Specificity for the Exchange of Phospholipids Through Polymyxin B Mediated Intermembrane Molecular Contacts

Abstract

Structural specificity for the direct vesicle-vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (PxB) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O.G., & Jain, M.K. (1996) Biochemistry 35, 299-308], intermembrane molecular contacts between anionic vesicles are formed by a small number of PxB molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of PxB to vesicles, formation of stable vesicle-vesicle contacts, and exchange of phospholipids. Polycationic PxB binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through PxB contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. PxB does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange of covesicles is not mediated by PxB. Vesicles of dianionic phospholipids, like phosphatidic acid, bind PxB; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of PxB at the interface. PxB in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that PxB at the interface can exist in two forms depending on the lipid/PxB ratio. Additional studies show that the stable PxB-mediated vesicle-vesicle contacts may be structurally and functionally distinct from "stalks", the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundation in interfacial equilibria and catalysis.

Anionic Lipids Enriched at the ExPortal of Streptococcus pyogenes

“…For example, defensins are cationic peptides produced by numerous host tissues to protect against bacterial infection (13). Polymyxin B also has a high affinity for PG (2) and can greatly decrease the efficiency of protein translocation via the Sec pathway in inverted membrane vesicles (41). This raises the possibility that these compounds may directly interfere with ExPortal-mediated protein secretion via their abilities to recognize anionic lipids, a property that could be exploited for further analysis of protein secretion in the gram-positive cocci.…”

Membrane interaction of a new synthetic antimicrobial lipopeptide sp-85 with broad spectrum activity

“…Interestingly, no destabilization is observed in liposomes of zwitterionic POPC, a phospholipid that is characteristic of eukaryotic membranes. In contrast, we have previously shown that the parent peptide PxB interacts with the anionic membranes in a different way, with membrane fusion and leakage appearing only at concentrations well above the MIC of this peptide [23]. This has been discussed in the context of the selectivity of PxB for Gram negative bacteria, since PxB acts by promoting the mixing of lipids between the inner and outer membranes that form the cell wall of Gram negative bacteria [14,15].…”

“…The mixing of lipids between membranes induced by the lipopeptide was determined using small unilamelar vesicles, as described in detail elsewhere [23]. Unilamelar vesicles of POPG, POPE/POPG (6:4) or POPC, alone or with 30% of the fluorescently labeled phospholipids (pyPG or pyPC) were prepared by sonication, as described previously.…”

Prophylactic Intratracheal Polymyxin B/Surfactant Prevents Bacterial Growth in Neonatal Escherichia coli Pneumonia of Rabbits

“…This could be caused by a homogenous PxB distribution resulting in an effective topical concentration of PxB. PxB can crosslink between the surfactant phospholipids (17,18) and can be distributed with surfactant homogenously. The administered dose of PxB corresponds to the recommended daily doses of aerosolized PxB (2.5 mg/kg/d) (1), but the effective deposited dose may be increased compared with aerosolization when Ͻ5% of the nebulised drug reach the alveolar space.…”