Benn, Georgina

Hoogenboom, Bart W

Kleanthous, Colin

Silhavy, Thomas J

English

UCL Discovery

Matters arising https://doi.org/10.1038/s41467-023-40275-0Antibiotics and hexagonal order in thebacterial outer membraneGeorgina Benn 1, Thomas J. Silhavy 1, Colin Kleanthous 2 &Bart W. Hoogenboom 3,4ARISING FROM S. Manioglu et al. Nature Communications https://doi.org/10.1038/s41467-022-33838-0 (2022)Polymyxin is a last-resort antibiotic that targets Gram-negative bac-teria. It does so by binding lipopolysaccharide (LPS) in the outer leafletof the outer membrane by an ill-defined mechanism. Recently, ref. 1used atomic force microscopy (AFM) to image outer membrane vesi-cles ruptured and flattened ontomica in the presence of cations. Theythen added polymyxins, which resulted in the appearance of a hex-agonal lattice in the membrane (Fig. 1A). They attributed this lattice toLPS-polymyxin crystallisation and suggested that such lattices arerelevant for the mechanism of action by which polymyxins inducebacterial cell death.Previous electron microscopy and AFM studies have shown thattrimeric outer membrane proteins (OMPs) also pack together in hex-agonal lattices, as observed in reconstituted 2D arrays2,3, in purifiedouter membranes4 and in living bacteria5–7 (Fig. 1B–D). These OMParrays have lattice constants virtually identical to those seen for thepolymyxin-induced hexagons reported by Manioglu et al. (Fig. 1E).Despite this similarity, Manioglu et al. proposed that polymyxinarranges LPS into such hexagonal arrays independent of OMP contentof the membrane1. To substantiate this hypothesis, they showed thatpolymyxin still formed hexagonal arrays in patches derived fromouterFig. 1 | Comparison of ordered configurations in the outer membrane, asimaged by AFM. A When polymyxin was applied to purified outer membranesfrom MG1655 E. coli, ref. 1 saw a hexagonal lattice with a spacing of 9.8 ± 3.9 nm.B Benn et al. 7 showed that the native, untreated outer membrane of living MG1655E. coliwas dominated by trimeric OMPs arranged in an imperfect hexagonal lattice,with a nearest-neighbour distance of 9.4 ± 2.1 nm. C Jaroslawski et al. 4 observedhexagonal arrangements of trimeric porins with a lattice constant of 8.1 ± 0.3 nm inpurified outer membranes from R. Denitrificans. D Oestreicher et al. 6 showedhexagonal lattices (spacing 10.5 ± 1.7 nm) on the surface of living E. coli, which theyattributed to trimeric OMPs. E Comparison of lattice-spacing distributions forpolymyxin-induced hexagons1 and for OMP lattices in native (untreated) MG1655 E.coli7, showing no significant difference by a two-sided t test with unequal variance(p =0.16). Values are mean ± SD. Scale bar is 20nm. Images A-D were reproducedfrom references as indicated, with permission.Received: 9 November 2022Accepted: 20 July 2023Check for updates1Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA. 2Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK.3LondonCentre for Nanotechnology, University College London, LondonWC1H0AH, UK. 4Department of Physics andAstronomy, University College London,London WC1E 6BT, UK. e-mail: b.hoogenboom@ucl.ac.ukNature Communications |         (2023) 14:4772 11234567890():,;1234567890():,;membrane vesicles from BL21 (DE3) omp8 E. coli cells that did notexpress the primary trimeric porins OmpF, OmpC, and LamB, or themonomeric OmpA, but were enriched in other OMPs8. However, wenowknow thatmanyOMPs9, not just trimeric porins, have apropensityto form heterogenous clusters in the presence of LPS. More extensivenegative controlswouldbeneeded todismissOMPs as thebasis for thehexagonal lattices that are observed following polymyxin treatment ofouter membrane vesicles.Conversely, the polymyxin-induced lattice was affected by LPSlength and cation concentration, from which Manioglu et al. con-cluded that the lattice was determined only by polymyxin–LPScomplexes1, aidedby the tendencyof LPSmolecules to formcrystallinedomains. They cite LPS crystallinity seen inmolecular dynamics and inmodel membranes10–12, to suggest that the polymyxin-induced hex-agonal geometry is related to LPS packing. However, the hexagonalLPS arrays referenced have lattice constants of approximately oneorder of magnitude smaller11 than the arrays seen by ref. 1.Based on their observations, Manioglu et al. conclude with thesuggestion that “a local ordering of LPS by polymyxin must lie atthe core of the [hexagonal] arrangement”1. In contrast, based on theobservations above, we propose an alternative conclusion, assigningtheobservedhexagonal order to the symmetryofOMPnetworks in theuntreated E. coli membrane7. The OMP–OMP interactions in suchnetworks are mediated by LPS9, which is thereby expected to followsimilar local ordering. Hence in our interpretation, the polymyxin doesnot order the LPS, but binds to the already ordered LPS and therebyreveals existing OMP lattices at a higher contrast than is the case forthe untreated membranes.References1. Manioglu, S. et al. Antibiotic polymyxin arranges lipopolysacchar-ide into crystalline structures to solidify the bacterial membrane.Nat. Commun. 13, 6195 (2022).2. Dorset, D. L., Engel, A., Häner, M., Massalski, A. & Rosenbusch, J. P.Two-dimensional crystal packing of matrix porin. J. Mol. Biol. 165,701–710 (1983).3. Schabert, F. A., Henn, C. & Engel, A. Native Escherichia coli OmpFporin surfaces probed by atomic force microscopy. Science 268,92–94 (1995).4. Jaroslawski, S., Duquesne, K., Sturgis, J. N. & Scheuring, S. High-resolution architecture of the outer membrane of the Gram-negative bacteria Roseobacter denitrificans. Mol. Microbiol. 74,1211–1222 (2009).5. Yamashita, H. et al. Single-molecule imaging on living bacterial cellsurface by high-speed AFM. J. Mol. Biol. 422, 300–309(2012).6. Oestreicher, Z., Taoka, A. & Fukumori, Y. A comparison of the sur-face nanostructure from two different types of gram-negative cells:Escherichia coli and Rhodobacter sphaeroides. Micron 72,8–14 (2015).7. Benn, G. et al. Phase separation in the outer membrane of Escher-ichia coli. Proc. Natl Acad. Sci. 118, e2112237118 (2021).8. Thoma, J. et al. Protein-enriched outer membrane vesicles as anative platform for outermembrane protein studies.Commun. Biol.1, 23 (2018).9. Webby,M. N. et al. Lipidsmediate supramolecular outermembraneprotein assembly in bacteria. Sci. Adv. 8, eadc9566 (2022).10. Jefferies, D., Hsu, P.-C. & Khalid, S. Through the lipopolysaccharideglass: a potent antimicrobial peptide induces phase changes inmembranes. Biochemistry 56, 1672–1679 (2017).11. Le Brun, A. P. et al. Structural characterization of a model gram-negative bacterial surface using lipopolysaccharides from roughstrains of Escherichia coli. Biomacromolecules 14, 2014–2022 (2013).12. Paracini, N., Clifton, L. A., Skoda, M. W. A. & Lakey, J. H. Liquidcrystalline bacterial outer membranes are critical for antibioticsusceptibility. Proc. Natl Acad. Sci. USA 115, E7587–E7594 (2018).AcknowledgementsThe authors acknowledge funding by the UK Engineering and PhysicalSciences Research Council (EP/N509577/1 to G.B. and B.W.H.), theNational Institute of General Medical Sciences of the NIH (R35-GM118024 to T.J.S.), the UK Biotechnology and Biological SciencesResearch Council (BB/V008056/1 to C.K. and BB/R000042/1 to B.W.H.),Wellcome Trust Collaborative Award (201505/Z/16/Z to C.K.), and theEuropean Research Council (Advanced Grant 742555, OMPorg, to C.K.).The content is solely the responsibility of the authors and does notnecessarily represent the official views of the NIH or other funders.Author contributionsG.B., T.S., C.K. and B.H. contributed equally to the interpretation ofresults and writing of the manuscript.Competing interestsThe authors declare no competing interests.Additional informationCorrespondence and requests for materials should be addressed toBart W. Hoogenboom.Peer review information Nature Communications thanks the anon-ymous reviewers for their contribution to the peer review of this work.Reprints and permissions information is available athttp://www.nature.com/reprintsPublisher’s note Springer Nature remains neutral with regard to jur-isdictional claims in published maps and institutional affiliations.Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in any medium or format, aslong as you give appropriate credit to the original author(s) and thesource, provide a link to the Creative Commons license, and indicate ifchanges were made. The images or other third party material in thisarticle are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is notincluded in the article’s Creative Commons license and your intendeduse is not permitted by statutory regulation or exceeds the permitteduse, you will need to obtain permission directly from the copyrightholder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.© The Author(s) 2023Matters arising https://doi.org/10.1038/s41467-023-40275-0Nature Communications |         (2023) 14:4772 2

Antibiotics and hexagonal order in the bacterial outer membrane

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Antibiotics and hexagonal order in the bacterial outer membrane

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