This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ?-hemolysin (HlyA) from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin), a group of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin cause the lysis of several cells as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, in a previous visit to the LFD we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation and fluorescence lifetime imaging microscopy (FLIM). In that opportunity we present evidences that, at sublytic concentrations, HlyA induces an increase of calcium concentration in rabbit erythrocytes in the first 10 s.
The aim of the present visit to the LFD is to study if the influx of calcium to the erythrocytes is through the pore that the toxin forms or if the toxin activates preexisting cation channels in the erythrocyte.
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