RESUMEN
Panton-Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus that has recently been associated with necrotizing pneumonia. In the present study, we report that in vitro, PVL induces polymorphonuclear cell death by necrosis or by apoptosis, depending on the PVL concentration. PVL-induced apoptosis was associated with a rapid disruption of mitochondrial homeostasis and activation of caspase-9 and caspase-3, suggesting that PVL-induced apoptosis is preferentially mediated by the mitochondrial pathway. Polymorphonuclear cell exposure to PVL leads to mitochondrial localization of the toxin, whereas Bax, 1 of the 2 essential proapoptotic members of the Bcl-2 family, was still localized in the cytosol. Addition of PVL to isolated mitochondria induced the release of the apoptogenic proteins cytochrome c and Smac/DIABLO. Therefore, we suggest that PVL, which belongs to the pore-forming toxin family, could act at the mitochondrion level by creating pores in the mitochondrial outer membrane. Furthermore, LukS-PV, 1 of the 2 components of PVL, was detected in lung sections of patients with necrotizing pneumonia together with DNA fragmentation, suggesting that PVL induces apoptosis in vivo and thereby is directly involved in the pathophysiology of necrotizing pneumonia.
Asunto(s)
Apoptosis/efectos de los fármacos , Leucocidinas/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neutrófilos/citología , Neutrófilos/efectos de los fármacos , Proteína X Asociada a bcl-2/fisiología , Proteínas Reguladoras de la Apoptosis , Toxinas Bacterianas , Membrana Celular/fisiología , Células Cultivadas , Citocromos c/metabolismo , Exotoxinas , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Cinética , Pulmón/microbiología , Pulmón/patología , Mitocondrias/fisiología , Proteínas Mitocondriales/metabolismo , Necrosis , Neutrófilos/microbiología , Neumonía Estafilocócica/microbiología , Neumonía Estafilocócica/patología , Staphylococcus aureus/fisiologíaRESUMEN
BACKGROUND: Staphylococcus aureus superantigens are associated with the pathogenesis of toxic shock syndrome, but their involvement in septic shock is unknown. METHODS: We compared the distribution of 11 superantigen genes in S. aureus blood culture isolates obtained from patients with sepsis who did and did not have septic shock (19 and 61 patients, respectively), as well as from patients with suppurative infections (101 patients) and patients with colonization (25 patients). RESULTS: The prevalence of the enterotoxin A gene (sea) increased significantly with the severity of infection (P<.001), whereas the prevalence of the enterotoxin gene cluster (egc) decreased significantly (P=.009). CONCLUSION: Enterotoxin A (SEA) might play a key role in sea-positive S. aureus sepsis by triggering over-expression of inflammatory mediators associated with shock. Novel treatments targeting superantigens, especially the sea gene, might be beneficial in the treatment of S. aureus sepsis.
Asunto(s)
Sepsis/microbiología , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Superantígenos/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Enterotoxinas/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infecciones Estafilocócicas/diagnósticoRESUMEN
The analysis of the different amino acid sequences deduced from the complete genome sequence of the gram-positive bacterium Staphylococcus aureus suggested the presence of two eukaryotic-protein-like low-molecular-mass phosphotyrosine protein phosphatases, which are usually found in gram-negative bacteria. To check this prediction, the corresponding genes were cloned and overexpressed in an Escherichia coli system. Two distinct proteins with an apparent molecular mass of 23 kDa each, PtpA and PtpB, were produced and then purified by affinity chromatography and assayed for enzymatic properties. As expected, they both exhibited phosphatase activity in vitro, with a maximum value at a pH of around 6.2 and at a temperature of 40 degrees C. In addition, their kinetic constants, their specificity for phosphotyrosine residues, and their sensitivity to two phosphatase inhibitors, N-ethylmaleimide and orthovanadate, matched those of acid low-molecular-mass phosphotyrosine protein phosphatases.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Proteínas Tirosina Fosfatasas/aislamiento & purificación , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Datos de Secuencia Molecular , Peso Molecular , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Análisis de Secuencia de ADN , Staphylococcus aureus/genética , Especificidad por SustratoRESUMEN
Panton Valentine leukocidin (PVL) may be responsible for pulmonary necrosis in necrotizing Staphylococcus aureus pneumonia, a highly lethal infection. Commercial intravenous immunoglobulin (IVIg) preparations containing antibodies against PVL might have therapeutic value in this setting, as an adjunct to antimicrobial chemotherapy. To test this possibility, we determined anti-PVL antibody titers in commercial IVIg and the capacity of IVIg to prevent the cytopathic effects of PVL in vitro. Specific enzyme-linked immunosorbent assays based on purified recombinant PVL (rPVL) showed that IVIg contained specific anti-PVL antibodies. The cytotoxicity of rPVL and of crude culture supernatants of PVL-producing S. aureus strains were investigated by measuring ethidium-bromide incorporation by polymorphonuclear neutrophils (PMNs) in flow cytometric assays, as well as PMN ultrastructural changes by transmission electron microscopy. IVIg was found to neutralize pore formation and the cytopathic effect of both rPVL and S. aureus culture supernatants.