Effects of Erythrocytes Treated with Alpha Hemolysin of E.Coli on Endothelial Cells

LEAL DENIS, MARIA FLORENCIA; ENRIQUE, NICOLAS; OSTUNI, MARIANO; HERLAX, VANESA; LEFEVRE, SOPHIE DENISE; MILESI, MARIA VERÓNICA; ALVAREZ, CORA; MATE, SABINA; SCHWARZBAUM, PABLO

New Orleans

Congreso; Biophysical Society - 61st Annual Meeting; 2017

Biophysical Society

Uropathogenic strains of E. coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It was reported that at high concentrations, the toxin forms pores in eukaryotic membranes, leading to cell lysis, while lower concentrations might interfere with host-cell-signaling pathways, causing apoptosis. In the present investigation we demonstrate that a relatively low concentration of HlyA induces morphological changes and phosphatidylserine (PS) externalization of human erythrocytes. On the other hand, the unacylated nonhemolytic form of HlyA, ProHlyA induces similar morphological changes but no PS externalization. We performed osmoscan experiments to test the effect of both proteins on erythrocytes structure. HlyA treated erythrocytes show increased membrane fragility and cell volume as well as diminished cytoplasmic viscosity and S/V ration. ProHlyA-treated erythrocyte are not different from control ones. Since PS exposure of erythrocytes is known to induce cell adhesion, we used a dynamic cell adhesion platform to study the consequences of HlyA vs ProHlya exposure of erythrocytes on their adhesion to human endothelial cells (HMEC). Results indicate that HlyA-treated erythrocytes adhere more to endothelial cells than Pro-treated erythrocytes at low flux (0.5 din). At higher fluxes (1 and 2 din), however, HlyA-treated erythrocytes detached easily than control ones, indicating that the adherence is weak. We also study the efflux of ATP from erythrocytes treated with both toxins by luciferin-luciferase luminescence. Results demonstrate that HlyA induces the efflux of ATP while ProHlyA does not. Since PS exposure was suggested to simultaneously increase extracellular ATP and adhesion to the vascular endothelium, and erythrocyte derived ATPe can alter the caliber of the vascular lumen, future experiments will be designed to relate HlyA induced efflux of ATP of erythrocytes with their adhesion and interaction with endothelial cells.