Evaluation of Oxidative Metabolism in Leukocytes during Phagocytosis of Escherichia coli Carrying Genetic Constructs soxS::lux or katG::lux.

We studied ROS generation by human peripheral blood monocytes and granulocytes during phagocytosis of Escherichia coli soxS::lux or katG::lux responding by luminescence (bioluminescence) to the development of oxidative stress. Initially high sensitivity of the bioluminescent reaction of E. coli katG::lux strain to the effects of model ROS (KO2 and H2O2) and pronounced induction of luminescence upon contact with granulocytes, whereas E. coli soxS::lux demonstrated less pronounced reaction to chemical oxidants and bioluminescence was observed primarily upon contact with monocytes. A correlation was found between quantitative characteristics of E. coli katG::lux bioluminescence and luminol-dependent chemiluminescence of leukocytes in some patients, but no dependence of this kind was noted for E. coli soxS::lux. The results can provide experimental substantiation of a new approach for evaluation of ROS production by leukocytes during phagocytosis and choosing the optimal object for these studies.

[Reactive oxygen and nitrogen species' effect on lux-biosensors based on Escherichia coli and Salmonella typhimurium].

The effect of reactive oxygen and nitrogen species on lux-biosensors based on the Escherichia coli K12 MG1655 and Salmonella typhimurium LT2 host strains was investigated. The bioactivity of exogenous free radicals to the constitutively luminescent E. coli strain with plasmid pXen7 decreased in the order H2O2 > OCl­ > NO• > RОO• > ONOO­> O 2 •- while the bioluminescence of S. typhimurium strain transformed with this plasmid decreased in the order NO• > H2O2 > ONOO­ > RОO• > OCl­ > O 2 •- The cross-reactivity of induced lux-biosensors to reactive oxygen and nitrogen species, the threshold sensitivity and the luminescence amplitude dependences from the plasmid specificity and the host strain were indicated. The biosensors with plasmid pSoxS'::lux possessed a wider range of sensitivity, including H2O2 and OCl­, along with O 2 •- and NO•. Among the used reactive oxygen and nitrogen species, H2O2 showed the highest induction activity concerning to the plasmids pKatG'::lux, pSoxS'::lux and pRecA'::lux. The inducible lux-biosensors based on S. typhimurium host strain possessed a higher sensitivity to the reactive oxygen and nitrogen species in comparison than the biosensors based on E. coli. .