RESUMO
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.
Assuntos
Anticorpos Antibacterianos/imunologia , Bacillus cereus/enzimologia , Bacillus cereus/imunologia , Sepse/microbiologia , Esfingomielina Fosfodiesterase/metabolismo , Animais , Anticorpos Antibacterianos/farmacologia , Bacillus cereus/crescimento & desenvolvimento , Sequência de Bases , Cromatografia em Camada Fina , Peróxido de Hidrogênio/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Fagocitose/efeitos dos fármacos , Fotodegradação , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Análise de Sequência de DNA , Microbiologia do Solo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A characteristic feature of gas gangrene with Clostridium perfringens (C. perfringens) is the absence of neutrophils within the infected area and the massive accumulation of neutrophils at the vascular endothelium around the margins of the necrotic region. Intravenous injection of C. perfringens alpha-toxin into mice resulted in the accumulation of neutrophils at the vascular endothelium in lung and liver, and release of GRO/KC, a member of the CXC chemokine family with homology to human interleukin-8 (IL-8). Alpha-toxin triggered activation of signal transduction pathways causing mRNA expression and production of IL-8, which activates migration and binding of neutrophils, in A549 cells. K252a, a tyrosine kinase A (TrkA) inhibitor, and siRNA for TrkA inhibited the toxin-induced phosphorylation of TrkA and production of IL-8. In addition, K252a inhibited the toxin-induced phosphorylation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK). PD98059, an ERK1/2 inhibitor, depressed phosphorylation of ERK1/2 and nuclear translocation of nuclear factor kappa B (NF-κB) p65, but SB203580, a p38 MAPK inhibitor, did not. On the other hand, PD98059 and SB203580 suppressed the toxin-induced production of IL-8. Treatment of the cells with PD98059 resulted in inhibition of IL-8 mRNA expression induced by the toxin and that with SB203580 led to a decrease in the stabilization of IL-8 mRNA. These results suggest that alpha-toxin induces production of IL-8 through the activation of two separate pathways, the ERK1/2/NF-κB and p38 MAPK pathways.