In search of a receptor for alpha hemolysin of E. coli in Human erythrocytes

CANÉ, L. ; GENETET, S.; DAZA MILLONE, M. A.; VELA, M. E.; OSTUNI, M. ; SCHWARZBAUM, P.J. ; MOURE-CHANTELOUP, I. , HERLAX, V

Congreso; Reunion Anual de Sociedades de Biociencia; 2020

INTRODUCTION Alpha-hemolysin (HlyA) is a hemolytic proteinsecreted by uropathogenic strains of E. coli. The binding of HlyAto a putative toxin-specific receptor produced contradictory results.Glycophorins (GPs) were characterized as presumed receptors inhorse red blood cells (RBCs), though other studies indicated HlyAdid not interact with a specific protein receptor in rabbitRBCs. Conversely,we previously demonstrated that HlyA induces a decreasein humanRBCs (hRBCs) deformability and the release of ATP, effectsusually associated to the interaction of ligands with GPs.OBJECTIVES Study the contribution of GPs and alternative membraneproteins in mediating the hemolytic effects of HlyA on hRBCs.METHODS We tested the lytic activity of HlyA on hRBCs pretreatedwith different antibodies to block the binding of HlyA to GPs (anti-GPA/GPB, anti-GPA and nanoantibody iH4), and also on the rareclinical mutant GPA/GPB null hRBCs. We measured the dissociationconstant between GPA and HlyA, ProHlyA (inactive protoxin) andHlyAΔ914-936 (HlyA mutant lacking the binding domain to GPA) by SurfacePlasmon Resonance (SPR). Finally, we performed Far WesternBlot assays plus mass spectroscopy analysis, to explore whetherHlyA binds to a specific hRBC membrane protein other than GPs.RESULTS AND CONCLUSIONS The hemolytic activity was slightlyinhibited by high concentration of anti-GPA/GPB. Surprisingly, thehemolytic activity of the toxin on the rare clinical mutant GPA/GPBnull was similar to control RBCs, indicating that GPs are not necessaryfor HlyA activity. SPR measurements indicated that the threeproteins variants bind with similar strength to GPA, and to humanseroalbumin, showing that the binding of the proteins to GPA is unspecific.Far Western Blot results showed that HlyA interacts withtwo hRBCs membrane proteins. The next step is to study the specificinteraction between HlyA and membrane proteins on hRBCs in amore biological environment using a Pull Down assay.