Research Article: rBPI21 Promotes Lipopolysaccharide Aggregation and Exerts Its Antimicrobial Effects by (Hemi)fusion of PG-Containing Membranes

Date Published: December 22, 2009

Publisher: Public Library of Science

Author(s): Marco M. Domingues, Miguel A. R. B. Castanho, Nuno C. Santos, Stefan Bereswill.

Abstract: Antimicrobial peptides (AMPs) are important potential alternatives to conventional therapies against bacterial infections. rBPI21 is a 21 kDa peptide based on the N-terminal region of the neutrophil bactericidal/permeability-increasing protein (BPI). This AMP possesses highly selective bactericidal effects on Gram-negative bacteria and have affinity for lipopolysaccharide (LPS) which is believed to be at the origin of its neutralizing effect of the LPS segregated into the bloodstream. We aim at understanding the molecular bases of rBPI21 bactericidal and LPS neutralization actions, using biomembrane model systems. Using dynamic light scattering spectroscopy we demonstrate that rBPI21 promotes aggregation of negatively charged large unilamellar vesicles (LUV), even in the absence of LPS, and LPS aggregates, while for zwitterionic phosphatidylcholine (POPC) LUV the size remains unchanged. The peptide also promotes the fusion (or hemifusion) of membranes containing phosphatidylglycerol (POPG). The aggregation and fusion of negatively charged LUV are peptide concentration-dependent until massive aggregation is reached, followed by sample flocculation/precipitation. Concomitantly, there is a progressive change in the zeta-potential of the LUV systems and LPS aggregates. LUV systems composed of phosphatidylglycerol (POPG) and lipid mixtures with POPG have higher zeta-potential variations than in the absence of POPG. The interaction of rBPI21 with lipid vesicles is followed by leakage, with higher effect in POPG-containing membranes. LPS aggregation can be related with a decreased toxicity, possibly by facilitating its clearance by macrophage phagocytosis and/or blocking of LPS specific receptor recognition. Our data indicate that rBPI21 mechanism of action at the molecular level involves the interaction with the LPS of the outer membrane of Gram-negative bacteria, followed by internalization and leakage induction through the (hemi)fusion of the bacterial outer and inner membranes, both enriched in phosphatidylglycerol.

Partial Text: The innate immune system acts as a first line in the defense of many organisms against pathogens [1]. Antimicrobial peptides (AMPs) are part of the innate immune system of several organisms and usually have a broad spectrum of action against pathogens. During infection they are produced by the cells of the innate immune system and exert their effects on Gram-negative and Gram-positive bacteria, as well as on fungi, viruses and parasites [2]. AMPs have defined properties that ensure selective interaction with microbial plasma membranes [2]–[5]. Usually they are hydrophobic and have a positive net charge. Gram-negative bacteria have two membrane bilayers, the asymmetric outer membrane, mainly composed of lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet, and the inner bilayer, composed by phospholipids [6]. The LPS molecules are very compact due to the salt bridge formed by the presence of Mg2+ and Ca2+ ions. This maintains an impermeable barrier to certain antibiotics and contributes to bacterial resistance.

Phosphate buffer saline (20 mM sodium phosphate, 150 mM NaCl) pH 7.4 was used in all the measurements, except when indicated otherwise.



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