Relationship between intracellular calcium and morphologic changes in rabbit erythrocytes: Effects of the acylated and unacylated forms of E. coli alpha-hemolysin

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

BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES

ELSEVIER SCIENCE BV

Lugar: Amsterdam; Año: 2016 vol. 1858 p. 1944 - 1953

0005-2736

Alpha-hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic Escherichia coli strains,whose expression correlates with the severity of the infections produced. HlyA is synthesized as a protoxin,ProHlyA, that becomes matured to the active form in the cytosol by hemolysin-C?directed fatty acylation atthe ε-amino residues of Lys 564 and Lys 690, before export from the toxin-producing bacteria. This posttranslationalmodificationis remarkable because the nature of the protein is changed by the lipidicmoiety froma benignprotein to a frank toxin.In the present work, we demonstrated for the first time that, despite being hemolitically inactive, ProHlyA inducedcellular morphologic changes in rabbit erythrocytes. A discocyte-to-echinocyte transformation was triggeredby the protoxin in the absence of any accompanying increase in intracellular-Ca2+ levels. In addition,the Ca2+-influx kinetics in HlyA-treated erythrocytes indicated that the active toxin induced an elevation inintraerythrocyte-Ca2+ content in a biphasic manner,with an initial rise in Ca2+ that depended on the associationof the protein with the target membrane and a second increment corresponding to an activation of purinergicchannels. The first increase was sufficient to trigger a discocyte-echinocyte-spherocyte transition in erythrocyteshape, though the subsequent rise mediated by purinergic signalling was essential for the occurrence of hemolysis.The results presented here provide new insights into the mechanism of action of this toxin.