{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T08:30:52Z","timestamp":1774945852676,"version":"3.50.1"},"reference-count":63,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2021,6,28]],"date-time":"2021-06-28T00:00:00Z","timestamp":1624838400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Many bacteria secrete toxic protein complexes that modify and disrupt essential processes in the infected cell that can lead to cell death. To conduct their action, these toxins often need to cross the cell membrane and reach a specific substrate inside the cell. The investigation of these protein complexes is essential not only for understanding their biological functions but also for the rational design of targeted drug delivery vehicles that must navigate across the cell membrane to deliver their therapeutic payload. Despite the immense advances in experimental techniques, the investigations of the toxin entry mechanism have remained challenging. Computer simulations are robust complementary tools that allow for the exploration of biological processes in exceptional detail. In this review, we first highlight the strength of computational methods, with a special focus on all-atom molecular dynamics, coarse-grained, and mesoscopic models, for exploring different stages of the toxin protein entry mechanism. We then summarize recent developments that are significantly advancing our understanding, notably of the glycolipid\u2013lectin (GL-Lect) endocytosis of bacterial Shiga and cholera toxins. The methods discussed here are also applicable to the design of membrane-penetrating nanoparticles and the study of the phenomenon of protein phase separation at the surface of the membrane. Finally, we discuss other likely routes for future development.<\/jats:p>","DOI":"10.3390\/toxins13070449","type":"journal-article","created":{"date-parts":[[2021,6,28]],"date-time":"2021-06-28T13:39:22Z","timestamp":1624887562000},"page":"449","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Computational Approaches to Explore Bacterial Toxin Entry into the Host Cell"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5509-0996","authenticated-orcid":false,"given":"Weria","family":"Pezeshkian","sequence":"first","affiliation":[{"name":"Groningen Biomolecular Sciences and Biotechnology Institute and Zernike Institute for Advanced Materials, University of Groningen, 9712 Groningen, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7885-735X","authenticated-orcid":false,"given":"Julian C.","family":"Shillcock","sequence":"additional","affiliation":[{"name":"Blue Brain Project, Laboratory of Molecular and Chemical Biology of Neurodegeneration, Ecole Polytechnique F\u00e9d\u00e9rale de Lausanne, 1015 Lausanne, Switzerland"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"John H.","family":"Ipsen","sequence":"additional","affiliation":[{"name":"MEMPHYS\/PhyLife, Department of Physics, Chemistry and Pharmacy (FKF), University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,6,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"530","DOI":"10.1038\/35080089","article-title":"The bacterial toxin toolkit","volume":"2","author":"Schiavo","year":"2001","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1614","DOI":"10.1039\/C9SM01913H","article-title":"Membrane rolling induced by bacterial toxins","volume":"16","author":"Klenow","year":"2020","journal-title":"Soft Matter"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-019-09890-8","article-title":"Cryo-EM structures of the pore-forming A subunit from the Yersinia entomophaga ABC toxin","volume":"10","author":"Piper","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1034","DOI":"10.1529\/biophysj.104.040519","article-title":"Fluorescence Correlation Spectroscopy Relates Rafts in Model and Native Membranes","volume":"87","author":"Bacia","year":"2004","journal-title":"Biophys. J."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"670","DOI":"10.1038\/nature05996","article-title":"Shiga toxin induces tubular membrane invaginations for its uptake into cells","volume":"450","author":"Berland","year":"2007","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"488","DOI":"10.1016\/S0021-9258(19)41923-6","article-title":"Membrane receptors as general markers for plasma membrane isolation procedures. The use of 125-I-labeled wheat germ agglutinin, insulin, and cholera toxin","volume":"250","author":"Chang","year":"1975","journal-title":"J. Biol. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"9182","DOI":"10.1074\/jbc.M009414200","article-title":"Cholera Toxin Is Found in Detergent-insoluble Rafts\/Domains at the Cell Surface of Hippocampal Neurons but Is Internalized via a Raft-independent Mechanism","volume":"276","author":"Shogomori","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"3675","DOI":"10.1038\/s41467-021-23961-9","article-title":"Ceramide structure dictates glycosphingolipid nanodomain assembly and function","volume":"12","author":"Arumugam","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"6320","DOI":"10.1073\/pnas.0405654102","article-title":"Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes","volume":"102","author":"Hammond","year":"2005","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4792","DOI":"10.1016\/j.jmb.2016.10.027","article-title":"Glycolipids and Lectins in Endocytic Uptake Processes","volume":"428","author":"Johannes","year":"2016","journal-title":"J. Mol. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Johannes, L. (2017). Shiga Toxin\u2014A Model for Glycolipid-Dependent and Lectin-Driven Endocytosis. Toxins, 9.","DOI":"10.3390\/toxins9110340"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"11010","DOI":"10.1021\/acs.langmuir.7b01461","article-title":"Membrane Tubulation in Lipid Vesicles Triggered by the Local Application of Calcium Ions","volume":"33","author":"Doosti","year":"2017","journal-title":"Langmuir"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/j.ceb.2021.02.009","article-title":"Simulating realistic membrane shapes","volume":"71","author":"Pezeshkian","year":"2021","journal-title":"Curr. Opin. Cell Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"5607","DOI":"10.1021\/acs.chemrev.8b00538","article-title":"Multiscale Simulations of Biological Membranes: The Challenge To Understand Biological Phenomena in a Living Substance","volume":"119","author":"Enkavi","year":"2019","journal-title":"Chem. Rev."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"4467","DOI":"10.1039\/C6NR08036G","article-title":"Probing the structure and in silico stability of cargo loaded DNA icosahedra using MD simulations","volume":"9","author":"Joshi","year":"2017","journal-title":"Nanoscale"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"21083","DOI":"10.1039\/D0CP03282D","article-title":"Structural characterization of supramolecular hollow nanotubes with atomistic simulations and SAXS","volume":"22","author":"Patmanidis","year":"2020","journal-title":"Phys. Chem. Chem. Phys."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.ijbiomac.2015.12.072","article-title":"Current updates on computer aided protein modeling and designing","volume":"85","author":"Khan","year":"2016","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-020-17437-5","article-title":"Protein\u2013ligand binding with the coarse-grained Martini model","volume":"11","author":"Souza","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1623","DOI":"10.1038\/s41467-017-01743-6","article-title":"Annexin A4 and A6 induce membrane curvature and constriction during cell membrane repair","volume":"8","author":"Boye","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"308","DOI":"10.1039\/D0SM00241K","article-title":"Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles","volume":"17","author":"Florentsen","year":"2021","journal-title":"Soft Matter"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"5164","DOI":"10.1039\/C6SM00464D","article-title":"Membrane Invagination Induced by Shiga toxin B-subunit: From Molecular Structure to Tube Formation","volume":"12","author":"Pezeshkian","year":"2016","journal-title":"Soft Matter"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"314","DOI":"10.1021\/acsnano.6b05706","article-title":"Mechanism of Shiga Toxin Clustering on Membranes","volume":"11","author":"Pezeshkian","year":"2017","journal-title":"ACS Nano"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1038\/nmeth.4067","article-title":"CHARMM36m: An improved force field for folded and intrinsically disordered proteins","volume":"14","author":"Huang","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"044130","DOI":"10.1063\/5.0014475","article-title":"Scalable molecular dynamics on CPU and GPU architectures with NAMD","volume":"153","author":"Phillips","year":"2020","journal-title":"J. Chem. Phys."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers","volume":"1\u20132","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1157","DOI":"10.1002\/jcc.20035","article-title":"Development and testing of a general amber force field","volume":"25","author":"Wang","year":"2004","journal-title":"J. Comput. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1638","DOI":"10.1002\/2211-5463.12321","article-title":"Cholera toxin B subunit induces local curvature on lipid bilayers","volume":"7","author":"Pezeshkian","year":"2017","journal-title":"FEBS Open Bio"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"15244","DOI":"10.1021\/la5036618","article-title":"Insights into Binding of Cholera Toxin to GM1 Containing Membrane","volume":"30","author":"Basu","year":"2014","journal-title":"Langmuir"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1352","DOI":"10.1039\/C4SM02456G","article-title":"The effects of globotriaosylceramide tail saturation level on bilayer phases","volume":"11","author":"Pezeshkian","year":"2015","journal-title":"Soft Matter"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"12074","DOI":"10.1021\/acs.jpcb.5b07909","article-title":"Microsecond Simulations of the Diphtheria Toxin Translocation Domain in Association with Anionic Lipid Bilayers","volume":"119","author":"Kurnikova","year":"2015","journal-title":"J. Phys. Chem. B"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1303","DOI":"10.3390\/toxins7041303","article-title":"Role of Acidic Residues in Helices TH8\u2013TH9 in Membrane Interactions of the Diphtheria Toxin T Domain","volume":"7","author":"Ghatak","year":"2015","journal-title":"Toxins"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Flores-Canales, J.C., Simakov, N.A., and Kurnikova, M. (2019). Microsecond Molecular Dynamics Simulations of Diphtheria Toxin Translocation T-Domain pH-Dependent Unfolding in Solution. bioRxiv, 572040.","DOI":"10.1101\/572040"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"1362","DOI":"10.3390\/toxins5081362","article-title":"pH-triggered conformational switching along the membrane insertion pathway of the diphtheria toxin T-domain","volume":"5","author":"Ladokhin","year":"2013","journal-title":"Toxins"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"252","DOI":"10.3389\/fphys.2017.00252","article-title":"Phase Partitioning of GM1 and Its Bodipy-Labeled Analog Determine Their Different Binding to Cholera Toxin","volume":"8","author":"Rissanen","year":"2017","journal-title":"Front. Physiol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"260","DOI":"10.1038\/nchembio.551","article-title":"Cholesterol modulates glycolipid conformation and receptor activity","volume":"7","author":"Lingwood","year":"2011","journal-title":"Nat. Chem. Biol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1016\/j.bbamem.2017.01.033","article-title":"Long-chain GM1 gangliosides alter transmembrane domain registration through interdigitation","volume":"1859","author":"Manna","year":"2017","journal-title":"Biochim. Biophys. Acta (BBA) Biomembr."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1043","DOI":"10.1006\/jmbi.1998.2076","article-title":"The 1.25 \u00c5 resolution refinement of the cholera toxin B-pentamer: Evidence of peptide backbone strain at the receptor-binding site","volume":"282","author":"Merritt","year":"1998","journal-title":"J. Mol. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1038\/nrm3968","article-title":"Building endocytic pits without clathrin","volume":"16","author":"Johannes","year":"2015","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"14978","DOI":"10.1073\/pnas.2001119117","article-title":"Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin","volume":"117","author":"Kabbani","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"17467","DOI":"10.1073\/pnas.2011359117","article-title":"Membrane deformation by the cholera toxin beta subunit requires more than one binding site","volume":"117","author":"Groza","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"11","DOI":"10.1038\/ncb1999","article-title":"GM1 structure determines SV40-induced membrane invagination and infection","volume":"12","author":"Ewers","year":"2009","journal-title":"Nat. Cell Biol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s42003-021-01693-2","article-title":"Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes","volume":"4","author":"Ivashenka","year":"2021","journal-title":"Commun. Biol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"592","DOI":"10.1038\/ncb2970","article-title":"Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers","volume":"16","author":"Lakshminarayan","year":"2014","journal-title":"Nat. Cell Biol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-020-15303-y","article-title":"Endophilin-A3 and Galectin-8 control the clathrin-independent endocytosis of CD166","volume":"11","author":"Renard","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1002\/wcms.1169","article-title":"The power of coarse graining in biomolecular simulations","volume":"4","author":"Lopez","year":"2014","journal-title":"Wiley Interdiscip. Rev. Comput. Mol. Sci."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1146\/annurev-biophys-083012-130348","article-title":"Coarse-Graining Methods for Computational Biology","volume":"42","author":"Saunders","year":"2013","journal-title":"Annu. Rev. Biophys."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"S2885","DOI":"10.1088\/0953-8984\/17\/31\/016","article-title":"Wetting, budding, and fusion\u2014morphological transitions of soft surfaces","volume":"17","author":"Lipowsky","year":"2005","journal-title":"J. Phys. Condens. Matter"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"405","DOI":"10.1016\/j.tcb.2018.01.009","article-title":"Clustering on Membranes: Fluctuations and More","volume":"28","author":"Johannes","year":"2018","journal-title":"Trends Cell Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"461","DOI":"10.1038\/nature05840","article-title":"Aggregation and vesiculation of membrane proteins by curvature-mediated interactions","volume":"447","author":"Reynwar","year":"2007","journal-title":"Nature"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"011506","DOI":"10.1103\/PhysRevE.72.011506","article-title":"Tunable generic model for fluid bilayer membranes","volume":"72","author":"Cooke","year":"2005","journal-title":"Phys. Rev. E"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"7898","DOI":"10.1021\/acs.chemrev.6b00163","article-title":"Coarse-Grained Protein Models and Their Applications","volume":"116","author":"Kmiecik","year":"2016","journal-title":"Chem. Rev."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"382","DOI":"10.1038\/s41592-021-01098-3","article-title":"Martini 3: A general purpose force field for coarse-grained molecular dynamics","volume":"18","author":"Souza","year":"2021","journal-title":"Nat. Methods"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"7812","DOI":"10.1021\/jp071097f","article-title":"The MARTINI force field: Coarse grained model for biomolecular simulations","volume":"111","author":"Marrink","year":"2007","journal-title":"J. Phys. Chem. B"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.1016\/j.bpj.2019.08.037","article-title":"Gangliosides Destabilize Lipid Phase Separation in Multicomponent Membranes","volume":"117","author":"Liu","year":"2019","journal-title":"Biophys. J."},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"236","DOI":"10.1016\/j.jmgm.2016.07.007","article-title":"Multi-scale molecular dynamics study of cholera pentamer binding to a GM1-phospholipid membrane","volume":"68","author":"Sridhar","year":"2016","journal-title":"J. Mol. Graph. Model."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"2488","DOI":"10.1021\/acs.jctc.0c01145","article-title":"Multiscale Molecular Dynamics Studies Reveal Different Modes of Receptor Clustering by Gb3-Binding Lectins","volume":"17","author":"Kociurzynski","year":"2021","journal-title":"J. Chem. Theory Comput."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"529","DOI":"10.1007\/s00232-015-9771-3","article-title":"Membrane Association of the Diphtheria Toxin Translocation Domain Studied by Coarse-Grained Simulations and Experiment","volume":"248","author":"Ladokhin","year":"2015","journal-title":"J. Membr. Biol."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"jcs208884","DOI":"10.1242\/jcs.208884","article-title":"Galectins at a glance","volume":"131","author":"Johannes","year":"2018","journal-title":"J. Cell Sci."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"244705","DOI":"10.1063\/5.0006756","article-title":"Domain formation in bicomponent vesicles induced by composition-curvature coupling","volume":"152","author":"Cornet","year":"2020","journal-title":"J. Chem. Phys."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"59","DOI":"10.3389\/fmolb.2019.00059","article-title":"A Multi-Scale Approach to Membrane Remodeling Processes","volume":"6","author":"Pezeshkian","year":"2019","journal-title":"Front. Mol. Biosci."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"598","DOI":"10.1016\/j.bpj.2020.12.028","article-title":"Influence of membrane-cortex linkers on the extrusion of membrane tubes","volume":"120","author":"Paraschiv","year":"2021","journal-title":"Biophys. J."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"9974","DOI":"10.1039\/C9SM01762C","article-title":"Fluctuations and conformational stability of a membrane patch with curvature inducing inclusions","volume":"15","author":"Pezeshkian","year":"2019","journal-title":"Soft Matter"},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"2296","DOI":"10.1038\/s41467-020-16094-y","article-title":"Backmapping triangulated surfaces to coarse-grained membrane models","volume":"11","author":"Pezeshkian","year":"2020","journal-title":"Nat. Commun."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/13\/7\/449\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:26:06Z","timestamp":1760163966000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/13\/7\/449"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,6,28]]},"references-count":63,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2021,7]]}},"alternative-id":["toxins13070449"],"URL":"https:\/\/doi.org\/10.3390\/toxins13070449","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,6,28]]}}}