Effects of alpha-hemolysin on human erythrocytes. Part 1. Regulation of extracellular ATP and cell volume.

LAURI, NATALIA; LEAL DENIS, MARÍA F.; ALVAREZ, CORA L.; LEFEVRE, S.D.; GONZALEZ-LEBRERO, R.; ESPELT MARÍA VICTORIA; OSTUNI, MARIANO A.; HERLAX, VANESA; PABLO JULIO SCHWARBAUM

La Plata

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Sociedad Argentina de Biofísica

The hemolytic toxin α-hemolysin (HlyA) is a virulence factor produced by several strains of Escherichia coli. It is involved in urinary tract infections, peritonitis, meningitis and septicemia.We studied the regulation of extracellular ATP (ATPe), cell volume and hemolysis in human erythrocytes exposed to HlyA.HlyA induced an increase of [ATPe] (3-36 fold) and hemolysis (1-44 fold), which was compatible with simultaneous lytic and non-lytic ATP release. Elevated [ATPe] was partially counteracted by ATPe hydrolysis driven by ectoATPase activity, and intracellular ATPase activity of hemolyzed cells.A quantitative analysis showed that lytic and non-lytic ATP exit mainly governed ATPe kinetics, while ATPe hydrolysis was important at late times of toxin exposure.Several features of HlyA-induce ATP release from erythrocytes were characterized: 1- the un-acylated toxin ProHlyA and the deletion mutant protein HlyA∆914-936 were unable to induce ATP release or hemolysis, suggesting that acylation of HlyA and the ∆914-936 segment binding erythrocyte glycophorins were important for toxin activity; 2- treatment of ATP loaded liposomes with HlyA caused low ATP release, so that the lipid inserted toxin did not permeate ATP; 3- carbenoxolone, a pannexin 1 inhibitor, partially inhibited (43-67%) ATP exit, implying a role for this protein as an ATP conduit; 4- HlyA induced a 1.5 fold swelling, while blocking this swelling reduced ATP release by 77 %. Reciprocally, blocking ATPe activation of P2X receptors reduced HlyA dependent swelling by 60-80%. Thus a catalytic loop was identified where swelling activated ATP release, and subsequent [ATPe] increments activated further swelling.