R. Hertle et al., SPECIFIC PHOSPHATIDYLETHANOLAMINE DEPENDENCE OF SERRATIA-MARCESCENS CYTOTOXIN ACTIVITY, Molecular microbiology, 26(5), 1997, pp. 853-865
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.