{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,6]],"date-time":"2026-03-06T14:43:43Z","timestamp":1772808223720,"version":"3.50.1"},"reference-count":87,"publisher":"American Society for Microbiology","issue":"10","license":[{"start":{"date-parts":[[2017,5,15]],"date-time":"2017-05-15T00:00:00Z","timestamp":1494806400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100000002","name":"HHS | National Institutes of Health","doi-asserted-by":"publisher","award":["R21AI115187"],"award-info":[{"award-number":["R21AI115187"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["J Bacteriol"],"published-print":{"date-parts":[[2017,5,15]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n          <jats:p>\n            Gram-negative bacteria acquire ferric siderophores through TonB-dependent outer membrane transporters (TBDT). By fluorescence spectroscopic hgh-throughput screening (FLHTS), we identified inhibitors of TonB-dependent ferric enterobactin (FeEnt) uptake through\n            <jats:named-content content-type=\"genus-species\">Escherichia coli<\/jats:named-content>\n            FepA (EcoFepA). Among 165 inhibitors found in a primary screen of 17,441 compounds, we evaluated 20 in secondary tests: TonB-dependent ferric siderophore uptake and colicin killing and proton motive force-dependent lactose transport. Six of 20 primary hits inhibited TonB-dependent activity in all tests. Comparison of their effects on [\n            <jats:sup>59<\/jats:sup>\n            Fe]Ent and [\n            <jats:sup>14<\/jats:sup>\n            C]lactose accumulation suggested several as proton ionophores, but two chemicals, ebselen and ST0082990, are likely not proton ionophores and may inhibit TonB-ExbBD. The facility of FLHTS against\n            <jats:named-content content-type=\"genus-species\">E. coli<\/jats:named-content>\n            led us to adapt it to\n            <jats:named-content content-type=\"genus-species\">Acinetobacter baumannii<\/jats:named-content>\n            . We identified its FepA ortholog (AbaFepA), deleted and cloned its structural gene, genetically engineered 8 Cys substitutions in its surface loops, labeled them with fluorescein, and made fluorescence spectroscopic observations of FeEnt uptake in\n            <jats:named-content content-type=\"genus-species\">A. baumannii<\/jats:named-content>\n            . Several Cys substitutions in AbaFepA (S279C, T562C, and S665C) were readily fluoresceinated and then suitable as sensors of FeEnt transport. As in\n            <jats:named-content content-type=\"genus-species\">E. coli<\/jats:named-content>\n            , the test monitored TonB-dependent FeEnt uptake by AbaFepA. In microtiter format with\n            <jats:named-content content-type=\"genus-species\">A. baumannii<\/jats:named-content>\n            , FLHTS produced Z\u2032 factors 0.6 to 0.8. These data validated the FLHTS strategy against even distantly related Gram-negative bacterial pathogens. Overall, it discovered agents that block TonB-dependent transport and showed the potential to find compounds that act against Gram-negative CRE (carbapenem-resistant\n            <jats:named-content content-type=\"genus-species\">Enterobacteriaceae)<\/jats:named-content>\n            \/ESKAPE (\n            <jats:named-content content-type=\"genus-species\">Enterococcus faecium<\/jats:named-content>\n            ,\n            <jats:named-content content-type=\"genus-species\">Staphylococcus aureus<\/jats:named-content>\n            ,\n            <jats:named-content content-type=\"genus-species\">Klebsiella pneumoniae<\/jats:named-content>\n            ,\n            <jats:named-content content-type=\"genus-species\">Acinetobacter baumannii<\/jats:named-content>\n            ,\n            <jats:named-content content-type=\"genus-species\">Pseudomonas aeruginosa<\/jats:named-content>\n            , and\n            <jats:named-content content-type=\"genus-species\">Enterobacter<\/jats:named-content>\n            species) pathogens. Our results suggest that hundreds of such chemicals may exist in larger compound libraries.\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            Antibiotic resistance in Gram-negative bacteria has spurred efforts to find novel compounds against new targets. The CRE\/ESKAPE pathogens are resistant bacteria that include\n            <jats:named-content content-type=\"genus-species\">Acinetobacter baumannii<\/jats:named-content>\n            , a common cause of ventilator-associated pneumonia and sepsis. We performed fluorescence high-throughput screening (FLHTS) against\n            <jats:named-content content-type=\"genus-species\">Escherichia coli<\/jats:named-content>\n            to find inhibitors of TonB-dependent iron transport, tested them against\n            <jats:named-content content-type=\"genus-species\">A. baumannii<\/jats:named-content>\n            , and then adapted the FLHTS technology to allow direct screening against\n            <jats:named-content content-type=\"genus-species\">A. baumannii<\/jats:named-content>\n            . This methodology is expandable to other drug-resistant Gram-negative pathogens. Compounds that block TonB action may interfere with iron acquisition from eukaryotic hosts and thereby constitute bacteriostatic antibiotics that prevent microbial colonization of human and animals. The FLHTS method may identify both species-specific and broad-spectrum agents against Gram-negative bacteria.\n          <\/jats:p>","DOI":"10.1128\/jb.00889-16","type":"journal-article","created":{"date-parts":[[2017,2,28]],"date-time":"2017-02-28T01:40:27Z","timestamp":1488246027000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":24,"title":["Fluorescence High-Throughput Screening for Inhibitors of TonB Action"],"prefix":"10.1128","volume":"199","author":[{"given":"Brittany L.","family":"Nairn","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Olivia S.","family":"Eliasson","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dallas R.","family":"Hyder","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noah J.","family":"Long","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aritri","family":"Majumdar","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Somnath","family":"Chakravorty","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter","family":"McDonald","sequence":"additional","affiliation":[{"name":"Shankel Structural Biology Center, University of Kansas, Lawrence, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anuradha","family":"Roy","sequence":"additional","affiliation":[{"name":"Shankel Structural Biology Center, University of Kansas, Lawrence, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Salete M.","family":"Newton","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Phillip E.","family":"Klebba","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Biophysics, Kansas State University, Manhattan, Kansas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1001\/jama.1975.03240130021018","article-title":"Nutritional immunity","volume":"231","author":"Weinberg ED","year":"1975","unstructured":"Weinberg ED . 1975. 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