Glycophorin is not a receptor for HlyA in human erythrocytes

LUCÍA CANÉ, SANDRINE GENETET, ISABELLE MOURE-CHANTELOUP, MARIANO OSTUNI, PABLO SCHWARZBAUM AND VANESA HERLAX

Congreso; Postlatam; 2019

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. HlyA acts on a variety of cell types from several species, including human red blood cells (RBCs); and also binds to and disrupts protein-free liposomes. Studies that have explored the binding of HlyA to membranes and the characterization of a putative toxin-specific receptor have produced contradictory results. Regarding RBCs, glycophorins were characterized as putative receptors in horse RBCs, though other studies indicated that the binding of HlyA to rabbit RBCs occurred in a nonsaturable manner and that the toxin did not interact with a specific protein receptor. On the other hand, we have previously demonstrated that HlyA induces a diminish in human RBCs deformability, and the release of lytic and non-lytic ATP, effects that have been associated to the interaction of a ligands with glycophorin. In this context, the aim of our study was to characterize if the interaction of HlyA with human glycophorins mediate the hemolytic and sublytic effects of the toxin. For this purpose we measure the hemolytic activity of HlyA on RBCs pretreated with different antibodies (iH4 (nanoantibody against Y52PPE55), anti-GPA/GPB, anti-GPA) and on GPA/GPB null RBCs. For our surprise, the hemolytic activity of the toxin was similar to control RBCs in all the conditions tested, indicating that glycophorin is not necessary for HlyA lytic activity. Moreover, we performed FACS assays using antibodies anti-GPA(R18) and anti-band3 complexed with glycophorin (Wr), on treated and untreated RBCs. Results showed a slight increment in fluorescence intensity when toxin interact with RBCs indicating conformational changes of both GPA and Band 3 when the toxin interact with RBCs. In conclusion, results indicate that HlyA-GPA or GPB interaction is not necessary for HlyA lytic activity.