syringomycin and Hemolysis

Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.

Topics: Amino Acid Sequence; Animals; Bacterial Proteins; Bacterial Toxins; Hemolysis; Humans; In Vitro Techniques; Membrane Lipids; Membranes, Artificial; Molecular Sequence Data; Peptides, Cyclic; Pseudomonas; Rabbits; Sterols; Surface Properties

The phytopathogenic bacterium Pseudomonas syringae pv. syringae produces two classes of necrosis-inducing lipodepsipeptide toxins commonly referred to as the syringomycins and syringopeptins. Members of the syringomycins class are pore-forming cytotoxins that act by promoting passive transmembrane ion flux. In this study, we test the hypothesis that syringopeptin forms SP22A and SP22B likewise function as pore-forming cytotoxins and are similar in activity to syringomycin in artificial and plant membranes. Correspondingly, syringopeptin increased the conductance of black-lipid membranes in a manner indicative of ion channel formation. In tobacco protoplast assays, syringopeptin forms SP22A and SP22B were equivalent in activity causing lysis of protoplasts and measurable 45Ca2+ influx at a threshold concentration of 50 ng/ml. A mixture of three forms of syringomycin did not show cytotoxic activity appreciably different from that of SP22A or SP22B in tobacco protoplast assays. Both forms of syringopeptin also displayed potent biosurfactant properties demonstrated by lowering of the interfacial tension of high-pressure liquid chromatography-grade water to 36 and 34.5 nm/m, respectively; the critical micellar concentration was 0.8 mg/ml for both forms of toxin. These results demonstrate that both classes of pore-forming lipodepsipeptides secreted by P. syringae pv. syringae are cytotoxic to plant cells at nanomolar concentrations and cause necrosis by forming ion channels that are freely permeable to divalent cations.

Topics: Amino Acid Sequence; Bacterial Proteins; Calcium; Chromatography, High Pressure Liquid; Hemolysis; Ion Channels; Ion Transport; Lipid Bilayers; Molecular Sequence Data; Nicotiana; Peptides, Cyclic; Plants, Toxic; Protoplasts; Pseudomonas; Surface-Active Agents