Alpha hemolysin of E. coli induces hemolysis of human erythrocytes independently of toxin interaction with membrane proteins

Abstract

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli.The role of glycophorins (GPs) as putative receptors for HlyA binding to red blood cells (RBCs) has beendebated.Experiments using anti-GPA/GPB antibodies and a GPA-specific epitope nanobody to block HlyA-GPbinding on hRBCs, showed no effect on hemolytic activity. Similarly, the hemolysis induced by HlyAremained unaffected when hRBCs from a GPAnull/GPBnull variant were used. Surface Plasmon Resonanceexperiments revealed similar values of the dissociation constant between GPA and either HlyA, ProHlyA(inactive protoxin), HlyAD914-936 (mutant of HlyA lacking the binding domain to GPA) or human serumalbumin, indicating that the binding between the proteins and GPA is not specific.Although far Western blot followed by mass spectroscopy analyses suggested that HlyA interacts withBand 3 and spectrins, hemolytic experiments on spectrin-depleted hRBCs and spherocytes, indicatedthese proteins do not mediate the hemolytic process.Our results unequivocally demonstrate that neither glycophorins, nor Band 3 and spectrins mediatethe cytotoxic activity of HlyA on hRBCs, thereby challenging the HlyA-receptor hypothesis. This findingholds significant relevance for the design of anti-toxin therapeutic strategies, particularly in light of thegrowing antibiotic resistance exhibited by bacteria.