Role of Sphingomyelinase in Infectious Diseases Caused by Bacillus cereus

Bacillus cereus (B. cereus) is a pathogen in opportunistic infections. Here we show that Bacillus cereus sphingomyelinase (Bc-SMase) is a virulence factor for septicemia. Clinical isolates produced large amounts of Bc-SMase, grew in vivo, and caused death among mice, but ATCC strains isolated from soil did not. A transformant of the ATCC strain carrying a recombinant plasmid containing the Bc-SMase gene grew in vivo, but that with the gene for E53A, which has little enzymatic activity, did not. Administration of an anti-Bc-SMase antibody and immunization against Bc-SMase prevented death caused by the clinical isolates, showing that Bc-SMase plays an important role in the diseases caused by B. cereus. Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H2O2 and phagocytosis of macrophages induced by peptidoglycan (PGN), but no effect on the release of TNF-α and little release of LDH under our experimental conditions. Confocal laser microscopy showed that the treatment of mouse macrophages with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that the cells treated with Bc-SMase exhibited a reduction in membrane fluidity. The results suggest that Bc-SMase is essential for the hydrolysis of SM in membranes, leading to a reduction in phagocytosis.


Introduction
B. cereus is well-known for its role as a mediator of food-borne illness [1,2,3,4]. The microorganism, which forms spores, is found worldwide in dust, air, and water [5]. Therefore, B. cereus is ubiquitous in the hospital environment, indicating that contamination of dressings, intravenous catheters, and linen provides an opportunity for infection [5]. It is possible that B. cereus is a pathogen of nosocomial infections transmitted via towels, linen, and balloons to compromised patients [6,7]. In recent years, there has been an increasing appreciation for its potential as an opportunistic pathogen in immunocompromised hosts [5,6,8,9]. The microorganism secretes a wide variety of membrane-damaging toxins, phospholipases such as Bc-SMase, phosphatidylinositol-specific phospholipase C (PIPLC) and phosphatidylcholine-specific phospholipase C (PCPLC), and hemolysins such as cereolysin O, hemolysins and proteases [3,10,11,12,13,14]. However, there has been little research into the contributions of these enzymes and toxins to the infectious diseases caused by B. cereus.
The SMase produced by the intracellular pathogen Listeria ivanovii was shown to mediate bacterial escape from the phagocytic vacuole following internalization, thereby promoting intracellular survival and propagation [15]. Helicobacter pyloriderived SMase was found to contribute toward cytotoxicity for gastric cells [16]. b-Hemolysin containing SMase activity from methicillin-resistant Staphylococcus aureus was expressed by 91% of strains in a high-toxicity group [17]. A mutant strain with deletions of b-hemolysin and catalase was significantly less virulent to mice than the wild-type Staphylococcus aureus strain [18]. We reported that Bc-SMase lysed sheep erythrocytes containing large amounts of SM in the outer lipid layer of their plasma membranes [19]. However, the enzyme is known not to be lethal or cytotoxic. Bc-SMase belongs to a family of Mg 2+dependent neutral SMases (nSMase) that includes SMases produced by Staphylococcus aureus, and Listeria ivanovii [20]. The members of this family share a high degree of homology in amino acid sequence [20,21,22]. However, the role of Bc-SMase in the virulence of B. cereus remains controversial.
To investigate the relationship between Bc-SMase and B. cereus infections, we examined the relationship between Bc-SMase and the growth in vivo of clinical isolates of B. cereus.

Pathogenicity of the Clinical Isolates of B. cereus
To investigate if B. cereus JMU-06B-31 and JMU-06B-1, isolated from a patient with septicemia, and JMU-06B-35, isolated from a patient with endophthalmitis, grow in mice in vivo, six-to eightweek old male wild-type mice of the ICR mice were each injected intraperitoneally with 5610 8 CFU of the clinical isolates or ATCC21928, ATCC31429, and ATCC6464 isolated from soil. Mice administered with the clinical isolates began to die after 12 h, and all mice died within 30 h of the administration (Fig. 1A). Mice injected with ATCC21928, ATCC31429, and ATCC6464 did not die within 100 h (Fig. 1A). The number of microorganisms in the blood of mice about 12 h after the administration of JMU-06B-31, JMU-06B-35, and JMU-06B-1 was 300-400 CFU/100 mL, whereas the ATCC strains were not detected in blood (Fig. 1B).

Production of Phospholipases by the Clinical Isolates and the ATCC Strains of B. cereus
Phospholipases produced by bacteria such as Staphylococcus aureus, Clostridium perfringens, and Helicobacter pylori are reported to be associated with local infections and of importance in the establishment of systemic diseases [4,16,23,24]. To analyze the production of phospholipases by B. cereus, we measured the amount of phospholipases produced by the clinical isolates and the ATCC strains in Luria Broth medium. These strains were cultured to an optical density at 620 nm of 0.8 in the medium. The enzyme samples fractionated from the culture supernatants were subjected to SDS-PAGE and Western blotting using anti-Bc-SMase, -PCPLC, and -PIPLC antibodies. As shown in Fig. 2A, large amounts (.5 mg/ml) of Bc-SMase, PCPLC, and PIPLC were detected in the culture supernatants of the clinical isolates, but very small amounts or undetectable levels in those of the ATCC strains. These phospholipase C genes were detected in every clinical and ATCC strain ( Figure S1).
Next, we focused on the promoter sequence for the Bc-SMase gene (smase) or PLC gene (plc) of clinical isolates and ATCC isolates. The 235 and 210 promoter sequences of smase or plc from clinical isolates were almost the same as those of ATCC strains ( Fig. 2B and 2C). In B. cereus, the transcriptional regulator PlcR (Phospholipase C regulator) controls most known virulence factors [25,26], and activates gene expression by binding to a nucleotidic sequence called the 'PlcR box' [25]. As shown in Fig. 2B and 2C, there was no clear difference in the sequence of the PlcR box between clinical isolates and ATCC strains. In addition, the amino acid sequence of Bc-SMase was highly conserved in all strains ( Figure S2).

Effect of Anti-phospholipases on Growth of B. cereus in Mice
To provide clues regarding the growth of B. cereus in vivo, the effect of anti-phospholipases on the growth of JMU-06B-35 in mice was investigated. Mice were intraperitoneally injected with the clinical isolate (JMU-06B-35, 5610 8 CFU) 2 h after the intraperitoneally administration of 50 mg of anti-PCPLC, -PIPLC, or -SMase antibody. The anti-Bc-SMase antibody completely inhibited the growth of JMU-06B-35 in the bloodstream (Fig. 3A). In addition, the mice injected with the anti-Bc-SMase antibody did not die within 100 h (Fig. 3B). The administration of the anti-PIPLC and -PCPLC antibodies had no effect on the growth and lethality of JMU-06B-35 in mice ( Fig. 3A and 3B). The concentration of these antibodies was enough to neutralize the activity of the three enzymes (10 mg) in vitro (data not shown). It therefore appears that Bc-SMase plays an important role in the propagation of B. cereus in vivo in our experimental condition.
To confirm the relationship between Bc-SMase and the growth of B. cereus in vivo, we investigated the effect of immunization of mice with Bc-SMase, PCPLC, or PIPLC on the death induced by JMU-06B-35. The BALB/c mice were immunized with 25 mg mixture of PCPLC, PIPLC, or Bc-SMase with Complete Freund's adjuvant (CFA) two times at two-week intervals. Sham-immunized mice administered the clinical isolate began to die after approximately 10 h, and all mice died within 30 h of the administration (Fig. 3C). The survival rate of mice immunized against Bc-SMase, PCPLC, or PIPLC was 100%, 0%, and 0% 30 h after infection, respectively (Fig. 3C).

Effect of Bc-SMase on Infection of Mice with ATCC Strain Isolated from Soil
To investigate the role of Bc-SMase in B. cereus infections, we examined the effect of Bc-SMase on B. cereus-induced death in mice. The animals were intraperitoneally injected with mixtures of ATCC21928 (5.0610 7 CFU/mouse), which did not produce Bc-SMase in the culture supernatants, and various concentrations of Bc-SMase. As shown in Fig. 4A, the increase in the rate of death was dependent on the dose of Bc-SMase above 1.0 mg/mouse. On administration of ATCC21928 and 5.0 mg of Bc-SMase, the death rate was 100% within 30 h (Fig. 4A). Mice injected with ATCC21928 or Bc-SMase alone survived after 100 h under the experimental conditions (Fig. 4A). In addition, the number of microorganisms in blood 12 h after the administration of the mixture of ATCC21928 and 1.0 or 5.0 mg of Bc-SMase was 50-100 and 300-400 CFU/100 ml, respectively (Fig. 4B). On the other hand, the administration of ATCC21928 with PCPLC (5.0 mg/mouse) or PIPLC (5.0 mg/mouse) resulted in no death under the conditions (data not shown).

subtilis in Mice
To investigate the effect of Bc-SMase on growth of B. cereus in vivo, we transfected a vector expressing smase or the gene for E53A (e53a), a variant which has little enzymatic activity [27] (Table S1), into ATCC21928 or Bacillus subtilis (ISW1215), which did not produce Bc-SMase in the culture supernatants. The ammonium sulfate precipitation fraction of the culture supernatant fluids of these transfected strains was subjected to SDS-PAGE and Western blotting using anti-Bc-SMase antibody. As shown in Fig. 5A, these proteins (.5.0 mg/ml) were detected in the culture supernatants of these transformants carrying smase or e53a, but not in each microorganism transformed with empty vector. When the mice intraperitoneally received ATCC21928 or ISW1215 transformants carrying the smase, the microorganisms were detected about 300-400 CFU/100 ml in bloodstream (Fig. 5B). However, administration of these bacteria carrying e53a had no effect on the growth of each microorganism in vivo (Fig. 5B). The results showed that overexpression of Bc-SMase in ATCC21928 or ISW1215 induced growth of these strains in vivo. In addition, the survival rate 100 h after administration of ATCC21928 transformants carrying the smase was approximately 50%, but that of ISW1215 was 100% (Fig. 5C).

Effect of Bc-SMase on Activation of Macrophages by Peptidoglycan
González Zorn et al. reported that the SMase from Listeria ivanovii mediates bacterial escape from phagocytic cells [15]. The activation of macrophages is known to be related to bactericidal action in vivo. To investigate the effect of Bc-SMase on the activation of macrophages, we assessed the effect of PGN, an activator of macrophages, on macrophages treated with Bc-SMase. Bc-SMase attenuated PGN-activated H 2 O 2 generation and phagocytosis of macrophages in a dose-dependent manner ( Fig. 6A  and 6B). However, Bc-SMase had no effect on the release of TNFa induced by PGN from macrophages and induced no release of lactate dehydrogenase (LDH) from the cells (Fig. 6C and 6D). It therefore is likely that Bc-SMase specifically influences H 2 O 2 generation and phagocytosis without impairing membranes of macrophages, suggesting that treatment of macrophages with Bc-SMase results in a change in function of the membranes. It was thought that the frustrated phagocytosis may be dependent on the formation of ceramide in macrophage membranes.

Localization of Ceramide in Membranes of Macrophages Treated with Bc-SMase
To determine the amount of ceramide formed in the macrophages treated with Bc-SMase, macrophages were incubated with Bc-SMase at 37uC for 30 min. The lipids extracted from the treated cells were phosphorylated by diacylglycerol kinase from Escherichia coli, and developed by reverse-phase thin layer chromatography (TLC). The level of ceramide in the cells treated with Bc-SMase increased in a dose-dependent manner (Fig. 7A). Using confocal microscopy, Montes et al. found that phospholipase C from P. aeruginosa caused the formation of ceramide-rich domains in biological membranes [28]. We reported that Bc-SMase induced the formation of ceramide-rich domains in membranes of sheep erythrocytes and a decrease in the fluidity of membranes, leading to destabilization under physical stimulation [19]. We investigated whether treatment of macrophages with Bc-SMase results in the local accumulation of BODIPY-ceramide formed in membranes of cells preincubated with BODIPY FL-C 12 -SM (BODIPY-SM). Fig. 7B (left) shows that the fluorescent substance in membranes of macrophages preincubated with BODIPY-SM was not localized. However, when BODIPY-SM -incubated macrophages were treated with Bc-SMase, the local accumulation of the fluorescent substance was found on membranes of the cells, as shown by the white arrows (Fig. 7B, right). To test whether the site where the substance accumulates coincides with a ceramide-rich site, BODIPY-SM-preincubated membranes treated with Bc-SMase were analyzed using Cy3-labeled anti-ceramide antibody. As shown in Fig. 7C, the distribution of the fluorescence of the antibody was different from that of BODIPY-SM in the untreated cells. In the case of BODIPY-SM-preincubated macrophages treated with Bc-SMase, the location of the fluorescence of Cy3-anti-ceramide antibody was consistent with that of BODIPY, as shown in Fig. 7B, suggesting that BODIPYceramide formed from BODIPY-SM in the macrophages treated with Bc-SMase is mostly located in ceramide-rich domains. Klein et al. reported that membrane fluidity of cells was evaluated by measurement of lateral diffusion of fluorescence-labeled SM by FRAP with a confocal laser microscopy [29]. A FRAP analysis revealed that the recovery of effective diffusion for the fluorescence of BODIPY in ceramide-rich domains of the macrophages treated with Bc-SMase decreased to about 70-80%, compared with that of BODIPY-SM in the untreated cells  (Fig. 7D). It therefore appears that the Bc-SMase-induced formation of ceramide from SM in membranes of macrophages results in a decrease in the fluidity of membranes.

Discussion
The present study showed that clinical isolates of B. cereus, which produced large amounts of Bc-SMase, from patients with sepsis and endophthalmitis were lethal to mice. The production of Bc-SMase from clinical isolates was greater than that from ATCC strains under our culture conditions. B. cereus produces several secreted toxins, the expression of which is controlled by the PlcR [25,26]. A difference in protein level from PlcR regulated proteins has been observed for pathogenic factors such as nonhemolytic enterotoxin and hemolysin BL [30,31]. In addition, variations of the InhA1, NprA, and HlyII, which regulation is independent on PlcR, between pathogenic and nonpathogenic B. cereus strains has also been observed [32]. In this study, the sequences of the PlcR box in clinical isolates were almost the same as that in ATCC strains. Therefore, it appears that various factors participate in the pathogenic expression of B. cereus and production of Bc-SMase.
Bc-SMase enhanced the growth of B. cereus in the peritoneal cavity, and in addition, invaded the bloodstream in mice, causing death. Furthermore, overexpression of Bc-SMase in B. subtilis, an avirulent strain, induced growth in mice. The administration of a mixture of ATCC21928 and Bc-SMase resulted in the death of mice, but that of PIPLC or PCPLC did not. In addition, the loss of PlcR-regulated factors, which include Bc-SMase, significantly attenuated the severity of Bacillus endophthalmitis [10]. Furthermore, mice administered the anti-Bc-SMase antibody or immunized with Bc-SMase were protected from the lethality of clinical isolates of B. cereus. Callegan et al., reported that intraocular infection with wild type B. cereus or isogenic mutants specifically deficient in PIPLC or PCPLC resulted in similar degrees of destruction of the retinal architecture, and a complete loss of retinal function [33,34]. These results suggested that Bc-SMase plays an important role in the propagation of B. cereus in vivo.
Antibody and immunization against Bc-SMase protected mice from B. cereus, suggesting Bc-SMase to be a candidate vaccine against infectious diseases caused by B. cereus. Furthermore, it has been reported that nSMase is secreted by several bacteria involved in infectious diseases [17,18]. Drobnik et al. found that an increase in ceramide levels mediated by nSMase in serum was associated with sepsis-related mortality [35]. Gonzalez-Zorn et al. reported that SMase from Listeria ivanovii was involved in avoiding phagocytic vacuoles in the bovine epithelial-cell line MDBK [15]. Heffernan et al. showed that the deletion of three phospholipase (PIPLC, PCPLC, and SMase) genes was required for the attenuation of virulence in a murine model of anthrax and that these enzymes play an important role in the growth of Bacillus anthracis in alveolar macrophages [36]. Lo et al. reported that a combination of b-hemolysin immunization and Christie Atkins Munch-Peterson factor neutralization cooperatively suppressed the skin lesions caused by a coinfection of Staphylococcus aureus and Propionibacterium acnes [37]. The result suggested the need for immunotherapy targeting the interaction of Staphylococcus aureus with a skin commensal. Therefore, our findings may provide significant opportunities for the development of new vaccines against infections caused by SMase-producing bacteria.
Treatment of macrophages with SMase resulted in a reduction in the generation of H 2 O 2 and phagocytosis of macrophages induced by PGN, but no effect on the release of TNF-a and little release of LDH under our experimental conditions. Therefore, it appears that Bc-SMase specifically inhibits the activation of macrophages induced by PGN without harmful effects on the cells. The treatment with Bc-SMase is known to induce changes in the lipid content of plasma membranes by generating ceramide upon the cleavage of SM [38]. Ceramide is reported to promote the coalescence of rafts, which have been termed ''membrane platforms'' [39]. We reported that Bc-SMase induced the formation of ceramide-rich domains in membranes of sheep erythrocytes, and a decrease in the fluidity of membranes, leading to destabilization under physical stimulation and finally the disruption of erythrocytes [19]. The treatment of mouse macrophages with Bc-SMase caused the formation of ceramide, and inhibition of the production of H 2 O 2 and phagocytosis induced by PGN. Treatment of macrophages with ceramide significantly inhibited the generation of H 2 O 2 and phagocytosis of macrophages activated by PGN, suggesting that the formation of ceramide induced by Bc-SMase is closely related to a reduction in the activation of macrophages. Nakabo and Pabst have reported that C2-or C6-ceramide inhibited the release of superoxides from monocytes primed with lipopolysaccharide, and induced the secretion of small amounts of TNF-a and IL-1b [40]. The treatment of macrophages with Bc-SMase resulted in the clustering of ceramide recognized by a Cy3-anti-ceramide antibody. Furthermore, using a confocal laser scanning microscope, we found that the effective diffusion rate for BODIPY-ceramide at ceramide-rich domains in macrophages treated with Bc-SMase was reduced about 70%, compared with that for BODIPY-SM, ceramide-poor domains, in control cells. Goni and Alonso reported that the ceramide-rich domains formed a rigid phase in membranes [28,41]. Therefore, it appears that the formation of ceramide in macrophages treated with Bc-SMase results in functional differences of the membranes through the coalescence of ceramide-rich domains and formation of the interface between rigid and fluid domains, leading specifically to inhibition of the production of H 2 O 2 and phagocytosis induced by PGN.
In conclusion, the hydrolysis of SM to form ceramide in the macrophage membrane treated with Bc-SMase induced the attenuation of membrane fluidity and the frustrated phagocytosis. Bc-SMase plays a crucial role in the evasion from immune response by macrophages during the early stages of infections of B. cereus.

Strains
The clinical isolates of B. cereus (JMU-06B-31, JMU-06B-35, and JMU-06B-1) were isolated at Jichi Medical University. These isolates, obtained from patients diagnosed with Bacillus bacteremia or endophthalmitis according to the CDC definition, were identified and characterized as B. cereus, as described previously [6]. The ATCC strains of B. cereus from soil (ATCC21928, ATCC31429, ATCC6464) were purchased from DS Pharma Biomedical, Tokyo, Japan. The characteristics of the clinical isolates were reported previously [6].

Mice
Six-to eight-week old male wild-type mice of the ICR and BALB/c strains (Nihon SLC, Japan) were used. Experimental protocols were approved by the Institute Animal Care and Use Committee at Tokushima Bunri University. The mice were housed in plastic cage under controlled environmental conditions (temperature 22uC, humidity 55%). Food and water were freely available.

Site-directed Mutagenesis
The transformer site-directed mutagenesis kit (LA PCR in vitro Mutagenesis Kit, Takara, Japan) was used with the primer E53A: 59-GTTATTTTAAATGCCGTGTTTGATAATAGC-39 to prepare the modified plasmid. The genetic sequence of Bc-SMase in each plasmid was confirmed with an ABI310 PRISM TM genetic analyzer (Life technologies, USA).

Preparation of Bc-SMase and Variants
Bc-SMase and E53A were overexpressed in B. subtilis ISW1214 or B. cereus ATCC21928 transformed with the plasmid vector pHY300PLK carrying smase or e53a. The expression and purification of the recombinant Bc-SMase and variants were performed as described previously [21].

Purification of PCPLC and PIPLC
PCPLC and PIPLC were overexpressed in B. subtilis ISW1214 transformed with pHY300PLK carrying the gene of PCPLC or PIPLC cloned from B. cereus IAM1029. The PCPLC and PIPLC were secreted into the culture medium. The 80% (w/v) ammonium sulfate fraction of the Luria Broth was fractionated by chromatography using a Cu 2+ -column and then a DEAE-Sepharose column. The purity of samples was verified using SDS-PAGE, and staining with coomasie Brilliant Blue. The PCPLC and PIPLC were observed as a single band at 28 kDa and 34 kDa, respectively.

Preparation of Antibodies
Anti-Bc-SMase, -PCPLC, and -PIPLC antibodies were prepared by immunizing rabbits with 100 mg of the purified phospholipases. CFA (Difco, USA) (1.0 ml) was used for the preparation of antigens. Two hypodermic booster injections were made. Antiserum was obtained 2 weeks after the last injection. Purification of these antisera was performed using Ab-Rapid PuRe (Protenova, Japan).

ELISA Procedure
The purified recombinant PCPLC, PIPLC, or Bc-SMase was diluted to 5 mg/ml in a carbonate buffer (0.05, pH 9.5) and used to coat the wells of polystyrene plates (100 ml/well: Nunc-Immuno plates with a Maxisorp surface) The plates were incubated overnight at 4uC, and the next morning washed three times with PBST (PBS/0.05% Tween-20). The remaining sites of absorption were blocked by the addition of 200 ml/well in PBS containing 3% BSA for 2 h at 37uC. The plates were washed three times with PBST. Serum from each group of immunized animals was serially diluted 2-fold (1:500 to 1:128,000) and examined in triplicate wells (100 ml/well) of the blocked antigencoated plates and incubated for 1 h at 37uC. The plates were then washed five times with PBST and further incubated at 37uC for 1 h with HRP-conjugated anti-mouse IgG (1:2000). The plates were washed five times with PBST and developed with 100 ml of ortho-pheylenediamine (0.4 mg/ml) in a fleshly prepared citrate phosphate buffer (0.1 M, pH5.0) and H 2 O 2 (0.4 mg/ml). The reaction was terminated by the addition of 50 ml of 2.5 N H 2 SO 4 /well. Absorbance was read at 492 nm with a microtiter plate reader.

Determination of ELISA Titer by Endpoint Dilution
The serum was diluted 2-fold from 1:500 to 1:128,000, and an absorbance value was determined for each dilution. The cut-off value for the assay was calculated from the reference curve for the control serum. The titer of immune serum was calculated as the reciprocal of the highest dilution yielding a specific optical density above the cut-off value. A significant (P,0.05) value of IgG antibody against recombinant PCPLC, PIPLC, and Bc-SMase was 128,000, 64,000, and 64,000, respectively, when compared with CFA-treated mouse serum.

Measurement of Cytokines
The concentration of TNF-a was determined with enzymelinked absorbent assay kits (R&D systems, USA).

Culture of Macrophages
Mouse macrophages were isolated from cells in peritoneal exudates with 2 ml of phenol red-free RPMI1640 medium (Wako Pure Chemical Industries, Japan) supplemented with 5% fetal bovine serum (FBS) (Biowest, USA). After centrifugation at 1706g for 10 min at 4uC, the cell pellet was resuspended in phenol redfree RPMI1640 medium supplemented with 5% FBS. Adherent macrophage monolayers were obtained by plating the cells in 96or 48-well plastic trays (Falcon, USA).
Preparation of sheep erythrocytes. Sheep erythrocytes were suspended in 0.02 M Tris-HCl buffer (pH 7.5) containing 0.9% NaCl (TBS), and centrifuged at 1,1006g for 3 min. The erythrocytes were washed by the centrifugation three times. The number of erythrocytes was determined with a cell counter (Celltac; Nihon Kohden, Japan).

Determination of Hemolytic Activity
Bc-SMase and E53A was incubated with sheep erythrocytes (12610 10 cells/ml) in TBS at 37uC for 30 min, and the cells were chilled at 4uC. The hemolysis of the erythrocytes was measured, as described previously [42]. Hemolysis was expressed as a percentage of the amount of hemoglobin released from 0.1 ml of erythrocytes suspended in 0.4 ml of 0.4% NaCl.

Preparation of Liposomes
SM (Nacalai Tesque, Japan) from bovine brain and cholesterol (1:1) in chloroform-methanol (2:1 v/v) were dried with N 2 gas, resuspended in TBS containing 0.1 M calboxyfluoroscein (CF). The liposome suspensions were centrifuged at 22,0006g for 15 min at 4uC to remove the nonencapsulated marker, and washed three times by centrifugation. The resulting liposomes were suspended in 200 ml of TBS.
The SM-liposome-disruption activity. The SM-liposomedisruption activity was evaluated at the amount of released-CF in the test aliquot. The SM-liposomes in TBS containing 1 mM MgCl 2 were incubated with Bc-SMase or E53A for 30 min at 37uC. The wavelengths for excitation and measurement were 490 and 530 nm, respectively.

Measurement of Intracellular H 2 O 2
Mouse macrophages (80% confluent in 48-well plates) isolated from mouse peritoneal exudates were activated with 5 mg/ml PGN (Sigma, USA) for 60 min in the presence of phenol red-free RPMI1640 medium (supplemented with 5% FBS). H 2 O 2 was measured in the supernatants using an H 2 O 2 assay kit (Oxis International, USA).

Assay of Phagocytosis
Phagocytic activity was determined by measuring the uptake of fluorescent microspheres (Fluoresbrite Carboxylate Microspheres, 1.75 mm in diameter, Polysciences), as described [34]. Mouse macrophages (80% confluent in 48-well plates) were stimulated by PGN in the presence of 5.0610 5 fluorescent microspheres per ml. After 3 h incubation, cells were washed, and fluorescent intensity in the cells was determined with a fluorescence imaging analyzer (FLA-1000, Fujifilm, Japan).

Measurement of LDH
LDH activity was determined with LDH assay kits (Wako Pure Chemical Industries, Japan), according to the manufacturer's instructions.

Determination of Ceramide
Mouse macrophages were incubated with various concentrations of Bc-SMase at 37uC for 60 min in phenol red-free RPMI1640 medium supplemented with 5% FBS. The isolation and the measurement of ceramide were performed as described previously [42,43]. The ceramide, which is from bovine brain, used as stimulants or standard was purchased from Sigma, USA.

Immunofluorescence Staining and Confocal Imaging
Mouse macrophages stained with 2 mM BODIPY-SM were plated on 35-mm glass-bottomed dishes (MatTek, USA). The cells were incubated with Bc-SMase in phenol red-free RPMI 1640 medium supplemented with 5% FBS at 37uC for 60 min, and the reaction was stopped by 1.0% paraformaldehyde at room temperature. For antibody labeling, the cells were incubated in 50 mM NH 4 Cl in phosphate-buffered saline (PBS) at room temperature for 10 min. After being washed with PBS, the cells were incubated in PBS containing 4% BSA at room temperature for 60 min, followed by mouse Cy3-labeled-anti-ceramide antibody in PBS for 60 min.

Fluorescence Microscopy
A confocal fluorescence microscope (A1; Nikon, Japan) was used. The excitation wavelength was 488 nm for BODIPY FL C 12 -SM (Molecular probes, USA). The fluorescence signals were simultaneously collected using NIS-Elements C (Nikon software, Japan) into a channel using bandpass filters of 525/50. The objective used in all experiments was a 606oil immersion, CFI Plan Apo VC 606oil/1.40 objective (Nikon). The objective lens was used with a zoom factor of 2. The experiments were performed at room temperature.

Fluorescence Recovery after Photobleaching
Fluorescence recovery after photobleaching (FRAP), a technology used to measure the lateral mobility of membranes, was performed with a Nikon A1R confocal laser scanning microscope, according to the manual. Mouse macrophages stained with 2 mM BODIPY-SM were plated on 35-mm glass-bottomed dishes (MatTek, USA). The photobleaching was performed in a 1.5 mm, visually uniform region of the cell membranes. Bleaching was performed with 5% laser intensity for a duration of approximately 1 s (10 scans of the laser) to achieve 20% bleaching of the BODIPY fluorescence. After photobleaching, images were acquired 200 times at 1 s intervals.

Statistics
Results were expressed as the mean 6 SEM. n equals the sample size. Statistical comparisons were performed using an unpaired t-test or one-way analysis of variance (ANOVA) with Bonferroni correction. p Values less than 0.05 were considered statistically significant. Table S1 Biological activities of E53A. Activity (%) was expressed as the percentage of each activity in the wild-type enzyme. Each value is the mean of five experiments. (DOCX)