'IN SEARCH OF A RECEPTOR FOR ALPHA HEMOLYSIN OF E.COLI IN HUMAN ERYTHROCYTES?

LUCÍA CANÉ; SANDRINE GENETET; MARÍA ANTONIETA DAZA-MILLONE; MARIA ELENA VELA; MARIANO OSTUNI; PABLO SCHWARZBAUM; ISABELLE MOURO-CHANTELOUP; VANESA HERLAX

Congreso; Reunion anual de Sociedades de Biociencias; 2020

INTRODUCTION Alpha-hemolysin (HlyA) is a hemolytic protein secreted by uropathogenic strains of E. coli. The binding of HlyA to a putative toxin-specific receptor produced contradictory results. Glycophorins (GPs) were characterized as presumed receptors in horse red blood cells (RBCs), though other studies indicated HlyA did not interact with a specific protein receptor in rabbitRBCs. Conversely, we previously demonstrated that HlyA induces a decrease in humanRBCs (hRBCs) deformability and the release of ATP, effects usually associated to the interaction of ligands with GPs. OBJECTIVES Study the contribution of GPs and alternative membrane proteins in mediating the hemolytic effects of HlyA on hRBCs. METHODS We tested the lytic activity of HlyA on hRBCs pretreated with different antibodies to block the binding of HlyA to GPs (anti-GPA/GPB, anti-GPA and nanoantibody iH4), and also on the rare clinical mutant GPA/GPB null hRBCs. We measured the dissociation constant between GPA and HlyA, ProHlyA (inactive protoxin) and HlyAΔ914-936 (HlyA mutant lacking the binding domain to GPA) by Surface Plasmon Resonance (SPR). Finally, we performed Far Western Blot assays plus mass spectroscopy analysis, to explore whether HlyA binds to a specific hRBC membrane protein other than GPs. RESULTS AND CONCLUSIONS The hemolytic activity was slightly inhibited by high concentration of anti-GPA/GPB. Surprisingly, the hemolytic activity of the toxin on the rare clinical mutant GPA/GPB null was similar to control RBCs, indicating that GPs are not necessary for HlyA activity. SPR measurements indicated that the three proteins variants bind with similar strength to GPA, and to human seroalbumin, showing that the binding of the proteins to GPA is unspecific. Far Western Blot results showed that HlyA interacts with two hRBCs membrane proteins. The next step is to study the specific interaction between HlyA and membrane proteins on hRBCs in a more biological environment using a Pull Down assay.