Effects of erythrocytes treated with alpha hemolysin of E.coli on endothelial cells

HERLAX, VANESA; LEAL DENIS, MARÍA FLORENCIA, MARGINEDAS FREIXA, IRENE, ENRIQUE, NICOLÁS; MATE, SABINA MARÍA; MILESI, VERÓNICA, OSTUNI, MARIANO ANIBAL; AND SCHWARZBAUM, PABLO JULIO

Congreso; 61st Annual Meeting of the Biophysical Society; 2017

Uropathogenic strains of E. coli deliver the toxin alpha-hemolysin (HlyA) tooptimize the host environment for the spread of infection. It was reported thatat high concentrations, the toxin forms pores in eukaryotic membranes, leadingto cell lysis, while lower concentrations might interfere with host-cell-signalingpathways, causing apoptosis. In the present investigation we demonstrate that arelatively 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 morphologicalchanges but no PS externalization. Weperformed osmoscan experimentsto test the effect of both proteins on erythrocytes structure. HlyA treated erythrocytesshow increased membrane fragility and cell volume as well as diminishedcytoplasmic viscosity and S/V ration. ProHlyA-treated erythrocyte arenot different from control ones. Since PS exposure of erythrocytes is knownto induce cell adhesion, we used a dynamic cell adhesion platform to studythe consequences of HlyA vs ProHlya exposure of erythrocytes on their adhesionto human endothelial cells (HMEC). Results indicate that HlyA-treated erythrocytesadhere 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 detachedeasily than control ones, indicating that the adherence is weak. We alsostudy the efflux of ATP from erythrocytes treated with both toxins by luciferinluciferaseluminescence. Results demonstrate that HlyA induces the efflux ofATP while ProHlyA does not. Since PS exposure was suggested to simultaneouslyincrease extracellular ATP and adhesion to the vascular endothelium,and erythrocyte derived ATPe can alter the caliber of the vascular lumen, futureexperiments will be designed to relate HlyA induced efflux of ATP of erythrocyteswith their adhesion and interaction with endothelial cells.