RESUMO
A comparative analysis of the four commercially available and laboratory luminescent sensor strains to the toxic effect of 10 carbon-based nanomatherials (CBNs) and 10 metal nanoparticles (MNPs) was carried out in this study. The bioluminescence inhibition assays with marine Photobacterium phosphoreum and recombinant Escherichia coli strains were varied in minimal toxic concentrations and EC50 values but led to well correlated biotoxicity evaluation for the most active compounds were ranked as Cu > (MgO, CuO) > (fullerenol, graphene oxide). The novel sensor strain Bacillus subtilis EG 168-1 exhibited the highest sensitivity to CBNs and MNPs that increased significantly number of toxic compounds causing the bacterial bioluminescence inhibition effect.
Assuntos
Antibacterianos , Bacillus subtilis/crescimento & desenvolvimento , Carbono , Escherichia coli/crescimento & desenvolvimento , Nanopartículas Metálicas/química , Photobacterium/crescimento & desenvolvimento , Antibacterianos/química , Antibacterianos/farmacologia , Carbono/química , Carbono/farmacologiaRESUMO
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.
Assuntos
Catalase/genética , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Leucócitos Mononucleares/fisiologia , Fagocitose , Transativadores/genética , Células Cultivadas , Interações Hospedeiro-Patógeno , Humanos , Leucócitos Mononucleares/microbiologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Luminescence intensity of recombinant Escherichia coli and Bacillus subtilis strains with cloned luxCD(AB)E genes of the natural luminescent microorganism Photobacterium leiognathi was studied under the influence of 30 individual samples of human blood serum of different component composition. A relationship was found between the level of residual bioluminescence and degree of the bactericidal effect. Moreover, the inhibition of E. coli lux+ luminescence was shown to be related to activity of the complement-lysozyme system. The reaction of B. subtilis lux+ primarily depended on the presence of ß-lysin in the blood serum. These data provide an experimental substantiation of a new method of differential analysis of humoral factors of nonspecific innate immunity with recombinant luminescent bacteria.
Assuntos
Peptídeos Catiônicos Antimicrobianos/sangue , Bacillus subtilis/imunologia , Atividade Bactericida do Sangue/fisiologia , Escherichia coli/imunologia , Luciferases Bacterianas/genética , Aciltransferases/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Atividade Bactericida do Sangue/genética , Proteínas Sanguíneas/metabolismo , Proteínas do Sistema Complemento/imunologia , Proteínas do Sistema Complemento/metabolismo , Escherichia coli/genética , Humanos , Imunidade Inata , Luminescência , Muramidase/metabolismo , Oxirredutases/genética , Photobacterium/metabolismo , Soro/imunologia , Soro/microbiologiaRESUMO
The authors show it possible to evaluate the phagocytic activity of human peripheral blood neutrophils from their impact on the fluorescence intensity of recombinant luminescent bacteria. They have established the preferred use of recombinant luminescent Escherichia coli bacteria with cloned luxCDABE genes OF Photobacterium leiognathii as phagocyted objects and substantiated the optimal method of their opsonization by normal human immunoglobulin. The variants of separate consideration of the luminal-dependent chemiluminescence of phagocytes and the bioluminescence of bacterial target in the phagocytic system, which are based on the study of two tests of different component composition or one test at two wavelengths of less than 420 nm for the assessment of chemiluminescence and more than 540 nm for the assessment of bioluminescence.
Assuntos
Escherichia coli/fisiologia , Neutrófilos/fisiologia , Fagocitose , Diabetes Mellitus Tipo 2/sangue , Escherichia coli/genética , Humanos , Imunoglobulinas , Medições Luminescentes , Proteínas Opsonizantes , Photobacterium/genética , Photorhabdus/genéticaRESUMO
It was found that divalent iron ions have alternative effects on the bioluminescence of the natural marine microorganism Photobacterium phosphoreum and the recombinant Escherichia coli strain with a cloned lux operon of P. leiognathi. In the presence of 0.25-5.0 mM FeSO4, the bioluminescence intensity of the former and the latter increased and decreased, respectively. To establish the causes of these differences, we studied the characteristics of the fatty acid composition of the compared microorganisms. The fatty acid profile of E. coli was characterized by a high proportion of unsaturated 11-octadecenoic (vaccenic) acid. A study of this acid in a cell-free enzyme system used for bioluminescence generation showed that it is a potent inhibitor of bacterial bioluminescence. It was found that such effects are enhanced if 11-octadecenoid acid is preincubated with Fe2+.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , Compostos Ferrosos/farmacologia , Luminescência , Substâncias Luminescentes/metabolismo , Photobacterium/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Proteínas de Bactérias/genética , Técnicas Biossensoriais , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Ácidos Oleicos/metabolismo , Óperon/genética , Photobacterium/genética , Photobacterium/metabolismo , Proteínas Recombinantes/genéticaRESUMO
The spectra of luminol-dependent chemiluminescence of leukocytes, forming as a result of their oxygen-dependent bactericidal systems activation, and bacterial bioluminescence of Escherichia coli recombinant strain with cloned lux operon, used as the object of phagocytosis, are not identical. Mutual overlapping of these spectra reaches 87%, including overlapping of the photoemission maximums. However these spectra can be evaluated separately in the short-wave "arm" of the chemiluminescence spectrum (<420 nm) and the long-wave "arm" of the bioluminescence spectrum (>560). The kinetics of luminol-dependent chemiluminescence of phagocytes and of bacterial bioluminescence in their mixtures is characterized by mutually dependent phase-wise changes in the intensity of the analyzed parameters.
Assuntos
Luminescência , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Fagócitos/metabolismo , Escherichia coli/metabolismo , Humanos , Luminol/química , Fagocitose/fisiologiaRESUMO
The Microbiosensor B 17677 F test system was applied to make a space-time monitoring of the biotoxicity of water used for production and everyday purposes at the Iriklinsk hydroelectric power station (IHEPS) and to identify the leading causes determining the biotoxicity of tested samples. There were seasonal variations in the biotoxicity with the maximum in spring and with minimum in winter and spring and a relationship of the spring rise in the biotoxicity to water pH changes. There was also an association of the certain values of the biotoxicity of industrial water with the concentration of petroleum products that are major pollutants at the IHEPS. The datum points that characterize the maximum level of technogenic exposure were identified.
