ABSTRACT
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.
Subject(s)
Bacterial Proteins/toxicity , Bacterial Toxins/toxicity , Peptides, Cyclic/toxicity , Pseudomonas/pathogenicity , Amino Acid Sequence , Animals , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Hemolysis/drug effects , Humans , In Vitro Techniques , Membrane Lipids/chemistry , Membranes, Artificial , Molecular Sequence Data , Peptides, Cyclic/chemistry , Peptides, Cyclic/genetics , Pseudomonas/chemistry , Pseudomonas/genetics , Rabbits , Sterols/chemistry , Surface PropertiesABSTRACT
Syringopeptin 25A, a pseudomonad lipodepsipeptide, can form ion channels in planar lipid membranes. Pore conductance is around 40 pS in 0.1 M NaCl. Channel opening is strongly voltage dependent and requires a negative potential on the same side of the membrane where the toxin was added. These pores open and close with a lifetime of several seconds. At negative voltages, an additional pore state of around 10 pS and a lifetime of around 30 ms is also present. The voltage dependence of the rates of opening and closing of the stable pores is exponential. This allows estimation of the equivalent charge that is moved across the membrane during the process of opening at about 2.6 elementary charges. When NaCl is present, the pore is roughly 3 times more permeant for anions than for cations. The current voltage characteristic of the pore is nonlinear, i.e., pore conductance is larger at negative than at positive voltages. The maximal conductance of the pore depends on the concentration of the salt present, in a way that varies almost linearly with the conductivity of the solution. From this, an estimate of a minimal pore radius of 0.4 nm was derived.
Subject(s)
Bacterial Toxins/toxicity , Ion Channels/drug effects , Ion Channels/metabolism , Membrane Lipids/chemistry , Peptides, Cyclic/toxicity , Pseudomonas/pathogenicity , Amino Acid Sequence , Animals , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Electric Conductivity , In Vitro Techniques , Ion Channel Gating/drug effects , Membrane Potentials , Molecular Sequence Data , Peptides, Cyclic/chemistry , Peptides, Cyclic/genetics , Pseudomonas/chemistry , Pseudomonas/geneticsABSTRACT
Pseudomonas aeruginosa exotoxin A (ETA) must be proteolytically nicked by furin at Arg279 before being translocated into the cytosol of target cells. A similar cleavage can also be obtained with trypsin. Using this assay we could show that the interaction with lipid bilayers can strongly influence the extent of nicking. We found that in the presence of vesicles containing negatively charged lipids ETA is cleaved into its two fragments A and B at enzyme concentrations approximately 50 times lower, or at pH values higher by 1.5 units, than in the absence of lipids. We suggest that the interaction with the lipid bilayer of the positively charged loop containing Arg279 provides the energy for its partial unfolding and makes it more accessible for proteolysis.
Subject(s)
ADP Ribose Transferases , Bacterial Toxins/chemistry , Exotoxins/metabolism , Lipid Bilayers/chemistry , Pseudomonas aeruginosa/metabolism , Trypsin/metabolism , Virulence Factors , Bacterial Toxins/metabolism , Hydrogen-Ion Concentration , Lipid Bilayers/metabolism , Pseudomonas aeruginosa Exotoxin AABSTRACT
The interaction of Pseudomonas aeruginosa exotoxin A (ETA) with lipid monolayers was studied by measuring the variation in surface pressure. ETA adsorbs to the monolayer, occupying an average area of approximately 4.6 nm2 per molecule, up to a maximum density of one molecule per 28 nm2 of lipid film, which corresponds roughly to the cross-sectional area of the toxin. This suggests that ETA molecules adsorb until they contact each other, but insert only a small portion into the lipid film. The kinetic process could be described by a Langmuir adsorption isotherm. The apparent association and dissociation rate constants were determined, as were their dependence upon toxin concentration, membrane composition, pH, and ionic strength. Two parameters were found to be paramount for this interaction: pH and surface potential of the lipid. It appears that ETA binding occurs only in a conformational state induced by low pH and is promoted by an electrostatic interaction between a positively charged region of the protein and the negative charge of acidic phospholipids. On the basis of a simple model, the salient features of ETA involved in its adsorption were derived: 1) the existence of a conformational state induced by the protonation of a group with pK 4.5 +/- 0.2; 2) a positive charge of 1.9 +/- 0.3 e.u. able to interact with the surface potential of the membrane; 3) the fraction of potential experienced by the protein in the activated state that precedes binding, approximately 80%; 4) the intrinsic adsorption and desorption rate constants, k(a)0 = (4.8 +/- 0.3) x 10(3) M(-1) s(-1) and k(d)0 = (4.4 +/- 0.4) x 10(-4) s(-1). These rate constants are independent of pH and lipid and buffer composition, and provide a dissociation constant Kd approximately 90 nM.
Subject(s)
ADP Ribose Transferases , Exotoxins/chemistry , Liposomes/chemistry , Virulence Factors , Adsorption , Bacterial Toxins/chemistry , Biophysics/methods , Hydrogen-Ion Concentration , Kinetics , Models, Chemical , Phosphatidic Acids , Phosphatidylcholines , Phosphatidylglycerols , Pseudomonas aeruginosa , Surface Properties , Thermodynamics , Pseudomonas aeruginosa Exotoxin AABSTRACT
This case report on one stage surgical treatment of meningoencephalocele offers the opportunity for a revision of the most recent trends expressed by other authors on this topic. Surgical technique involved a system of rigid fixations by means of microplates which, according to our experience in other cases, is particularly advisable in pediatric age. We find that the simultaneous utilization of calvarial bone grafts facilitates a better reconstruction of the cranial base, and also offers greater stability in results.
