Early effects produced by the interaction of sublytical concentration of alpha hemolysin of E.coli with erythrocytes.

ROMINA VAZQUEZ; FERNANDA CARRIZO; SABINA MATÉ; LAURA BAKÁS; VANESA HERLAX

Congreso; XLII Reunión Anual de la Sociedad Argentina de Biofisica; 2013

α-hemolysin (HlyA) is one of the key virulence factors released by invading E. coli strains. It causes lysis of various mammalian cells, including erythrocytes of different animal species. HlyA is a calcium dependant toxin that at high concentration forms pores in target cells causing lysis of them. Nevertheless, what is not clear is how often HlyA reaches levels that are high enough to lyse host target cells during the course of an infection. In fact, sublytic concentrations of HlyA may even be more physiologically relevant. The aim of the present work is to study the intracellular effects triggered by the interaction of sublytical concentration of HlyA with rabbit and sheep erythrocytes. These animal species erythrocytes were chosen due to their differences in the lipid composition of their membranes. The calcium concentration inside the erythrocytes was monitored while incubating with sublytic concentrations of HlyA by lifetime of the calcium indicator Green 1 using fluorescence lifetime imaging microscopy (FLIM). HlyA induces calcium concentration raise in both erythrocytes, but the increment in rabbit is 4 fold higher and faster than in sheep erythrocytes. To study this thoughtfully, the hemolytic assays in the presence and absence of different ionic channel inhibitors (amiloride, PPADS and KN 62) were done. Results demonstrated that the influx of calcium in both erythrocytes species seems to be through purinergic channels. The morphological changes observed where quite different, while rabbit erythrocytes shrinkage before lysis, sheep erythrocytes did not. To further study the differences observed in both animal species more experiments are needed, as measurement of the kinetic released of ATP, hemolytic activity in the presence of clotrimidazole (agonist of Ca+2 activated K+ efflux) and measure of erythrocytes volumes changes by flow cytometry of both animal species erythrocytes treated with sublytical concentration of HlyA