RESUMEN
We examined the viability of Escherichia coli transformants harboring various carotenoids synthesizing genes in a medium containing an enzymatic singlet oxygen generating system, which contained myeloperoxidase, hydrogen peroxide and Br(-) at pH 4.5. Singlet oxygen quenching activities of various carotenoids in phosphatidyl choline micelles in aqueous media were also studied using the same enzymatic singlet oxygen generating system. Viability of the transformants producing carotenoids was higher than that of the wild type E. coli in the singlet oxygen generation mixture. Of the transformants tested, the viability of zeaxanthin-diglucoside producing transformant was the highest. Carotenoids in increasing order of k(q) values were beta-carotene, a cyclic caroteneAsunto(s)
Carotenoides/metabolismo
, Escherichia coli/fisiología
, Oxígeno/metabolismo
, Peroxidasa/metabolismo
, Adenosina Trifosfato/análisis
, Carotenoides/biosíntesis
, Transporte de Electrón
, Erwinia/enzimología
, Erwinia/genética
, Escherichia coli/genética
, Técnicas de Transferencia de Gen
, Micelas
, Oxidorreductasas/metabolismo
, Fosfatidilcolinas
, Fotoquímica
, Rhizobium/enzimología
, Rhizobium/genética
, Oxígeno Singlete
Asunto(s)
Antibacterianos/farmacología , Endotelio Vascular/citología , Escherichia coli/efectos de los fármacos , Peróxidos Lipídicos/química , Oxígeno/farmacología , Propionatos/química , Escualeno/química , Escualeno/farmacología , Carotenoides/genética , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Escherichia coli/genética , Calor , Humanos , Hidrólisis , Indicadores y Reactivos , Cinética , Licopeno , Oxígeno/toxicidad , Fosfolípidos/química , Fotoquímica , Oxígeno Singlete , Solventes , Venas UmbilicalesRESUMEN
Intraphagosomal viability of wild type E. coli and lycopene (a powerful (1)O(2) quencher)-producing transformant E. coli was investigated using human polymorphonuclear leukocytes as the cells for phagocytosis of opsonized viable bacteria. While the viability of both wild type and the transformant E. coli decreased very rapidly in the phagosome, but the viability of the lycopene-transformant in phagosomes was about 1.7 times higher than that of wild type E. coli after 5 min of incubation. The results were very similar to the results obtained when E. coli strains were exposed to (1)O(2) generated in myeloperoxidase-H(2)O(2)-Br(-) system (a pure (1)O(2) generating system) at pH 4.5. The reason for HOCl, which may be generated in the myeloperoxidase-H(2)O(2)-Cl(-) system under physiological conditions but does not become involved in bactericidal action, could be explained by the near neutral pH in phagosomes at which bacterial killing by chlorination is extensively attenuated. This is the first report which proved (1)O(2)-mediated bacterial killing in neutrophil-bacterial phagosomal system.
Asunto(s)
Carotenoides/sangre , Escherichia coli/fisiología , Neutrófilos/fisiología , Oxígeno/sangre , Peroxidasa/sangre , Fagosomas/fisiología , Carotenoides/genética , Escherichia coli/genética , Humanos , Peróxido de Hidrógeno/sangre , Técnicas In Vitro , Licopeno , Neutrófilos/microbiología , Oxidantes , Fosforilación Oxidativa , Consumo de Oxígeno , Fagosomas/microbiología , Oxígeno Singlete , Transformación BacterianaRESUMEN
To distinguish the bactericidal action of singlet oxygen (1O2) from hypohalous acids, wild-type and lycopene transformant E. coli strains were exposed to each of the oxidants and then bacterial viability was investigated. 1O2 was generated by chemical and enzymatic systems at pH 4.5. ExpoSure of wild-type E. coli to 1O2 caused a significant loss of E. coli viability due to inactivation of membrane respiratory chain enzymes by 1O2. This action of 1O2 could be attenuated by lycopene in the bacterial cell membrane. In the lycopene transformant strain of E. coli, inactivation of NADH oxidase and succinate oxidase by hypohalous acids were significantly suppressed, but E. coli viability was unaffected. Based on these findings, we suggest that phagocytic leukocytes produce 1O2 as a major bactericidal oxidant in the phagosome.
Asunto(s)
Escherichia coli/metabolismo , Oxígeno/metabolismo , Peroxidasa/antagonistas & inhibidores , Bromuros/metabolismo , Carotenoides/genética , Carotenoides/metabolismo , Transporte de Electrón , Represión Enzimática , Escherichia coli/enzimología , Escherichia coli/genética , Peróxido de Hidrógeno/metabolismo , Peróxidos Lipídicos/síntesis química , Licopeno , NAD/metabolismo , Propionatos/síntesis química , Oxígeno SingleteRESUMEN
3-(4'-Methyl-1'-naphthyl)-propionic acid, 1',4'-endoperoxide (NEPO) provides singlet state of oxygen (1O2, 1delta g) at 37 degrees C in sodium phosphate buffer (pH 7.2), acetate buffer (pH 4.5), methanol or chloroform, through the retro-Diels-Alder reaction. The total amount of 1O2 generated by NEPO was calculated using the following equation: [1O2]= [NEPO]0[1-exp(-kt)], where [1O2], [NEPO]0 and k are the total amount of 1O2 produced during the time t, initial concentration of NEPO and the first-order reaction rate constant, respectively. When squalene was exposed to 1O2 which was generated thermolytically from NEPO, it was oxidized to three hydroperoxides, mono-, di- and tri-hydroperoxides, in amounts proportional to the dose of NEPO. The oxidizability of squalene was much more extensive compared with unsaturated phospholipids. Additionally, when wild-type E. coli and lycopene-producing mutant E. coli were exposed to NEPO-derived 1O2, there was significant loss of viability of wild-type E. coli but no significant loss of viability in lycopene-producing strain, suggesting that lycopene by scavenging 1O2 protected E. coli against 1O2 toxicity.