Differential Interactions of Piscidins with Phospholipids and Lipopolysaccharides at Membrane Interfaces

Abstract

Piscidins 1 and 3 (P1 and P3) are potent antimicrobial peptides isolated from striped bass. Their mechanism of action involves formation of amphipathic α-helices on contact with phospholipids and destabilization of the microbial cytoplasmic membrane. The peptides are active against both Gram-positive and Gram-negative bacteria, suggesting easy passage across the outer membrane. Here, we performed a comparative study of these two piscidins at the air–water interface on lipopolysaccharide (LPS) monolayers modeling the outer bacterial surface of Gram-negative organisms and on phospholipid monolayers, which mimic the inner membrane. The results show that P1 and P3 are highly surface active (log K AW ∼ 6.8) and have similar affinities to phospholipid monolayers (log K lip ≈ 7.7). P1, which is more potent against Gram negatives, exhibits a much stronger partitioning into LPS monolayers (log K LPS = 8.3). Pressure–area isotherms indicate that under increasing lateral pressures, inserted P1 repartitions from phospholipid monolayers back to the subphase or to a more shallow position with in-plane areas of ∼170 Å2 per peptide, corresponding to fully folded amphipathic α-helices. In contrast, peptide expulsion from LPS occurs with areas of ∼35 Å2, suggesting that the peptides may not form the similarly oriented, rigid secondary structures when they avidly intercalate between LPS molecules. Patch-clamp experiments on Escherichia coli spheroplasts show that when P1 and P3 reach the outer surface of the bacterial cytoplasmic membrane, they produce fluctuating conductive structures at voltages above 80 mV. The data suggests that the strong activity of these piscidins against Gram-negative bacteria begins with the preferential accumulation of peptides in the outer LPS layer followed by penetration into the periplasm, where they form stable amphipathic α-helices upon contact with phospholipids and attack the energized inner membrane.

Partitioning of Seven Different Classes of Antibiotics into LPS Monolayers Supports Three Different Permeation Mechanisms through the Outer Bacterial Membrane

“…Both Rd and Re-LPS isolation was performed using a modified phenol-chloroform-petroleum ether method following a previously published protocol . Re-LPS was extracted from the D31m4 deep rough (Re) mutant strain of E.…”

“…Both Rd and Re-LPS isolation was performed using a modified phenol-chloroform-petroleum ether method following a previously published protocol. 42 Re-LPS was extracted from the D31m4 deep rough (Re) mutant strain of E. coli whose core oligosaccharide chain is truncated at the second KDO group. The longer form, Rd -PS, was extracted from a ΔwaaG rough mutant strain of E. coli (CWG303; E. coli F470 with waaG::aacC1 originating from C. Whitfield, University of Guelph) whose LPS inner core extends through HepII.…”

“…The longer form, Rd -PS, was extracted from a ΔwaaG rough mutant strain of E. coli (CWG303; E. coli F470 with waaG::aacC1 originating from C. Whitfield, University of Guelph) whose LPS inner core extends through HepII. 42 Bacteria were grown in 4−6 L of culture for LPS extraction in shaking broth culture (225 rpm, 37 °C) under aerobic conditions in Lennox LB media (AmericanBio, Canton, MA) supplemented with 1 mM MgCl 2 (Sigma-Aldrich). The mutant E. coli ΔwaaG was grown under the same conditions with the addition of 15 μg/mL gentamicin (Gibco).…”

“…Experiments using the Re-LPS form followed protocols described previously. 42 Briefly, Re-LPS was dissolved in a 1:9 methanol/ chloroform solution to a concentration of 1.6 mg/mL before deposition on the subphase. These experiments were done using a stainless steel trough with a depth of 1 mm (Kibron).…”

Structural Organization of Cardiolipin-Containing Vesicles as Models of the Bacterial Cytoplasmic Membrane

“…To the best of our knowledge, only a few studies have been reported in which ternary mixtures of PE, PG, and CL lipids were used. These latter studies focused on the thermotropic behavior of PE/PG/CL mixtures, the structure of the lipid monolayer at the air/water interface, or directly implemented PE/PG/CL mixtures to investigate the activity of an antimicrobial agent. ,, Therefore, a detailed structural and dynamic characterization of PE/PG/CL mixtures is still missing.…”