Loop Diuretics Diminish Hemolysis Induced by α-Hemolysin from Escherichia coli

Uropathogenic Escherichia coli often produce the virulence factor α-hemolysin (HlyA), and the more severe the infection, the likelier it is to isolate HlyA-producing E. coli from patients. HlyA forms pores upon receptor-independent insertion of the toxin into biological membranes and it has been substantiated that HlyA-induced hemolysis is amplified by toxin-induced ATP release and activation of P2X receptors. Thus, hemolysis inflicted by HlyA is a protracted process involving signal transduction. It consists of early, marked cell shrinkage followed by swelling and eventually lysis. The initially shrinkage is a consequence of a substantial Ca²⁺-influx and activation of Ca²⁺-sensitive K⁺ and Cl^- channels (K_Ca3.1/TMEM16A). The shrinkage is followed by gradual cell swelling, which ultimately lyses the cells. These findings clearly show that the HlyA pore provides a substantial volume challenge for the cells, and the fate of the given cell is co-determined by intrinsic erythrocytal volume regulation. We therefore speculated that other mechanisms involved in erythrocyte volume regulation may influence the hemolytic process inflicted by HlyA. Strikingly, HlyA-induced hemolysis is markedly reduced in erythrocytes isolated from NKCC1-deficient (NKCC1^-/-) mice compared to controls. The NKCC1 inhibitors furosemide and bumetanide concentration-dependently inhibit HlyA-induced lysis of human and murine erythrocytes. However, in high concentrations bumetanide further reduced hemolysis in erythrocytes from NKCC1^-/- mice and, thus, also exhibit indirect effects on hemolysis. The effect of loop diuretics on the hemolysis is not unique to HlyA but is similarly seen in LtxA- and α-toxin-induced hemolysis. Bumetanide clearly potentiates HlyA-induced volume reduction and delays the following erythrocyte swelling. This allows increased phagocytosis of damaged erythrocytes by THP-1 cell as a result of prolonged cell shrinkage. These data suggest that erythrocyte susceptibility to cytolysins is modified by NKCC1 and signifies intrinsic volume regulators as important determinants of cellular outcome of pore-forming toxins.