rBPI(21) promotes lipopolysaccharide aggregation and exerts its antimicrobial effects by (hemi)fusion of PG-containing membranes

Abstract

Antimicrobial peptides (AMPs) are important potential alternatives to conventional therapies against bacterial infections. rBPI(21) 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 rBPI(21) bactericidal and LPS neutralization actions, using biomembrane model systems. Using dynamic light scattering spectroscopy we demonstrate that rBPI(21) 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 rBPI(21) 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 rBPI(21) 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.

Figure 1. Size distributions of LPS aggregates in the presence of different concentration of rBPI₂₁.

Figure 2. Aggregation of LPS promoted by rBPI₂₁. Hydrodynamic diameter variation of LPS aggregates with increasing rBPI₂₁ concentration.

Bars represent the size range from three independent experiments.

Figure 3. Zeta-potential for LPS aggregates in the presence of rBPI₂₁.

Bars represent the zeta-potential range from two independent experiments.

Figure 4. Aggregation of LUV caused by rBPI₂₁.

Hydrodynamic diameter variation of lipid vesicles with increasing rBPI₂₁ concentration. Bars represents the size range from three independent experiments. Larger ranges are intrinsically associated to vesicles flocculation/precipitation (marked *). POPC (black circles); POPG (blue circles); POPC∶POPG 55∶45 (orange circles); POPC∶POPG 80∶20 (green circles); POPC∶LPS 80∶20 (red circles); POPC∶POPG∶LPS 60∶20∶20 (brown circles). The lipid concentration was kept constant at 50 µM. The differences in hydrodynamic size for [rBPI₂₁]/[Lipid] = 0.0048 were statistically significant for the following data pairs: POPC/POPC∶POPG (80∶20), POPC/POPC∶POPG (55∶45), POPC/POPG, POPC/POPC∶POPG∶LPS (60∶20∶20), POPC∶LPS (80∶20)/POPC∶POPG (80∶20), POPC∶LPS (80∶20)/POPC∶POPG (55∶45), POPC∶LPS (80∶20)/POPG and POPC∶LPS (80∶20)/POPC∶POPG∶LPS (60∶20∶20).

Figure 5. Zeta-potential for membrane model systems in the presence of rBPI₂₁.

Bars represent the zeta-potential range from at least two independent experiments. POPC (black circles); POPG (blue circles); POPC∶POPG 55∶45 (orange circles); POPC∶POPG 80∶20 (green circles); POPC∶LPS 80∶20 (red circles); POPC∶POPG∶LPS 60∶20∶20 (brown circles). The lipid concentration was kept constant at 200 µM. The differences in potential-zeta for [rBPI₂₁]/[Lipid]  = 0.015 were statistically significant for the following data pairs: POPC/POPC∶LPS (80∶20), POPC/POPC∶POPG (80∶20), POPC/POPC∶POPG (55∶45), POPC/POPG, POPC/POPC∶POPG∶LPS (60∶20∶20), POPC∶LPS (80∶20)/POPG; POPC∶POPG (80∶20)/POPG, POPC∶POPG (55∶45)/POPG and POPC∶POPG∶LPS (60∶20∶20)/POPG.

Figure 6. Fusion or hemifusion of membrane model systems promoted by rBPI₂₁.

LUV labeled with 0.6% mol of RhB-PE and NBD-PE were mixed with unlabeled liposomes to a final proportion of 1∶4 and the energy transfer assay was evaluated in the presence of rBPI₂₁ after 10 min of incubation. Bars represent the range from three independent experiments. POPC (black circles); POPG (blue circles); POPC∶POPG 55∶45 (orange circles); POPC∶POPG 80∶20 (green circles); POPC∶LPS 80∶20 (red circles); POPC∶POPG∶LPS 60∶20∶20 (brown circles). The differences in (hemi)fusion percentage for rBPI₂₁ 9 µM were statistically significant between all data pairs, except POPC/POPC∶LPS (80∶20).

Figure 7. rBPI₂₁-induced membrane leakage.

Carboxyfluorescein fluorescence increase measured 20 min after rBPI₂₁ addition in POPC (black circles), POPC∶LPS 80∶20 (red circles), POPC∶POPG 80∶20 (green circles), POPC∶POPG 55∶45 (orange circles), POPC∶POPG∶LPS 60∶20∶20 (brown circles) and POPG (blue circles) liposomes. Bars represent the range from at least two independent experiments. The differences in leakage percentages for rBPI₂₁ 9 µM were statistically significant for the following data pairs: POPC/POPC∶LPS (80∶20), POPC/POPC∶POPG (80∶20), POPC/POPC∶POPG (55∶45), POPC/POPG, POPC/POPC∶POPG∶LPS (60∶20∶20), POPC∶LPS (80∶20)/POPG∶POPG (55∶45), POPC∶LPS (80∶20)/POPG, POPC∶POPG (80∶20)/POPC∶POPG (55∶45), POPC∶POPG (80∶20)/POPG, POPC∶POPG (55∶45)/POPC∶POPG∶LPS (60∶20∶20) and POPC∶POPG∶LPS (60∶20∶20)/POPG.

Figure 8. Schematic representation of the proposed mechanism of action of rBPI₂₁ protein.

A) The protein interacts with LPS aggregates promoting their aggregation, which allow LPS phagocytosis by innate immune cells and reduce LPS availability for its receptor. B) rBPI₂₁ interacts electrostatically with the LPS of the outer leaflet of outer membrane. This interaction disrupts the higher tight bond of the LPS, which allow the peptide insertion and translocation to the outer membrane inner leaflet and to the intermembrane space. rBPI₂₁ induces the fusion (or hemifusion) of the inner leaflet of the outer membrane and the inner membrane (both rich in PG), following a process similar to the occurring at the Bayer junctions . These membrane fusion events promote an increased membrane permeability, culminating with the leakage of the bacterial content.