Polymyxins induce lipid scrambling and disrupt the homeostasis of Gram-negative bacteria membrane

Abstract

Polymyxins are increasingly used as the last-line therapeutic option for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, efforts to address the resistance in superbugs are compromised by a poor understanding of the bactericidal modes because high-resolution detection of the cell structure is still lacking. By performing molecular dynamics simulations at a coarse-grained level, here we show that polymyxin B (PmB) disrupts Gram-negative bacterial membranes by altering lipid homeostasis and asymmetry. We found that the binding of PmBs onto the asymmetric outer membrane (OM) loosens the packing of lipopolysaccharides (LPS) and induces unbalanced bending torque between the inner and outer leaflets, which in turn triggers phospholipids to flip from the inner leaflet to the outer leaflet to compensate for the stress deformation. Meanwhile, some LPSs may be detained on the inner membrane (IM). Then, the lipid-scrambled OM undergoes phase separation. Defects are created at the boundaries between LPS-rich domains and phospholipid-rich domains, which consequently facilitate the uptake of PmB across the OM. Finally, PmBs target LPSs detained on the IM and similarly perturb the IM. This lipid Scramble, membrane phase Separation, and peptide Translocation model depicts a novel mechanism by which polymyxins kill bacteria and sheds light on developing a new generation of polymyxins or antibiotic adjuvants with improved killing activities and higher therapeutic indices.

Figure 1

Schematic illustration of lipid scramble, membrane phase separation, and peptide translocation mechanism model. To see this figure in color, go online.

Figure 2

(A) Time sequences of an asymmetric OM model bound by PmBs at concentration defined by molar ratio between peptide and phospholipid, P:PL, of 3%. For a concerned PmB molecule, the charged beads are in purple; uncharged but hydrophilic beads are in gold; and the hydrophobic beads of the fatty acid tail, Phe residue, and Leu residue are in black, blue, and green, respectively. Other PmBs are in red and transparent, POPE are in blue, POPG are in green, and Re-LPSs are in yellow and transparent. (B) Side views of a-OM models bound by PmBs at different concentrations. (C) Density distribution profiles of typical functional groups of lipids, residues of PmB, and Ca²⁺ ions along the direction of the bilayer normal. See Fig. S1 for the definition of bead types. To see this figure in color, go online.

Figure 3

(A–C) Area per LPS (A), stress profile (B), and bending torque (C) of a-OM models bound by different numbers of PmBs. (D) Potential of mean force for a PmB molecule translocating across an a-OM unbound or bound by PmBs. The traction molecule was pulled from bulk water to the phospholipid head interface of the inner leaflet in the bilayer normal direction. The average position of the terminal tail beads of the phospholipids in the inner leaflet was regarded as the reference zero point. (E) PMF for a PmB molecule translocating across IM models. (F) PMF for a POPE molecule flipping from the inner leaflet to the outer leaflet of a-OM models bound by different numbers of PmBs. To see this figure in color, go online.

Figure 4

Top and side views of snapshots of a-OM, s-OM with various deficiencies of LPS, and IM models in the absence (upper panel) and presence (lower panel) of PmBs binding on the outer leaflet. In the circled regions, PmBs penetrate more deeply into the membrane. To see this figure in color, go online.

Figure 5

Snapshots of PmB-induced phase separation of LPS-deficient s-OM with r_LPS = 10%. To see this figure in color, go online.

Figure 6

(A) Potential of mean force for a PmB molecule translocating across a-OM, LPS-deficient s-OM, and IM bound by PmBs at a concentration of P:PL = 5%. (B) Compressibility of the LPS-deficient s-OM model (r_LPS= 10%) bound by different numbers of PmBs. Light gray shadows are fluctuation errors. To see this figure in color, go online.

Figure 7

Schematic illustration of the steps by which polymyxins disrupt Gram-negative bacterial membrane. To see this figure in color, go online.