Cooperative, synergistic and antagonistic haemolytic interactions between haemolysin BL, phosphatidylcholine phospholipase C and sphingomyelinase from Bacillus cereus

Haemolysis of erythrocytes from different species (sheep, bovine, swine and human), caused by various combinations of phosphatidylcholine (PC)preferring phospholipase C (PC-PLC), sphingomyelinase (SMase) and the three-component, pore-forming toxin haemolysin BL (HBL) from Bacillus cereus was analysed. The lytic potency of HBL did not correlate with phospholipid (PL) content, but lysis by the individual or combined enzymes did. SMase alone lysed ruminant erythrocytes, which contain 46-53 % sphingomyelin (SM). The cooperative action of PC-PLC and SMase was needed to lyse swine and human erythrocytes (22-31 % PC and 28-25 % SM), SMase synergistically enhanced haemolysis caused by HBL for all erythrocytes tested, which all contained >25 % SM, PC-PLC enhanced HBL haemolysis only in cells containing significant amounts of PC (swine, 22 % PC; human, 31 % PC). Unexpectedly, PC-PLC inhibited HBL lysis of sheep erythrocytes (< 2 % PC) and enhanced the discontinuous haemolysis pattern that is characteristic of HBL in sheep blood agar. Inhibition and pattern enhancement was abolished by washing PC-PLC-treated erythrocytes or by adding EDTA, suggesting that enzymic alteration of the membrane is not involved, but that zinc in the active site is required, perhaps to facilitate binding. These observations highlight the potential for cooperative and synergistic interactions among virulence factors in B. cereus infections and dependence of these effects on tissue composition.