{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,8]],"date-time":"2026-10-08T16:15:46Z","timestamp":1791476146763,"version":"4.3.4"},"reference-count":36,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2021,12,6]],"date-time":"2021-12-06T00:00:00Z","timestamp":1638748800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["SFB807\/P18"],"award-info":[{"award-number":["SFB807\/P18"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Antibiotics"],"abstract":"<jats:p>Gram-negative Tripartite Resistance Nodulation and cell Division (RND) superfamily efflux pumps confer various functions, including multidrug and bile salt resistance, quorum-sensing, virulence and can influence the rate of mutations on the chromosome. Multidrug RND efflux systems are often characterized by a wide substrate specificity. Similarly to many other RND efflux pump systems, AcrAD-TolC confers resistance toward SDS, novobiocin and deoxycholate. In contrast to the other pumps, however, it in addition confers resistance against aminoglycosides and dianionic \u03b2-lactams, such as sulbenicillin, aztreonam and carbenicillin. Here, we could show that AcrD from Salmonella typhimurium confers resistance toward several hitherto unreported AcrD substrates such as temocillin, dicloxacillin, cefazolin and fusidic acid. In order to address the molecular determinants of the S. typhimurium AcrD substrate specificity, we conducted substitution analyses in the putative access and deep binding pockets and in the TM1\/TM2 groove region. The variants were tested in E. coli \u0394acrB\u0394acrD against \u03b2-lactams oxacillin, carbenicillin, aztreonam and temocillin. Deep binding pocket variants N136A, D276A and Y327A; access pocket variant R625A; and variants with substitutions in the groove region between TM1 and TM2 conferred a sensitive phenotype and might, therefore, be involved in anionic \u03b2-lactam export. In contrast, lower susceptibilities were observed for E. coli cells harbouring deep binding pocket variants T139A, D176A, S180A, F609A, T611A and F627A and the TM1\/TM2 groove variant I337A. This study provides the first insights of side chains involved in drug binding and transport for AcrD from S. typhimurium.<\/jats:p>","DOI":"10.3390\/antibiotics10121494","type":"journal-article","created":{"date-parts":[[2021,12,6]],"date-time":"2021-12-06T22:18:42Z","timestamp":1638829122000},"page":"1494","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Characterization and Molecular Determinants for \u03b2-Lactam Specificity of the Multidrug Efflux Pump AcrD from Salmonella typhimurium"],"prefix":"10.3390","volume":"10","author":[{"given":"Jenifer","family":"Cuesta Bernal","sequence":"first","affiliation":[{"name":"Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jasmin","family":"El-Delik","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephan","family":"G\u00f6ttig","sequence":"additional","affiliation":[{"name":"Institute of Medical Microbiology and Infection Control, Hospital of the Goethe University, Paul-Ehrlich-Stra\u00dfe 40, D-60596 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9035-3827","authenticated-orcid":false,"given":"Klaas M.","family":"Pos","sequence":"additional","affiliation":[{"name":"Institute of Biochemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,12,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1555","DOI":"10.2165\/11317030-000000000-00000","article-title":"Efflux-mediated drug resistance in bacteria: An update","volume":"69","author":"Li","year":"2009","journal-title":"Drugs"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1128\/CMR.00117-14","article-title":"The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria","volume":"28","author":"Li","year":"2015","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"143","DOI":"10.2217\/fmb-2019-0235","article-title":"RND efflux pumps in Gram-negative bacteria; regulation, structure and role in antibiotic resistance","volume":"15","author":"Colclough","year":"2020","journal-title":"Future Microbiol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"5479","DOI":"10.1021\/acs.chemrev.1c00055","article-title":"Structure, assembly, and function of tripartite efflux and type 1 secretion systems in gram-negative bacteria","volume":"121","author":"Alav","year":"2021","journal-title":"Chem. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2003","DOI":"10.1093\/jac\/dky042","article-title":"Role of bacterial efflux pumps in biofilm formation","volume":"73","author":"Alav","year":"2018","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"7376","DOI":"10.1128\/AEM.01310-08","article-title":"Inactivation of efflux pumps abolishes bacterial biofilm formation","volume":"74","author":"Kvist","year":"2008","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2386","DOI":"10.1073\/pnas.0502890102","article-title":"Quorum sensing and multidrug transporters in Escherichia coli","volume":"103","author":"Yang","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2512","DOI":"10.1128\/jb.179.8.2512-2518.1997","article-title":"Active efflux of bile salts by Escherichia coli","volume":"179","author":"Thanassi","year":"1997","journal-title":"J. Bacteriol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"e00968-17","DOI":"10.1128\/mBio.00968-17","article-title":"Lack of AcrB efflux function confers loss of virulence on Salmonella enterica Serovar Typhimurium","volume":"8","author":"Blair","year":"2017","journal-title":"mBio"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1126\/science.aar7981","article-title":"Heterogeneity in efflux pump expression predisposes antibiotic-resistant cells to mutation","volume":"362","author":"Dunlop","year":"2018","journal-title":"Science"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"363","DOI":"10.1016\/j.resmic.2018.03.001","article-title":"RND transporters in the living world","volume":"169","author":"Nikaido","year":"2018","journal-title":"Res. Microbiol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"D320","DOI":"10.1093\/nar\/gkw1068","article-title":"TransportDB 2.0: A database for exploring membrane transporters in sequenced genomes from all domains of life","volume":"45","author":"Elbourne","year":"2017","journal-title":"Nucleic Acids Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"5378","DOI":"10.1021\/acs.chemrev.0c00621","article-title":"Structural and functional diversity of resistance-nodulation-cell division transporters","volume":"121","author":"Klenotic","year":"2021","journal-title":"Chem. Rev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1111\/j.1365-2958.2005.04940.x","article-title":"Virulence and drug resistance roles of multidrug efflux systems of Salmonella enterica serovar Typhimurium","volume":"59","author":"Nishino","year":"2006","journal-title":"Mol. Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1038\/35101614","article-title":"Complete genome sequence of Salmonella enterica serovar Typhimurium LT2","volume":"413","author":"McClelland","year":"2001","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1145","DOI":"10.1128\/AAC.48.4.1145-1150.2004","article-title":"Expression of acrB, acrF, acrD, marA, and soxS in Salmonella enterica serovar Typhimurium: Role in multiple antibiotic resistance","volume":"48","author":"Eaves","year":"2004","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"e01916-16","DOI":"10.1128\/mBio.01916-16","article-title":"Beyond antimicrobial resistance: Evidence for a distinct role of the AcrD Efflux pump in Salmonella biology","volume":"7","author":"Buckner","year":"2016","journal-title":"mBio"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"382","DOI":"10.1128\/CMR.19.2.382-402.2006","article-title":"Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria","volume":"19","author":"Piddock","year":"2006","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1093\/jac\/dku380","article-title":"Expression of homologous RND efflux pump genes is dependent upon AcrB expression: Implications for efflux and virulence inhibitor design","volume":"70","author":"Blair","year":"2015","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"433","DOI":"10.1038\/ja.2011.28","article-title":"AcrA dependency of the AcrD efflux pump in Salmonella enterica serovar Typhimurium","volume":"64","author":"Yamasaki","year":"2011","journal-title":"J. Antibiot."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"10680","DOI":"10.1074\/jbc.M114.547794","article-title":"beta-Lactam selectivity of multidrug transporters AcrB and AcrD resides in the proximal binding pocket","volume":"289","author":"Kobayashi","year":"2014","journal-title":"J. Biol. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1923","DOI":"10.1128\/JB.187.6.1923-1929.2005","article-title":"Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli","volume":"187","author":"Aires","year":"2005","journal-title":"J. Bacteriol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1754","DOI":"10.1128\/JB.182.6.1754-1756.2000","article-title":"AcrD of Escherichia coli is an aminoglycoside efflux pump","volume":"182","author":"Rosenberg","year":"2000","journal-title":"J. Bacteriol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"8075","DOI":"10.1038\/s41598-017-08747-8","article-title":"Molecular rationale behind the differential substrate specificity of bacterial RND multi-drug transporters","volume":"7","author":"Ramaswamy","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"13819","DOI":"10.1038\/ncomms13819","article-title":"Transport of lipophilic carboxylates is mediated by transmembrane helix 2 in multidrug transporter AcrB","volume":"7","author":"Oswald","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.1111\/j.1348-0421.2007.tb04001.x","article-title":"Molecular cloning and characterization of all RND-type efflux transporters in Vibrio cholerae non-O1","volume":"51","author":"Rahman","year":"2007","journal-title":"Microbiol. Immunol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1038\/nprot.2007.521","article-title":"Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances","volume":"3","author":"Wiegand","year":"2008","journal-title":"Nat. Protoc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"8225","DOI":"10.1128\/JB.00912-08","article-title":"Site-directed mutagenesis reveals putative substrate binding residues in the Escherichia coli RND efflux pump AcrB","volume":"190","author":"Bohnert","year":"2008","journal-title":"J. Bacteriol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"5687","DOI":"10.1073\/pnas.1114944109","article-title":"Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop","volume":"109","author":"Eicher","year":"2012","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Lu, W., Zhong, M., Chai, Q., Wang, Z., Yu, L., and Wei, Y. (2014). Functional relevance of AcrB Trimerization in pump assembly and substrate binding. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0089143"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"3889","DOI":"10.1038\/s41467-021-24151-3","article-title":"Allosteric drug transport mechanism of multidrug transporter AcrB","volume":"12","author":"Tam","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5722","DOI":"10.1073\/pnas.0802190105","article-title":"Quality control of overexpressed membrane proteins","volume":"105","author":"Geertsma","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3079","DOI":"10.2174\/15680266113136660220","article-title":"RND efflux pumps: Structural information translated into function and inhibition mechanisms","volume":"13","author":"Ruggerone","year":"2013","journal-title":"Curr. Top. Med. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"4686","DOI":"10.1128\/JB.180.17.4686-4692.1998","article-title":"Multidrug efflux pump AcrAB of Salmonella typhimurium excretes only those beta-lactam antibiotics containing lipophilic side chains","volume":"180","author":"Nikaido","year":"1998","journal-title":"J. Bacteriol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4767","DOI":"10.1128\/AAC.02733-13","article-title":"Switch-loop flexibility affects transport of large drugs by the promiscuous AcrB multidrug efflux transporter","volume":"58","author":"Cha","year":"2014","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1038\/nature10641","article-title":"Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket","volume":"480","author":"Nakashima","year":"2011","journal-title":"Nature"}],"container-title":["Antibiotics"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-6382\/10\/12\/1494\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T07:40:10Z","timestamp":1760168410000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-6382\/10\/12\/1494"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,12,6]]},"references-count":36,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2021,12]]}},"alternative-id":["antibiotics10121494"],"URL":"https:\/\/doi.org\/10.3390\/antibiotics10121494","relation":{},"ISSN":["2079-6382"],"issn-type":[{"value":"2079-6382","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,12,6]]}}}