• “Effects of alpha hemolysin from E.coli on erythrocytes from different Species”.

SUSANA A. SANCHEZ; ROMINA VAZQUEZ; SABINA MATE; LAURA BAKÁS; ENRICO GRATTON; VANESA HERLAX

Baltimore

Congreso; 55th Annual Meeting of the Biophysical Society; 2011

Alpha-hemolysin (HlyA) is one of the key virulence factors released by E. coli strains. This toxin causes lysis of various mammalian cells, including erythrocytes of different animal species. The hemolytic activity of the toxin on rabbit and sheep erythrocytes was determined, showing that rabbit is the most susceptible specie. Calcium concentration inside the erythrocytes while incubated with sublytic concentrations of HlyA was monitored using twophoton fluorescence Lifetime Imaging (FLIM), the calcium indicator Calcium Green 1 and the phasor analysis method. HlyA induces an increase in calcium concentration in both erythrocytes, but the increment in rabbit is 4 times higher and faster than in sheep. Twophoton Laurdan Generalized Polarization (GP) was used to determine the fluidity of the membrane (measured as the membrane water content) in the presence and absence of HlyA in live erythrocytes. The GP value for the sheep erythrocytes membranes was higher that the ones from rabbit and after incubation of the erythrocytes with sublytic concentration of Hlyn, an increase on the GP value was observed only on the rabbit membranes. Membrane lipid composition showed similar content of Cholesterol and PE in the two cell type, however the content of Phosphatidylcholine (PC) and sphingomyelin (SM) showed differences: sheep erythrocytes contained 28% of SM and not PC and Rabbit erythrocyte membranes present 10% SM and 18% of PC. We concluded that at sublytical concentration, the initial interaction of HlyA with the erythrocyte and the mechanisms of calcium influx strongly depend on the membrane composition and fluidity of the target cell.E. coli strains. This toxin causes lysis of various mammalian cells, including erythrocytes of different animal species. The hemolytic activity of the toxin on rabbit and sheep erythrocytes was determined, showing that rabbit is the most susceptible specie. Calcium concentration inside the erythrocytes while incubated with sublytic concentrations of HlyA was monitored using twophoton fluorescence Lifetime Imaging (FLIM), the calcium indicator Calcium Green 1 and the phasor analysis method. HlyA induces an increase in calcium concentration in both erythrocytes, but the increment in rabbit is 4 times higher and faster than in sheep. Twophoton Laurdan Generalized Polarization (GP) was used to determine the fluidity of the membrane (measured as the membrane water content) in the presence and absence of HlyA in live erythrocytes. The GP value for the sheep erythrocytes membranes was higher that the ones from rabbit and after incubation of the erythrocytes with sublytic concentration of Hlyn, an increase on the GP value was observed only on the rabbit membranes. Membrane lipid composition showed similar content of Cholesterol and PE in the two cell type, however the content of Phosphatidylcholine (PC) and sphingomyelin (SM) showed differences: sheep erythrocytes contained 28% of SM and not PC and Rabbit erythrocyte membranes present 10% SM and 18% of PC. We concluded that at sublytical concentration, the initial interaction of HlyA with the erythrocyte and the mechanisms of calcium influx strongly depend on the membrane composition and fluidity of the target cell.