Membrane-permeabilizing activities of cyclic lipodepsipeptides, syringopeptin 22A and syringomycin E from Pseudomonas syringae pv. syringae in human red blood cells and in bilayer lipid membranes.

The pore-forming activities of cyclic lipodepsipeptides (CLPs), syringopeptin 22A (SP22A) and syringomycin E (SRE) were compared on the human red blood cell (RBC) membrane and on bilayer lipid membranes (BLMs). SP22A above a concentration of 4 x 10(5) molecules/cell significantly increased the RBC membrane permeability for 86Rb. With electric current measurements on BLM, it was proved that like SRE, the SP22A formed two types of ion channels in the membrane, small and large, the latter having six times larger conductance and longer dwell time. Both CLPs formed clusters consisting of six small channels, and the channel-forming activity of SP22A is about one order of magnitude higher than that of SRE. A Hill coefficient of 2-3 estimated from the concentration dependence of these CLPs-induced lysis gave a proof of the pore oligomerization on RBCs. Transport kinetic data also confirmed that SP22A pores were oligomers of at least three monomers. While SRE pores were inactivated in time, no pore inactivation was observed with SP22A. The 86Rb efflux through SP22A-treated RBCs approached the tracer equilibrium distribution with a constant rate; a constant integral current was measured on the BLM for as long as 2.5 h as well. The partition coefficient (Kp = 2 x 10(4) l/mol) between the RBC membrane and the extracellular space was estimated for SRE to be at least six times higher than that for SP22A. This finding suggested that the higher ion permeability of the SP22A-treated cells compared to that of SRE was the result of the higher pore-forming activity of SP22A.

Effect of temperature on the formation and inactivation of syringomycin E pores in human red blood cells and bimolecular lipid membranes.

The effects of temperature on the formation and inactivation of syringomycin E (SRE) pores were investigated with human red blood cells (RBCs) and lipid bilayer membranes (BLMs). SRE enhanced the RBC membrane permeability of 86Rb and monomeric hemoglobin in a temperature dependent manner. The kinetics of 86Rb and hemoglobin effluxes were measured at different temperatures and pore formation was found to be only slightly affected, while inactivation was strongly influenced by temperature. At 37 degrees C, SRE pore inactivation began 15 min after and at 20 degrees C, 40 min after SRE addition. At 6 degrees C, below the phase transition temperature of the major lipid components of the RBC membrane, no inactivation occurred for as long as 90 min. With BLMs, SRE induced a large current that remained stable at 14 degrees C, but at 23 degrees C it decreased over time while the single channel conductance and dwell time did not change. The results show that the temperature dependent inactivation of SRE pores is due to a decrease in the number of open pores.

Membrane sterol composition modulates the pore forming activity of syringomycin E in human red blood cells.

The effect of lipopeptide antifungal agent, syringomycin E (SRE) on the membrane permeability of human red blood cells (RBCs) was studied. SRE added to RBCs above a concentration of 2x106 molecules/cell (50 microgram/ml RBCs) caused a rapid and concentration dependent lysis of a small subpopulation of RBCs; the extent of this lysis remained unchanged as long as 100 min. During this time period the membranes of the unlysed cells had enhanced permeability for ions which was monitored by direct measurement of 86Rb flux. Both the extent of cell lysis and ion transport rate showed linear relationships with SRE concentration demonstrating a random distribution of SRE molecules in red blood cells. The kinetics of the 86Rb efflux suggested pore formation by syringomycin E. The pores had discrete life times and were eventually inactivated. The pores were also a pathway for efflux of monomeric haemoglobin. Alteration of the membrane sterol composition, i.e. depletion of cholesterol by 50% or partial ergosterol substitution of the cholesterol increased the SRE induced membrane permeability for 86Rb by two orders compared to membranes with unaltered sterol composition. This modification of the sterol composition promotes the pore forming activity of this lipopeptide in the membrane.