RBPI 21 Causes (HEMI)Fusion and Leakage of Membranes and Promotes Massive Lipopolysaccharide Aggregation

Abstract

Bacteria are infectious agents that seem to have difficulties in acquiring resistance against natural occurring antimicrobial peptides (AMPs). These positively charged polypeptides were then developed as an alternative to conventional therapy against bacterial infections. rBPI21 is a 21 kDa peptide based on the N-terminal region of the neutrophil bactericidal/permeability-increasing protein (BPI). It has bactericidal effects on Gram-negative bacteria and higher affinity for lipopolysaccharide (LPS), neutralizing its effect. rBPI21 is in phase III clinical trials against meningitis. This work aimed at elucidating the mechanism of action of rBPI21 at the molecular level. We demonstrate that rBPI21 promotes the aggregation of negatively charged large unilamellar vesicles (LUV) and LPS aggregates, by dynamic light scattering, while for zwitterionic phosphatidylcholine (POPC) LUV the size remains unchanged. The aggregation increases with peptide concentration until promotion of massive aggregation followed by sample flocculation/precipitation [1,2]. Through a Förster resonance energy transfer (FRET) assay, the aggregation is followed by (hemi)fusion of the negatively charged vesicles, culminating at their leakage (also assessed by fluorescence spectroscopy). With the rBPI21-lipid interaction, there is a progressive change in the zeta-potential of the LUV systems and LPS aggregates [2]. LUV systems composed of phosphatidylglycerol (POPG) and POPC:POPG mixtures have higher zeta-potential variations than POPC LUV. For the LPS aggregates, rBPI21 neutralizes the surface charge and, at higher peptide concentrations, overcompensates it [2]. The results demonstrate 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. [1] Domingues, MM et al., Biophys. J. 2009, 96, 987-996. [2] Domingues, MM et al., PLoS ONE 2009, 4, e8385.