SPECIFIC PHOSPHATIDYLETHANOLAMINE DEPENDENCE OF SERRATIA-MARCESCENS CYTOTOXIN ACTIVITY

The cytolytic and haemolytic activity of Serratia marcescens is determ ined by the ShlA protein, which is secreted across the outer membrane with the aid of the ShlB protein, In the absence of ShlB, inactive Shl A remains in the periplasm of Escherichia coil transformed with an sh lA-encoding plasmid, which indicates that Shia converts ShlA to activ e ShlA. ShlA in a periplasmic extract and partially purified ShlA* we re activated in vitro by partially purified ShlB. When both proteins w ere highly purified, ShlA was only activated by ShlB when phosphatidy lethanolamine (PE) or phosphatidylserine was added to the assay, while phosphatidylglycerol contributed little to ShlA activation. Lyso-PE, cardiolipin, phosphatidylcholine, phosphatidic acid, lipopolysacchari de and various detergents could not substitute for PE, Although radioa ctively labelled PE was so tightly associated with ShlA that it remain ed bound to ShlA after heating and SDS-PAGE, it was not covalently lin ked to ShlA as PE could be removed by thin-layer chromatography with o rganic solvents. The number of PE molecules associated per molecule of ShlA was 3.9 +/- 2.2, Active ShlA was inactivated by treatment with p hospholipase A,, which indicated that PE is also required for ShIA act ivity. ShlA-255 (containing the 255 N-terminal amino acids of ShlA) re versibly complemented ShlA to active ShlA and was inactivated by phos pholipase A(2), which demonstrated that PE binds to the N-terminal por tion of ShlA; this region has previously been found to be involved in ShlA secretion and activation, Electrospray mass spectroscopy of ShlA- 255 determined a molar mass that corresponded to that of unmodified Sh lA-255. An E. coli mutant that synthesized only minute amounts of PE d id not secrete ShIA but contained residual cell-bound haemolytic activ ity. Since PE binds strongly to ShlA in the absence of ShlB without c onverting ShlA to haemolytic ShlA, ShlB presumably imposes a conforma tion on ShlA that brings PE into a position to mediate interaction of the hydrophilic haemolysin with the lipid bilayer of the eukaryotic me mbrane.