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Role of sphingomyelinase in infectious diseases caused by Bacillus cereus.

Oda M, Hashimoto M, Takahashi M, Ohmae Y, Seike S, Kato R, Fujita A, Tsuge H, Nagahama M, Ochi S, Sasahara T, Hayashi S, Hirai Y, Sakurai J - PLoS ONE (2012)

Bottom Line: Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H(2)O(2) 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan.

ABSTRACT
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 H(2)O(2) 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.

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Effect of antibody and immunization against Bc-SMase, PCPLC, or PIPLC on lethality of B. cereus.Mice intraperitoneally received 50 µg of anti-SMase, -PCPLC, or -PIPLC antibodies and 2 h after the injection, were intraperitoneally administered B. cereus (JMU-06B-35). A) B. cereus in blood was cultured on Luria Broth agar plates 12 h after the intraperitoneal injection. Values represent the mean ± SEM; n = 3 independent experiments. ND: not detected. B) Mice were monitored every five hours after the injection of B. cereus. The duration of the experiment was set at 100 h. ▪, B. cereus; ▴, anti-PIPLC antibody + B. cereus; •, anti-PCPLC antibody + B. cereus; ○, anti-Bc-SMase antibody + B. cereus. C) Mice subcutaneously received an emulsion of the enzyme (Bc-SMase (○), PCPLC (•), or PIPLC (▴)) and CFA (▪) 2 times every 2 weeks. The immunized mice received B. cereus (JMU-06B-35, 3×108 CFU/mouse). The duration of the experiment was set at 100 h.
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pone-0038054-g003: Effect of antibody and immunization against Bc-SMase, PCPLC, or PIPLC on lethality of B. cereus.Mice intraperitoneally received 50 µg of anti-SMase, -PCPLC, or -PIPLC antibodies and 2 h after the injection, were intraperitoneally administered B. cereus (JMU-06B-35). A) B. cereus in blood was cultured on Luria Broth agar plates 12 h after the intraperitoneal injection. Values represent the mean ± SEM; n = 3 independent experiments. ND: not detected. B) Mice were monitored every five hours after the injection of B. cereus. The duration of the experiment was set at 100 h. ▪, B. cereus; ▴, anti-PIPLC antibody + B. cereus; •, anti-PCPLC antibody + B. cereus; ○, anti-Bc-SMase antibody + B. cereus. C) Mice subcutaneously received an emulsion of the enzyme (Bc-SMase (○), PCPLC (•), or PIPLC (▴)) and CFA (▪) 2 times every 2 weeks. The immunized mice received B. cereus (JMU-06B-35, 3×108 CFU/mouse). The duration of the experiment was set at 100 h.

Mentions: 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, 5×108 CFU) 2 h after the intraperitoneally administration of 50 µg 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 µg) 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.


Role of sphingomyelinase in infectious diseases caused by Bacillus cereus.

Oda M, Hashimoto M, Takahashi M, Ohmae Y, Seike S, Kato R, Fujita A, Tsuge H, Nagahama M, Ochi S, Sasahara T, Hayashi S, Hirai Y, Sakurai J - PLoS ONE (2012)

Effect of antibody and immunization against Bc-SMase, PCPLC, or PIPLC on lethality of B. cereus.Mice intraperitoneally received 50 µg of anti-SMase, -PCPLC, or -PIPLC antibodies and 2 h after the injection, were intraperitoneally administered B. cereus (JMU-06B-35). A) B. cereus in blood was cultured on Luria Broth agar plates 12 h after the intraperitoneal injection. Values represent the mean ± SEM; n = 3 independent experiments. ND: not detected. B) Mice were monitored every five hours after the injection of B. cereus. The duration of the experiment was set at 100 h. ▪, B. cereus; ▴, anti-PIPLC antibody + B. cereus; •, anti-PCPLC antibody + B. cereus; ○, anti-Bc-SMase antibody + B. cereus. C) Mice subcutaneously received an emulsion of the enzyme (Bc-SMase (○), PCPLC (•), or PIPLC (▴)) and CFA (▪) 2 times every 2 weeks. The immunized mice received B. cereus (JMU-06B-35, 3×108 CFU/mouse). The duration of the experiment was set at 100 h.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368938&req=5

pone-0038054-g003: Effect of antibody and immunization against Bc-SMase, PCPLC, or PIPLC on lethality of B. cereus.Mice intraperitoneally received 50 µg of anti-SMase, -PCPLC, or -PIPLC antibodies and 2 h after the injection, were intraperitoneally administered B. cereus (JMU-06B-35). A) B. cereus in blood was cultured on Luria Broth agar plates 12 h after the intraperitoneal injection. Values represent the mean ± SEM; n = 3 independent experiments. ND: not detected. B) Mice were monitored every five hours after the injection of B. cereus. The duration of the experiment was set at 100 h. ▪, B. cereus; ▴, anti-PIPLC antibody + B. cereus; •, anti-PCPLC antibody + B. cereus; ○, anti-Bc-SMase antibody + B. cereus. C) Mice subcutaneously received an emulsion of the enzyme (Bc-SMase (○), PCPLC (•), or PIPLC (▴)) and CFA (▪) 2 times every 2 weeks. The immunized mice received B. cereus (JMU-06B-35, 3×108 CFU/mouse). The duration of the experiment was set at 100 h.
Mentions: 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, 5×108 CFU) 2 h after the intraperitoneally administration of 50 µg 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 µg) 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.

Bottom Line: Treatment of mouse macrophages with Bc-SMase resulted in a reduction in the generation of H(2)O(2) 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima, Japan.

ABSTRACT
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 H(2)O(2) 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.

Show MeSH
Related in: MedlinePlus