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The Interaction of Lipodepsipeptide Toxins from Pseudomonas syringae pv. syringae with Biological and Model Membranes: A Comparison of Syringotoxin, Syringomycin, and Two Syringopeptins

¹CNR-ITC Centro Fisica Stati Aggregati, Via Sommarive 18, I-38050 Povo (Trento), Italy; ²Dipartimento di Ingegneria dei Materiali, Università di Trento, Via Mesiano 77, I-38050 Mesiano (Trento), Italy; ³Dipartimento di Scienze Biochimiche e Centro CNR di Biologia Molecolare, Roma “La Sapienza,” P.le Aldo Moro 5, 00185 Roma, Italy

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.

Additional keywords: calcein release, detergents, lipid bilayer.