acidic or neutral sphingomyelinase enzyme activities

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ACCEPTED MANUSCRIPT After treatment with epsilon-toxin (5 g/ml), ACHN cells were lysed in neutral lysis buffer (20

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mM Tris-HCl buffer, pH 7.5, 1% Triton X-100, 1 mM EDTA) or acid lysis buffer (50 mM sodium

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acetate buffer, pH 5.0, 1% Triton X-100, 1 mM EDTA) with freshly added protease inhibitor cocktail

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(Nacalai, Kyoto, Japan). SMase activity was analyzed using a Amplex Red Sphingomyelinase Assay

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Kit, as described in the manufacturer’s protocol.

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2.5. Western blotting

ACHN cells (1.0 × 106 cells) were incubated with or without epsilon-toxin (5 g/ml) at 37°C in

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culture medium. The reaction was stopped by adding 0.5 ml ice-cold 7.5% trichloroacetic acid

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containing 0.1 mM Na3VO4, and kept on ice for 30 min. The precipitate was collected by centrifugation at 13,000 ×g for 20 min and heated in 2% SDS sample buffer at 99°C for 3 min. The

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samples were resolved by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and electrically transferred to Immobilon polyvinylidene difluoride membrane (Millipore, Tokyo, Japan). The membrane was blocked with 5% bovine serum albumin (BSA) in TBST buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2% Tween 20) for 1 h at room temperature, and then incubated for 1 h at room temperature with first with the primary antibody in TBST buffer containing 1% BSA. The membrane were then washed in TBST and incubated for 1 h at room temperature with a horseradish peroxidase-conjugated secondary antibody. After repeated washing, detection was conducted using an enhanced chemiluminescence kit. Quantitative analysis was

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performed by densitometry (LAS-4000, Fujifilm, Japan).

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2.6. Transfection with small interfering RNAs for neutral sphingomyelinase 2

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Small interfering RNAs (siRNA) targeting nSMase 2 and a siRNA negative control were obtained from Qiagen (Tokyo, Japan). ACHN cells (1.0 × 106 cells/ml) were mixed with 10 nM siRNA for

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nSMase 2 or negative control siRNA (NC-siRNA), and then electroporated using a Neon

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Transfection System (Life Technologies, Tokyo, Japan). After electroporation, cells were seeded into 24-well plates including prewarmed antibiotic-free media and incubated at 37°C for 2 days.

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Silencing of nSMase 2 was confirmed by western blotting with the anti-nSMase 2 antibody.

2.7. Immunofluorescence staining and confocal imaging

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MDCK cells were seeded on a poly-L-lysine-coated glass-bottomed dishes (Matsunami, Tokyo, Japan) and cultured at 37°C in a 5% CO2 incubator overnight in fetal bovine serum/DMEM. After incubation, cells were treated with epsilon-toxin (1 g/ml) at 37°C for 1 h, fixed with 4% paraformaldehyde in PBS at room temperature for 15 min, and then washed three times with PBS. The immunostaining was performed under non-permeablilized condition to detect cell surface ceraminde and epsilon-toxin. Fixed cells were treated with 4% BSA for 30 min, and then with primary antibodies (anti-epsilon-toxin antibody and anti-ceramide antibody) in 4% BSA at 4°C for 16 h. They were then washed with PBS, incubated with secondary antibodies (Alexa Fluor

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ACCEPTED MANUSCRIPT 488-conjugated anti-rabbit IgG and Alexa Fluor 546-conjugated anti-mouse IgM) in 4% BSA at

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room temperature for 1 h. Nuclei were stained with Hoechst 33342. Stained cells were observed

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under a Nikon A1 laser scanning confocal microscope (Nikon Instruments, Tokyo, Japan).

2.8. Statistical analysis

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One-way analysis of variance (ANOVA), followed by Bonferroni’s multiple-comparison post-test,

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was used to compare the means. Data are expressed as the mean ± standard deviation (SD). P value

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of 0.05 or less was considered statistical significant.

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3. Results

3.1. Epsilon-toxin induces ceramide formation through activation of neutral sphingomyelinase

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Epsilon-toxin binds to the plasma membrane of sensitive cells and forms an oligomer required for pore formation in plasma membrane lipid rafts [6, 19]. The lipid composition of the membrane and membrane fluidity have been shown to be critical factors for oligomerization and pore formation by epsilon-toxin [16]. Ceramide production in the plasma membrane results in ceramide clustering, thereby initiating the formation of microdomains [27]. Therefore, to examine the relationship between oligomer formation of the toxin and ceramide generation, we investigated whether epsilon-toxin induces ceramide production in ACHN cells. After the cells were treated with epsilon-toxin at 37°C for the indicated time periods, the levels of ceramide were determined. As

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ACCEPTED MANUSCRIPT shown in Fig. 1A, epsilon-toxin caused a time-dependent increase in the production of ceramide.

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Moreover, as shown in Fig. S1, epsilon-toxin caused the ceramide production in a dose-dependent

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manner. No ceramide production was evoked by heat-inactivated epsilon-toxin, and toxin-induced

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ceramide production was completely neutralized by an anti-epsilon-toxin antibody (data not shown). To test whether the treatment of ACHN cells with epsilon-toxin induced the activation of endogenous

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SMases, the activities of nSMase and aSMase were measured in cell lysates of the cells after

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treatment with epsilon-toxin at 37°C for the indicated time periods (Fig. 1B). An approximately 1.4-fold increase in the activity of nSMase was detected after treatment of ACHN cells with

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epsilon-toxin. In contrast, no remarkable change in the activity of aSMase was detected. Taken

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together, these results indicated that nSMase is involved in ceramide generation in ACHN cells induced by epsilon-toxin.

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3.2. Effect of neutral sphingomyelinase inhibitor and small interfering RNA for neutral sphingomyelinase on the action of epsilon-toxin in ACHN cells To further investigate the involvement of nSMase in epsilon-toxin-mediated cytotoxic activity, we examined the effect of GW4869, a specific inhibitor of nSMase, on ceramide generation induced by epsilon-toxin. As shown in Fig. 2A, epsilon-toxin treatment in the presence of 0.1% dimethyl sulfoxide (DMSO) (vehicle control) led to an increase in ceramide production. On the other hand, GW4869 inhibited ceramide formation induced by the toxin. Next, to test whether ceramide production contributed to epsilon-toxin induced cell death, we pretreated ACHN cells with GW4869.

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ACCEPTED MANUSCRIPT GW4869 attenuated the cytotoxicity induced by epsilon-toxin (Fig. 2B). The cytotoxicity of

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epsilon-toxin alone resembled that of the toxin in the presence of 0.1% DMSO (data not shown).

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Similarly, epsilon-toxin-induced activation of nSMase was inhibited by GW4869 (Fig. 2C). These

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results indicated that activation of nSMase contributed to epsilon-toxin-induced cytotoxicity. To further elucidate the role of nSMase in epsilon-toxin-induced cytotoxicity, an RNAi assay was

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employed to silence the native expression of nSMase in ACHN cells. Transfection of ACHN cells

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with nSMase-specific siRNA significantly reduced nSMase protein expression, whereas a negative control siRNA (NC-siRNA) had no effect (Fig. 2D). As shown in Fig. 2E, epsilon-toxin induced the

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the toxin-derived cytotoxicity.

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death of NC-siRNA transfected cells. On the other hand, the silencing of nSMase by siRNA blocked

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3.3. Activation of neutral sphingomyelinase promotes the oligomerization of epsilon-toxin The cytotoxicity of epsilon-toxin depends on the binding and oligomerization of the toxin to cells [9, 19]. To investigate whether activation of nSMase is involved in the oligomerization of epsilon-toxin, GW4869-treated ACHN cells were incubated with epsilon-toxin. As shown in Fig. 3A, epsilon-toxin formed oligomers in the presence of DMSO. The results showed two bands, a 28 kDa band that corresponded to the expected size of epsilon-toxin monomer and a ~180 kDa band that corresponded to the expected size of epsilon-toxin oligomer. On the other hand, GW4869 inhibited the oligomerization of epsilon-toxin but not the binding of the toxin monomer (Fig. 3A).

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ACCEPTED MANUSCRIPT Furthermore, we tested the binding and oligomerization of epsilon-toxin on NC-siRNA transfected or

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nSMase-siRNA-transfected cells (Fig. 3B). When NC-siRNA transfected ACHN cells were

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incubated with epsilon-toxin at 37°C for the indicated time period, an intense band of epsilon-toxin

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oligomer and a trace band of the toxin monomer were detected. On the other hand, epsilon-toxin oligomer in nSMase-siRNA-transfected cells decreased and the toxin monomer increased. These

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results indicated that nSMase contributes to oligomerization of epsilon-toxin.

3.4. Colocalization of ceramide and epsilon-toxin in the plasma membrane

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To further investigate the role of ceramide in the oligomerization of epsilon-toxin, we examined

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whether epsilon-toxin colocalized with ceramide in the cell membrane. Immunofluorescent staining was performed under non-permeabilized conditions to detect surface labeling. As shown in Fig. 4,

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control cells did not exhibit ceramide labeling (red) in the cells. Treatment of the cells with epsilon-toxin for 60 min induced ceramide accumulation in the cell membranes. Furthermore, epsilon-toxin (green) merged with the fluorescence of ceramide ion the plasma membrane. As shown in Fig. S2, about 40% colocalization was observed between epsilon-toxin and ceramide. Results of studies indicated that epsilon-toxin can interact with ceramide platforms.

4. Discussion Previous studies have shown that epsilon-toxin formed a heptamer in sensitive cells and that cytotoxicity was dependent on the formation of an oligomer [3, 4, 6, 12, 19]. However, the 13

ACCEPTED MANUSCRIPT mechanisms by which epsilon-toxin forms oligomers in the plasma membrane remained to be

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investigated. In this study, we first showed that epsilon-toxin induced the activation of nSMase in the

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plasma membrane, production of ceramide and oligomer formation of the toxin responsible for

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cytotoxicity. It has been reported that epsilon-toxin formed a functional oligomer, which was linked to its cytotoxicity, in the lipid rafts of MDCK cells [6, 9, 19]. Although the toxin monomer bound to

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non-lipid rafts in the plasma membrane, the oligomer was formed in lipid rafts. Movement of toxin

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monomers associated with the receptor in restricted areas (i.e. lipid rafts) leads to local clustering of toxin molecules and facilitates their interactions and subsequent oligomer formation [12]. Therefore,

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we demonstrated that epsilon-toxin (i) binds to the cell surface receptor in membrane non-lipid rafts,

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(ii) is concentrated at lipid rafts, and (iii) forms a functional oligomer. In the present study, epsilon-toxin induced the activation of nSMase, the production of ceramide,

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and cell death in sensitive cells. Furthermore, GW4869 or silencing of nSMase by siRNA inhibited the epsilon-toxin-induced activation of nSMase and cytotoxicity as well as oligomer formation of the toxin. Because ceramide molecules have the tendency to interact with each other, ceramide generation results in ceramide clustering and facilitates the formation of ceramide rich lipid rafts in which cellular receptors cluster [27]. Therefore, it was suggested that the ceramide generation via specific activation of nSMase with epsilon-toxin may promote oligomer formation of the toxin in lipid rafts. However, signaling pathway involving the toxin-induced endogeneous nSMase activation is still unknown. Further investigations are needed to elucidate how signal transduction system

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ACCEPTED MANUSCRIPT mediates cell death cuased by the toxin.

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In this work, we used epsilon-toxin-sensitive ACHN and MDCK cells. MDCK cells are about

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100-fold sensitive compared to the ACHN cells against the toxin [22]. Epsilon-toxin binds to lipid

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rafts and oligomerizes to heptameric complex in both cells. The toxin oligomer inserts into plasma membrane and forms an active pore. Epsilon-toxin leads to cell death in common mechanism of

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action to MDCK and ACHN cells. We confirmed that GW4869 or silencing of nSMase by siRNA

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blocked the epsilon-toxin-induced cytotoxicity in MDCK cells as well as in ACHN cells. Although it is well recognized that the toxin caused ceramide production in ACHN cells, ceramide metabolism

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was only slightly detected in MDCK cells because the toxin causes rapid cell death. When we

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examined the ceramide metabolism with a low concentration of the toxin in MDCK cells, the toxin had little or no effect on lipid metabolism. On the one hand, we have been reported that MDCK cells

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represent an excellent model for study of the toxin localization using confocal microscopy [14], as it is likely that the cells abundantly expressed the specific receptor(s) which epsilon-toxin can utilize. We performed an experiment of localization in ACHN cells, but detection of the toxin was difficult. MDCK cells have been widely used for the study of epsilon-toxin. We chose a sensitive cell good for checking an action of the toxin. We have been reported that epsilon-toxin monomer was detected in the non-lipid rafts of cells incubated with the toxin at 4 °C, indicating that the receptor of epsilon-toxin is distributed in non-lipid rafts of plasma membranes [14]. Subsequently, the oligomer of the toxin was detected in

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ACCEPTED MANUSCRIPT lipid rafts after incubation of the cells at 37 °C. In contrast, other reports have not been investigated

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the binding of the toxin at 4 °C and was examined at 37 °C. On the other hand, the temperature

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dependence of the cell disruption caused by epsilon-toxin seems to be due to the requirement of high

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temperature for increasing the membrane fluidity and activation of nSMase to allow the pore formation. From these findings, we conclude that epsilon-toxin monomer binds to the receptor in the

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presence of non-lipid rafts and gathers in lipid rafts through generating ceramide via activation of

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nSMase. Then, the toxin forms heptameric pore responsible for the potent cytotoxicity induced by the toxin.

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The pore-forming activity of epsilon-toxin has been examined using lipid bilayers (liposomes).

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The toxin has an ability to form pores in lipid bilayers in the absence of a receptor, albeit with less efficiency. Even if a variety of lipids have been used in previous studies, the toxin has not been

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shown to have any lipid binding preference. On the other hand, we have been reported that lipids with low melting points appear to favor membrane insertion under the same experimental condition [16]. Moreover, we also reported a 100-fold lower sensitivity of the toxin to carboxyfluorescein loaded liposomes compared with susceptible cells [16]. From present results, we consider that activation of nSMase caused by epsilon-toxin provides favorable binding site for promoting both toxin binding and insertion. Epsilon-toxin-sensitive cells possessing both a specific receptor and nSMase are highly susceptible to toxin-induced cell death. On the other hand, Gil et al. [17] have shown that membrane lipids represent receptors for epsilon-toxin and that sulfatides are involved in

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ACCEPTED MANUSCRIPT channel formation of the toxin. In the present study, the toxin caused the ceramide production. As a

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result, we think that the membrane lipids change in a state where the toxin is likely to bind. And, by

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toxin can easily forms an active pore in plasma membranes.

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the dynamic changes of localization of sulfatides during the toxin-induced ceramide production, the

We reported previously that epsilon-toxin is endocytosed and trafficked from early endosomes to

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late endosomes and lysosomes. Then, epsilon-toxin oligomers accelerate the formation of vacuoles in

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the late endosomes and lysosomes. In the present study, epsilon-toxin caused ceramide generation through the activation of nSMase. Ceramide accumulation in the plasma membrane initiates local

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membrane bending and invaginations [27]. Therefore, ceramide clustering induced by epsilon-toxin

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may act as an initial step in endocytosis of the toxin itself. Adherence of bacteria to host cells is considered to serve as a key step as intestinal infections

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caused by C. perfringens. NanI, the predominant secreted sialidase elaborated by type D strain CN3718, is related to the adherence of CN3718 cells to Caco-2 cells [28]. NanI may change the target cell membrane to promote type D cell adherence [28]. On the other hand, the host cell receptor for type D strain cells has not been confirmed. Ceramide-rich domains may have a critical role as bacterial adherence by P. aeruginosa and N. gonorrheae [29, 30]. During N. gonorrheae infection, carcinoem bryonic antigen cell adhesion molecule (CEACAM) receptor binding to Opa protein, a cell surface protein of N. gonorrheae, forms clusters in ceramide platforms [30]. From the present study, we speculated that epsilon-toxin-generated ceramide-rich microdomains in host cell

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ACCEPTED MANUSCRIPT membranes may play a role in the type D cell adherence. Namely, epsilon-toxin has the potential to

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affect bacterial adherence during the intestinal infection caused by type D strain cells.

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5. Conclusion

Our findings establish a new mechanism by which epsilon-toxin forms oligomers in the sensitive

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cells. Epsilon-toxin monomers bind to a specific receptor and then induce the activation of nSMase

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in host cells. The activation of nSMase leads to the formation of ceramide-rich microdomains in the plasma membrane. Epsilon-toxin binds to the receptor clusters in ceramide-rich microdomains and

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forms oligomers. The present study showed that GW4869, a potent inhibitor nSMase, blocks the

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cytotoxicity caused by epsilon-toxin. It will be interesting to determine if this or related drugs can be

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used in preventing infection in vivo.

Conflict of interest The authors have declared that there are no conflicts of interest.

Acknowledgements

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ACCEPTED MANUSCRIPT We thank M. Nao, T. Takamatsu, and E. Hayashi for technical assistance. This work was

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supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports,

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Science, and Technology of Japan, MEXT. SENRYAKU, 2015.

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Sakurai J, Nagahama M, Ochi S, Major toxins of Clostridium perfringens, J. Toxicol. Toxin Rev.

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mammalian genes contributing to intoxication by Clostridium perfringens epsilon-toxin, PLoS One 6, (2011) e17787.

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14 Nagahama M, Itohayashi Y, Hara H, Higashihara M, Fukatani Y, Takagishi T, Oda M, Kobayashi K, Nakagawa I, Sakurai J, Cellular vacuolation induced by Clostridium perfringens epsilon-toxin, FEBS J. 278, (2011) 3395-3407. 15 Ivie SE, McClain MS, Identification of amino acids important for binding of Clostridium perfringens epsilon-toxin to host cells and to HAVCR1, Biochemistry 51, (2012) 7588-7595. 16 Nagahama M, Hara H, Fernandez-Miyakawa M, Itohayashi Y, Sakurai J, Oligomerization of Clostridium perfringens epsilon-toxin is dependent upon membrane fluidity in liposomes, Biochemistry 45, (2006) 296-302.

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cytotoxic action depends on sulfatide, PLoS One, 10(10), (2015) e0140321.

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epsilon-toxin forms a heptameric pore within the detergent-insoluble microdomains of MDCK

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cells and rat synaptosomes, J. Biol. Chem. 277, (2002) 39463-39468. 20 Nagahama M, Hayashi S, Morimitsu S, Sakurai J, Biological activities and pore formation of

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Clostridium perfringens beta toxin in HL 60 cells, J. Biol. Chem. 278, (2003) 36934-36941.

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ACCEPTED MANUSCRIPT 25 Pettus BJ, Chalfant CE, Hannun YA, Ceramide in apoptosis: an overview and current

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perspectives, Biochim. Biophys. Acta. 1585, (2002) 114-125.

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26 Gómez-Muñoz A, Ceramide 1-phosphate/ceramide, a switch between life and death, Biochim.

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Biophys. Acta. 1758(12), (2006) 2049-2056.

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28 Li J, Sayeed S, Robertson S, Chen J, McClane BA, Sialidases affect the host cell adherence and

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Pathog. 7(12), (2011) e1002429.

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epsilon toxin-induced cytotoxicity of Clostridium perfringens type D strain CN3718, PLoS

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rafts, Nat. Med. 9, (2003) 322-30. 30 Hauck CR, Grassmé H, Bock J, Jendrossek V, Ferlinz K, Meyer TF, Gulbins E, Acid sphingomyelinase is involved in CEACAM receptor-mediated phagocytosis of Neisseria gonorrhoeae, FEBS Lett. 478, (2000) 260-266.

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ACCEPTED MANUSCRIPT Figure Legends

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Figure 1. Role of neutral sphingomyelinase on ceramide formation of epsilon-toxin in ACHN

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cells.

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(A) ACHN cells were treated with epsilon-toxin (5 g/ml) at 37°C for the indicated time periods. Ceramide was phosphorylated by 1,2-diacylglycerol kinase and separated by thin-layer

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chromatography (TLC) and analyzed by autoradiography. One representative experiment from three

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is shown. For quantitative analysis, the level of ceramide was measured by densitography. Control level of untreated cells was set to 1. Values represent means ± S.D. (n=3). *P<0.01, compared with

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the value of untreated cells. (B) ACHN cells were treated with epsilon-toxin (5 g/ml) at 37°C for

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the indicated time periods. Sphingomyelinase activities were measured as described in the Materials and Methods. Values represent means ± S.D. (n=3). *P<0.05, compared with the value of untreated

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cells.

Figure 2. Effect of neutral sphingomyelinase inhibitor and small interfering RNA for neutral sphingomyelinase on action of epsilon-toxin in ACHN cells. (A) ACHN cells were pretreated with 0.1 % dimethyl sulfoxide (DMSO) or 10M GW4869 at 37°C for 60 min, and incubated with epsilon-toxin (5 g/ml) at 37°C for the indicated time periods. Ceramide was phosphorylated by 1,2-diacylglycerol kinase and separated by thin-layer chromatography (TLC) and analyzed by autoradiography. One representative experiment from three

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ACCEPTED MANUSCRIPT is shown. For quantitative analysis, the level of ceramide was measured by densitography. Control

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level of untreated cells was set to 1. Values represent means ± S.D. (n=3). *P <0.05 and **P <0.01,

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compared with the value of DMSO-treated cells. (B) ACHN cells were pretreated with 0.1 % DMSO

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or 10M GW4869 at 37°C for 60 min, and incubated with epsilon-toxin (5 g/ml) at 37°C for 3 h. Cell viability was measured using the MTS test. The number of live cells is shown as a percentage of

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the values for the untreated control. Values represent means ± S.D. (n=3). *P <0.01, compared with

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the value of DMSO-treated cells. (C) ACHN cells were pretreated with 0.1 % DMSO or 10M GW4869 at 37°C for 60 min, and incubated with epsilon-toxin (5 g/ml) at 37°C for 15 min. Neutral

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sphingomyelinase (nSMase) activity was measured as described in the Materials and Methods.

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Values represent means ± S.D. (n=3). *P <0.05, compared with the value of DMSO-treated cells. (D) ACHN cells were transfected with negative control small interfering RNA (siRNA; NC-siRNA) (10

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nM) or siRNA against nSMase (10 nM). The cells were cultured for 2 days at 37°C. The lysates were subjected to SDS-PAGE and western blotting with anti-nSMase antibody. (E) ACHN cells were transfected with siRNA, as described above. The cells were incubated with epsilon-toxin (5 g/ml) at 37°C for 3 h. Cell viability was measured using the MTS test. The number of live cells is shown as a percentage of the values for untreated control. Values represent means ± S.D. (n=3). *P <0.05, compared with the value of NC-siRNA-transfected cells.

Figure 3. Role of neutral sphingomyelinase oligomerization of epsilon-toxin in ACHN cells.

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ACCEPTED MANUSCRIPT (A) ACHN cells were pretreated with 0.1 % dimethyl sulfoxide (DMSO) or 10M GW4869 at 37°C

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for 60 min, and incubated with epsilon-toxin (5 g/ml) at 37°C for the indicated times. The cells

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were subjected to SDS-PAGE and western blotting with anti-epsilon-toxin antibody. One

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representative experiment from three is shown. For quantitative analysis, the level of oligomer formation was measured by densitography. Values represent means ± S.D. (n=3). *P <0.05,

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compared with the value of DMSO- versus GW4869-treated cells. (B) ACHN cells were transfected

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with small interfering RNA (siRNA), as described above. The cells were incubated with epsilon-toxin (5 g/ml) at 37°C for the indicated times. The cells were subjected to SDS-PAGE and

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western blotting with anti-epsilon-toxin antibody. One representative experiment from three is shown.

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For quantitative analysis, the level of oligomer formation was measured by densitography. Values represent means ± S.D. (n=3). *P <0.05, compared with the value of NC-siRNA- versus

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siRNA-nSMase-transfected cells.

Figure 4. Localization of epsilon-toxin and ceramide on MDCK cells. MDCK cells were incubated with epsilon-toxin (1 g/ml) at 37°C for 60 min. The cells were fixed in 4% paraformaldehyde and stained with Hoechst33342 and antibodies against epsilon-toxin and ceramide. Epsilon-toxin (green), ceramide (red) and nuclei (blue) were viewed with the use of a confocal microscope. One representative experiment from three is shown. Scale bar: 10m.

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Graphical abstract

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Highlights

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1. We demonstrate that neutral sphigomyelinase (nSMase) activity is necessary for

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efficient oligomerization of epsilon-toxin in plasma membrane. 2. Epsilon-toxin-induced cell death is blocked by nSMase inhibitors and knock down of nSMase.

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3. nSMase is a new therapeutic target for the treatment of epsilon-toxin-producing organism

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infection.

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