Effects of alpha hemolysin from E.coli on erythrocytes from different species

SUSANA SANCHEZ; ROMINA VAZQUEZ; SABINA MATÉ; 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 by using 2-photon 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. Two-photon 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 one for rabbit, however only the GP value of rabbit increases  after incubation with sublytic concentration of HlyA, indicating insertion of the toxin in the membrane. Membrane lipid composition was determined: while the content of Cholesterol and PE was  similar in the two cell type, the content of Phosphatidylcholine (PC) and sphingomyelin (SM) showed differences: sheep erythrocytes do not have PC and contained 28% of SM and  Rabbit erythrocytes present 18% of PC and 10% SM. 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.