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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 overexpression of Bc-SMase on growth of B. cereus or B. subtilis in mice.A) B. cereus (ATCC21928) or B. subtilis (ISW1215) was transfected with the plasmid carrying smase or e53a. A) 50% ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) of each microorganism were subjected to SDS-PAGE and Western blotting using anti-Bc-SMase antibody. A representative result from one of three experiments is shown. B, C) Mice intraperitoneally received B. cereus or B. subtilis transformants (1×108 CFU/mouse) carrying empty vector (vector), smase, or e53a. The microorganisms in the blood of mice about 12 h after the administration of these strains were cultured on Luria Broth agar plates. Values represent the mean ± SEM; n = 5 independent experiments. C) Mice were monitored every five hours after the injection. The duration of the experiment was set at 100 h. •, vector (B. cereus); ▪, smase (B. cereus); ▴, e53a (B.cereus); ○, vector (B. subtilis); □, smase (B. subtilis); △, e53a (B. subtilis).
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pone-0038054-g005: Effect of overexpression of Bc-SMase on growth of B. cereus or B. subtilis in mice.A) B. cereus (ATCC21928) or B. subtilis (ISW1215) was transfected with the plasmid carrying smase or e53a. A) 50% ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) of each microorganism were subjected to SDS-PAGE and Western blotting using anti-Bc-SMase antibody. A representative result from one of three experiments is shown. B, C) Mice intraperitoneally received B. cereus or B. subtilis transformants (1×108 CFU/mouse) carrying empty vector (vector), smase, or e53a. The microorganisms in the blood of mice about 12 h after the administration of these strains were cultured on Luria Broth agar plates. Values represent the mean ± SEM; n = 5 independent experiments. C) Mice were monitored every five hours after the injection. The duration of the experiment was set at 100 h. •, vector (B. cereus); ▪, smase (B. cereus); ▴, e53a (B.cereus); ○, vector (B. subtilis); □, smase (B. subtilis); △, e53a (B. subtilis).

Mentions: 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 µg/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 µl 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).


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 overexpression of Bc-SMase on growth of B. cereus or B. subtilis in mice.A) B. cereus (ATCC21928) or B. subtilis (ISW1215) was transfected with the plasmid carrying smase or e53a. A) 50% ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) of each microorganism were subjected to SDS-PAGE and Western blotting using anti-Bc-SMase antibody. A representative result from one of three experiments is shown. B, C) Mice intraperitoneally received B. cereus or B. subtilis transformants (1×108 CFU/mouse) carrying empty vector (vector), smase, or e53a. The microorganisms in the blood of mice about 12 h after the administration of these strains were cultured on Luria Broth agar plates. Values represent the mean ± SEM; n = 5 independent experiments. C) Mice were monitored every five hours after the injection. The duration of the experiment was set at 100 h. •, vector (B. cereus); ▪, smase (B. cereus); ▴, e53a (B.cereus); ○, vector (B. subtilis); □, smase (B. subtilis); △, e53a (B. subtilis).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368938&req=5

pone-0038054-g005: Effect of overexpression of Bc-SMase on growth of B. cereus or B. subtilis in mice.A) B. cereus (ATCC21928) or B. subtilis (ISW1215) was transfected with the plasmid carrying smase or e53a. A) 50% ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) of each microorganism were subjected to SDS-PAGE and Western blotting using anti-Bc-SMase antibody. A representative result from one of three experiments is shown. B, C) Mice intraperitoneally received B. cereus or B. subtilis transformants (1×108 CFU/mouse) carrying empty vector (vector), smase, or e53a. The microorganisms in the blood of mice about 12 h after the administration of these strains were cultured on Luria Broth agar plates. Values represent the mean ± SEM; n = 5 independent experiments. C) Mice were monitored every five hours after the injection. The duration of the experiment was set at 100 h. •, vector (B. cereus); ▪, smase (B. cereus); ▴, e53a (B.cereus); ○, vector (B. subtilis); □, smase (B. subtilis); △, e53a (B. subtilis).
Mentions: 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 µg/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 µl 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).

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