{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T08:06:12Z","timestamp":1790409972316,"version":"4.1.0"},"reference-count":27,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2020,7,31]],"date-time":"2020-07-31T00:00:00Z","timestamp":1596153600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001665","name":"Agence Nationale de la Recherche","doi-asserted-by":"publisher","award":["ANR-15-CE05-0003-01"],"award-info":[{"award-number":["ANR-15-CE05-0003-01"]}],"id":[{"id":"10.13039\/501100001665","id-type":"DOI","asserted-by":"publisher"}]},{"name":"TIMC-Grant Emergence","award":["AO 2017"],"award-info":[{"award-number":["AO 2017"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA\/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA\/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA\/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA\/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.<\/jats:p>","DOI":"10.3390\/ijms21155496","type":"journal-article","created":{"date-parts":[[2020,8,3]],"date-time":"2020-08-03T06:16:47Z","timestamp":1596435407000},"page":"5496","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins"],"prefix":"10.3390","volume":"21","author":[{"given":"Claire","family":"Siebert","sequence":"first","affiliation":[{"name":"TheRex Team, TIMC-IMAG, CNRS, INP, Universit\u00e9 Grenoble Alpes, 38000 Grenoble, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4118-1904","authenticated-orcid":false,"given":"Corinne","family":"Mercier","sequence":"additional","affiliation":[{"name":"GEM Team, TIMC-IMAG, CNRS, INP, Universit\u00e9 Grenoble Alpes, 38000 Grenoble, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5913-2372","authenticated-orcid":false,"given":"Donald K.","family":"Martin","sequence":"additional","affiliation":[{"name":"SyNaBi Team, TIMC-IMAG, CNRS, INP, Universit\u00e9 Grenoble Alpes, 38000 Grenoble, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Patricia","family":"Renesto","sequence":"additional","affiliation":[{"name":"TheRex Team, TIMC-IMAG, CNRS, INP, Universit\u00e9 Grenoble Alpes, 38000 Grenoble, France"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3008-7309","authenticated-orcid":false,"given":"Beatrice","family":"Schaack","sequence":"additional","affiliation":[{"name":"TheRex Team, TIMC-IMAG, CNRS, INP, Universit\u00e9 Grenoble Alpes, 38000 Grenoble, France"},{"name":"CEA, IBS, CNRS, Universit\u00e9 Grenoble Alpes, 38044 Grenoble, France"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,7,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"967","DOI":"10.1038\/nrmicro1045","article-title":"Tularaemia: Bioterrorism defence renews interest in Francisella tularensis","volume":"2","author":"Oyston","year":"2004","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"415","DOI":"10.4049\/jimmunol.87.4.415","article-title":"Prophylactic effectiveness of live and killed tularemia vaccines. I. Production of vaccine and evaluation in the white mouse and guinea pig","volume":"87","author":"Eigelsbach","year":"1961","journal-title":"J. Immunol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"D48","DOI":"10.1016\/j.vaccine.2009.07.090","article-title":"Francisella tularensis vaccines","volume":"27","author":"Oyston","year":"2009","journal-title":"Vaccine"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"8345","DOI":"10.1128\/IAI.73.12.8345-8352.2005","article-title":"A mutant of Francisella tularensis strain SCHU S4 lacking the ability to express a 58-kilodalton protein is attenuated for virulence and is an effective live vaccine","volume":"73","author":"Twine","year":"2005","journal-title":"Infect. Immun."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"3424","DOI":"10.1128\/IAI.00196-09","article-title":"Reintroduction of two deleted virulence loci restores full virulence to the live vaccine strain of Francisella tularensis","volume":"77","author":"Salomonsson","year":"2009","journal-title":"Infect. Immun."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"4429","DOI":"10.1128\/IAI.00702-09","article-title":"The 58-kilodalton major virulence factor of Francisella tularensis is required for efficient utilization of iron","volume":"77","author":"Lindgren","year":"2009","journal-title":"Infect. Immun."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"25191","DOI":"10.1074\/jbc.M112.371856","article-title":"Paralogous outer membrane proteins mediate uptake of different forms of iron and synergistically govern virulence in Francisella tularensis tularensis","volume":"287","author":"Ramakrishnan","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"4276","DOI":"10.1128\/IAI.00503-10","article-title":"The fslE homolog, FTL-0439 (fupA\/B), mediates siderophore-dependent iron uptake in Francisella tularensis LVS","volume":"78","author":"Sen","year":"2010","journal-title":"Infect. Immun."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"808","DOI":"10.1080\/22221751.2019.1615848","article-title":"Francisella tularensis: FupA mutation contributes to fluoroquinolone resistance by increasing vesicle secretion and biofilm formation","volume":"8","author":"Siebert","year":"2019","journal-title":"Emerg. Microbes Infect."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"107","DOI":"10.3389\/fcimb.2017.00107","article-title":"Iron and Virulence in Francisella tularensis","volume":"7","author":"Ramakrishnan","year":"2017","journal-title":"Front. Cell. Infect. Microbiol."},{"key":"ref_11","unstructured":"(2020, July 30). BLAST Proteins, Available online: https:\/\/blast.ncbi.nlm.nih.gov\/Blast.cgi?PAGE=Proteins."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1038\/nrg3456","article-title":"Functional and evolutionary implications of gene orthology","volume":"14","author":"Koonin","year":"2013","journal-title":"Nat. Rev. Genet."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1099\/mic.0.072835-0","article-title":"The FupA\/B protein uniquely facilitates transport of ferrous iron and siderophore-associated ferric iron across the outer membrane of Francisella tularensis live vaccine strain","volume":"160","author":"Ramakrishnan","year":"2014","journal-title":"Microbiology"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"453","DOI":"10.1002\/mbo3.342","article-title":"Two parallel pathways for ferric and ferrous iron acquisition support growth and virulence of the intracellular pathogen Francisella tularensis Schu S4","volume":"5","author":"Johnson","year":"2016","journal-title":"Microbiologyopen"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1146\/annurev-micro-102215-095308","article-title":"The Power of Asymmetry: Architecture and Assembly of the Gram-Negative Outer Membrane Lipid Bilayer","volume":"70","author":"Henderson","year":"2016","journal-title":"Annu. Rev. Microbiol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"845","DOI":"10.1038\/nprot.2015.053","article-title":"The Phyre2 web portal for protein modeling, prediction and analysis","volume":"10","author":"Kelley","year":"2015","journal-title":"Nat. Protoc."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"21614","DOI":"10.1073\/pnas.0907315106","article-title":"Simulations of anion transport through OprP reveal the molecular basis for high affinity and selectivity for phosphate","volume":"106","author":"Pongprayoon","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1016\/j.pep.2018.01.006","article-title":"Functional characterization of cell-free expressed Kv1.3 channel using a voltage-sensitive fluorescent dye","volume":"145","author":"Cortes","year":"2018","journal-title":"Protein Expr. Purif."},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Crouch, C.H., Bost, M.H., Kim, T.H., Green, B.M., Arbuckle, D.S., Grossman, C.H., and Howard, K.P. (2018). Optimization of detergent-mediated reconstitution of influenza A M2 protein into proteoliposomes. Membranes, 8.","DOI":"10.3390\/membranes8040103"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/1472-6750-5-11","article-title":"Liposome retention in size exclusion chromatography","volume":"5","author":"Ruysschaert","year":"2005","journal-title":"BMC Biotechnol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/0031-6865(94)90009-4","article-title":"Separation of liposomes by a gel filtration chromatographic technique: A preliminary evaluation","volume":"69","author":"Vemuri","year":"1994","journal-title":"Pharm. Acta Helv."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"9988","DOI":"10.1021\/acs.langmuir.7b01731","article-title":"Functional Characterization of Cell-Free Expressed OprF Porin from Pseudomonas aeruginosa Stably Incorporated in Tethered Lipid Bilayers","volume":"33","author":"Maccarini","year":"2017","journal-title":"Langmuir"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1038\/nprot.2007.519","article-title":"Membrane reconstitution of ABC transporters and assays of translocator function","volume":"3","author":"Geertsma","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1590\/S0100-879X2002000700001","article-title":"Membrane proteins: Functional and structural studies using reconstituted proteoliposomes and 2-D crystals","volume":"35","author":"Rigaud","year":"2002","journal-title":"Braz. J. Med. Biol. Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1016\/bs.mie.2014.12.028","article-title":"Lipid reconstitution and recording of recombinant ion channels","volume":"556","author":"Stockbridge","year":"2015","journal-title":"Methods Enzymol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"32585","DOI":"10.1074\/jbc.M113.508838","article-title":"Characterization of a membrane-active peptide from the bordetella pertussis CyaA toxin","volume":"288","author":"Subrini","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1016\/j.bpj.2013.11.1121","article-title":"Transient potential gradients and impedance measures of tethered bilayer lipid membranes: Pore-forming peptide insertion and the effect of electroporation","volume":"106","author":"Cranfield","year":"2014","journal-title":"Biophys J."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/15\/5496\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T09:53:27Z","timestamp":1760176407000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/21\/15\/5496"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,7,31]]},"references-count":27,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2020,8]]}},"alternative-id":["ijms21155496"],"URL":"https:\/\/doi.org\/10.3390\/ijms21155496","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,7,31]]}}}