Study of calcium influx dynamics in erythrocytes treated with E.coli alpha hemolysin

ROMINA VAZQUEZ; SABINA MATÉ; LAURA BAKÁS; CARLOS MUÑOZ-GARAY; VANESA HERLAX

Salto

Congreso; Latin American Crosstalk in Biophysics and Physiology; 2015

Sociedad Argentina de Biofísica- Seccional Biofísica de la Sociedad Uruguaya de Biociencias

Alpha hemolysin(HlyA) is an exotoxin secreted by uropathogenic E. coli strains that targets many cell types causing lysis anddeath. HlyA is claimed to be a pore forming toxin although no pore structurehas been identified yet. After treating erythrocytes with HlyA a Ca2+influx occurs that ultimately leads to cell swelling and osmotic lysis.Recently it has been shown that Ca2+ influx initially triggerserythrocytes shrinkage through activation of K3.1 channels andsubsequent K+ effux1. It is not well established ifinitial Ca2+ entry occurs through the toxin pore or through cationchannels. Nevertheless, it has been demonstrated that purinergic channelsamplificate the signal2. Ca2+ entry in rabbiterythrocytes treated with HlyA shows a biphasic behavior, with a first slightincrease followed by a second increment that keeps rising until lysis occurs3.The aim of the present work was to further explore the mechanism of Ca2+entry. Inhibition of Ca2+ channels with Ni+2 or BrilliantBlue G impaired HlyA hemolytic activity in rabbit erythrocytes. Nevertheless,when Ca2+ influx was monitored by Fluo-4 fluorescence imaging ofindividual cells, the initial increase in Ca2+ concentration wasstill observed. In order to test if this Ca2+ increment was a resultof toxin insertion into the membrane, Ca+2 levels were monitored inerythrocytes treated with proHlyA, the unacylated nonhemolytic form of HlyA.ProHlyA showed insertion levels similar to those of HlyA in lipid monolayersmimicking the outer leaflet of erythrocytes membranes but did not cause Ca2+influx in erythrocytes. Strikingly, cell shrinkage was still observed. These results suggestthat monomer toxin insertion into the membrane may trigger cellular responses leadingto cell shrinkage independently of Ca2+ levels. Nevertheless, acylchains covalently bound to the toxin are necessary for initial Ca2+entry and amplification through Ca2+ channels activation is furtherneeded for lysis. [1] Skals M, etal. J Biol Chem 285:15557-65, 2010.[2] Skals M, etal. Proc Natl Acad Sci 106:4030-35, 2009.[3] Sanchez, S,et al. Plos One 6(6):e21127, 2011.