Depth-Resolved Temperature-Dependent Penetration of Polymyxin B in Phospholipids/Lipopolysaccharide Asymmetric Bilayers

Overview

Journal ACS Omega

Publisher American Chemical Society

Date 2025 Feb 3

PMID 39895715

Authors

Nicolo Paracini

Jeremy H Lakey

Luke A Clifton

Affiliations

Soon will be listed here.

Abstract

The lipid matrix of the outer membrane (OM) of Gram-negative bacteria consists of a highly asymmetric lipid bilayer containing phospholipids on the inner leaflet and lipopolysaccharides (LPS) in the outer layer. The latter ensures that harmful molecules do not permeate the bacterial cell, but polymyxin B (PmB), a last-resort antibiotic, is capable of interfering with the stability of the LPS layer and overcoming the OM barrier. We have previously shown that the efficacy of PmB in disrupting isotopically asymmetric OM models (H-phospholipids and H-LPS) is regulated by the gel-to-fluid phase transition of the LPS layer. Here, we employ fully deuterated OM models (H-phospholipids and H-LPS) to track the temperature-dependent penetration of PmB within the model membrane by using neutron reflectometry. We use a model-independent approach to quantify PmB penetration as a function of both concentration and temperature as well as a model-dependent analysis to localize PmB in the asymmetric bilayer. By leveraging the ability of neutrons to differentiate hydrogen from deuterium in structural biology we find that PmB hijacks LPS molecules and accumulates predominantly in the hydrophobic region of lipid A.

References

Fu L, Wan M, Zhang S, Gao L, Fang W . Polymyxin B Loosens Lipopolysaccharide Bilayer but Stiffens Phospholipid Bilayer. Biophys J. 2019; 118(1):138-150. PMC: 6950770. DOI: 10.1016/j.bpj.2019.11.008. View

Manioglu S, Modaresi S, Thoma J, Overall S, Upert G, Luther A . Reply to: Antibiotics and hexagonal order in the bacterial outer membrane. Nat Commun. 2023; 14(1):4773. PMC: 10412578. DOI: 10.1038/s41467-023-40276-z. View

Paracini N, Schneck E, Imberty A, Micciulla S . Lipopolysaccharides at Solid and Liquid Interfaces: Models for Biophysical Studies of the Gram-negative Bacterial Outer Membrane. Adv Colloid Interface Sci. 2022; 301:102603. DOI: 10.1016/j.cis.2022.102603. View

Nelson A, Prescott S . : neutron and X-ray reflectometry analysis in Python. J Appl Crystallogr. 2019; 52(Pt 1):193-200. PMC: 6362611. DOI: 10.1107/S1600576718017296. View

Needham B, Trent M . Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis. Nat Rev Microbiol. 2013; 11(7):467-81. PMC: 6913092. DOI: 10.1038/nrmicro3047. View

Paracini N, Clifton L, Lakey J . Studying the surfaces of bacteria using neutron scattering: finding new openings for antibiotics. Biochem Soc Trans. 2020; 48(5):2139-2149. PMC: 7609035. DOI: 10.1042/BST20200320. View

Slingerland C, Martin N . Recent Advances in the Development of Polymyxin Antibiotics: 2010-2023. ACS Infect Dis. 2024; 10(4):1056-1079. PMC: 11019560. DOI: 10.1021/acsinfecdis.3c00630. View

Clifton L, Skoda M, Le Brun A, Ciesielski F, Kuzmenko I, Holt S . Effect of divalent cation removal on the structure of gram-negative bacterial outer membrane models. Langmuir. 2014; 31(1):404-12. PMC: 4295546. DOI: 10.1021/la504407v. View

Jiang X, Sun Y, Yang K, Yuan B, Velkov T, Wang L . Coarse-grained simulations uncover Gram-negative bacterial defense against polymyxins by the outer membrane. Comput Struct Biotechnol J. 2021; 19:3885-3891. PMC: 8441625. DOI: 10.1016/j.csbj.2021.06.051. View

10.

Tran A, Lester M, Stead C, Raetz C, Maskell D, McGrath S . Resistance to the antimicrobial peptide polymyxin requires myristoylation of Escherichia coli and Salmonella typhimurium lipid A. J Biol Chem. 2005; 280(31):28186-94. DOI: 10.1074/jbc.M505020200. View

11.

Henderson J, Zimmerman S, Crofts A, Boll J, Kuhns L, Herrera C . The Power of Asymmetry: Architecture and Assembly of the Gram-Negative Outer Membrane Lipid Bilayer. Annu Rev Microbiol. 2016; 70:255-78. DOI: 10.1146/annurev-micro-102215-095308. View

12.

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

13.

Gerelli Y . Phase Transitions in a Single Supported Phospholipid Bilayer: Real-Time Determination by Neutron Reflectometry. Phys Rev Lett. 2019; 122(24):248101. DOI: 10.1103/PhysRevLett.122.248101. View

14.

Whitfield C, Williams D, Kelly S . Lipopolysaccharide O-antigens-bacterial glycans made to measure. J Biol Chem. 2020; 295(31):10593-10609. PMC: 7397119. DOI: 10.1074/jbc.REV120.009402. View

15.

Benn G, Mikheyeva I, Inns P, Forster J, Ojkic N, Bortolini C . Phase separation in the outer membrane of . Proc Natl Acad Sci U S A. 2021; 118(44). PMC: 8612244. DOI: 10.1073/pnas.2112237118. View

16.

Prasad N, Seiple I, Cirz R, Rosenberg O . Leaks in the Pipeline: a Failure Analysis of Gram-Negative Antibiotic Development from 2010 to 2020. Antimicrob Agents Chemother. 2022; 66(5):e0005422. PMC: 9112940. DOI: 10.1128/aac.00054-22. View

17.

Manioglu S, Modaresi S, Ritzmann N, Thoma J, Overall S, Harms A . Antibiotic polymyxin arranges lipopolysaccharide into crystalline structures to solidify the bacterial membrane. Nat Commun. 2022; 13(1):6195. PMC: 9587031. DOI: 10.1038/s41467-022-33838-0. View

18.

Weerakoon D, Petrov K, Pedebos C, Khalid S . Polymyxin B1 within the ell envelope: insights from molecular dynamics simulations. Biophys Rev. 2022; 13(6):1061-1070. PMC: 8724489. DOI: 10.1007/s12551-021-00869-8. View

19.

Pahil K, Gilman M, Baidin V, Clairfeuille T, Mattei P, Bieniossek C . A new antibiotic traps lipopolysaccharide in its intermembrane transporter. Nature. 2024; 625(7995):572-577. PMC: 10794137. DOI: 10.1038/s41586-023-06799-7. View

20.

Clifton L, Skoda M, Daulton E, Hughes A, Le Brun A, Lakey J . Asymmetric phospholipid: lipopolysaccharide bilayers; a Gram-negative bacterial outer membrane mimic. J R Soc Interface. 2013; 10(89):20130810. PMC: 3808558. DOI: 10.1098/rsif.2013.0810. View