Limits...
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.

Show MeSH

Related in: MedlinePlus

Expression of phospholipases and promoter sequences of smase from clinical isolates and ATCC strains of B. cereus.A) 50% Ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) were subjected to SDS-PAGE and Western blotting using anti-Bc-SMase, -PCPLC, and -PIPLC antibodies. Lane: 1, JMU-06B-31; 2, JMU-06B-35; 3, JMU-06B-1; 4, ATCC21928; 5, ATCC31429; 6, ATCC6464. A representative result from one of three experiments is shown. B, C) The sequences of the promoter region of plc and smase from clinical isolates and ATCC strains of B. cereus were aligned by the program T-Coffee [44]. Consensus sequences of regulatory elements are indicated in bold type. Gray areas indicate nucleotide sequence differences.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368938&req=5

pone-0038054-g002: Expression of phospholipases and promoter sequences of smase from clinical isolates and ATCC strains of B. cereus.A) 50% Ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) were subjected to SDS-PAGE and Western blotting using anti-Bc-SMase, -PCPLC, and -PIPLC antibodies. Lane: 1, JMU-06B-31; 2, JMU-06B-35; 3, JMU-06B-1; 4, ATCC21928; 5, ATCC31429; 6, ATCC6464. A representative result from one of three experiments is shown. B, C) The sequences of the promoter region of plc and smase from clinical isolates and ATCC strains of B. cereus were aligned by the program T-Coffee [44]. Consensus sequences of regulatory elements are indicated in bold type. Gray areas indicate nucleotide sequence differences.

Mentions: 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 µg/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).


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)

Expression of phospholipases and promoter sequences of smase from clinical isolates and ATCC strains of B. cereus.A) 50% Ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) were subjected to SDS-PAGE and Western blotting using anti-Bc-SMase, -PCPLC, and -PIPLC antibodies. Lane: 1, JMU-06B-31; 2, JMU-06B-35; 3, JMU-06B-1; 4, ATCC21928; 5, ATCC31429; 6, ATCC6464. A representative result from one of three experiments is shown. B, C) The sequences of the promoter region of plc and smase from clinical isolates and ATCC strains of B. cereus were aligned by the program T-Coffee [44]. Consensus sequences of regulatory elements are indicated in bold type. Gray areas indicate nucleotide sequence differences.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368938&req=5

pone-0038054-g002: Expression of phospholipases and promoter sequences of smase from clinical isolates and ATCC strains of B. cereus.A) 50% Ammonium sulfate precipitation fractions of the culture supernatants (1.0 mg protein) were subjected to SDS-PAGE and Western blotting using anti-Bc-SMase, -PCPLC, and -PIPLC antibodies. Lane: 1, JMU-06B-31; 2, JMU-06B-35; 3, JMU-06B-1; 4, ATCC21928; 5, ATCC31429; 6, ATCC6464. A representative result from one of three experiments is shown. B, C) The sequences of the promoter region of plc and smase from clinical isolates and ATCC strains of B. cereus were aligned by the program T-Coffee [44]. Consensus sequences of regulatory elements are indicated in bold type. Gray areas indicate nucleotide sequence differences.
Mentions: 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 µg/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).

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