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

LEAL DENIS MARÍA FLORENCIA; GONZALEZ-LEBRERO RODOLFO; HERLAX VANESA; ALVAREZ CORA LILIA; ESPELT MARÍA VICTORIA; SCHWARZBAUM PABLO JULIO; LAURI NATALIA; LEFEVRE SOPHIE DENISE; OSTUNI MARIANO ANIBAL

La Plata

Congreso; 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 severalstrains 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 inhuman erythrocytes exposed to HlyA.HlyA induced an increase of [ATPe] (3-36 fold) and hemolysis (1-44 fold), whichwas compatible with simultaneous lytic and non-lytic ATP release. Elevated [ATPe]was partially counteracted by ATPe hydrolysis driven by ectoATPase activity, andintracellular ATPase activity of hemolyzed cells.A quantitative analysis showed that lytic and non-lytic ATP exit mainly governedATPe 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 HlyAΔ914-936were unable to induce ATP release or hemolysis, suggesting that acylation of HlyAand the Δ914-936 segment binding erythrocyte glycophorins were important fortoxin activity; 2- treatment of ATP loaded liposomes with HlyA caused low ATPrelease, so that the lipid inserted toxin did not permeate ATP; 3- carbenoxolone, apannexin 1 inhibitor, partially inhibited (43-67%) ATP exit, implying a role for thisprotein as an ATP conduit; 4- HlyA induced a 1.5 fold swelling, while blocking thisswelling reduced ATP release by 77 %. Reciprocally, blocking ATPe activation ofP2X receptors reduced HlyA dependent swelling by 60-80%. Thus a catalytic loopwas identified where swelling activated ATP release, and subsequent [ATPe]increments activated further swelling.