{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,16]],"date-time":"2026-03-16T11:41:14Z","timestamp":1773661274565,"version":"3.50.1"},"reference-count":36,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2006,6,15]],"date-time":"2006-06-15T00:00:00Z","timestamp":1150329600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["J of Molecular Recognition"],"published-print":{"date-parts":[[2006,7]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p><jats:italic>Bacillus anthracis<\/jats:italic> has recently been shown to secrete a potently hemolytic\/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane\u2014binding properties of this protein. Recombinant anthrolysin O (rALO<jats:sub>35\u2013512<\/jats:sub>) and two N\u2010terminally truncated versions of ALO (rALO<jats:sub>390\u2013512<\/jats:sub> and rALO<jats:sub>403\u2013512<\/jats:sub>) from <jats:italic>B. anthracis<\/jats:italic> were overproduced in <jats:italic>Escherichia coli<\/jats:italic> and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage\u2010like lines, and also <jats:italic>Drosophila<\/jats:italic> Schneider 2 cells. Challenging the macrophage cells with rALO<jats:sub>35\u2013512<\/jats:sub>, but not rALO<jats:sub>390\u2013512<\/jats:sub> or rALO<jats:sub>403\u2013512<\/jats:sub>, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. <jats:italic>Drosophila<\/jats:italic> cells, which contain ergosterol as their major membrane sterol, were resistant to rALO\u2010mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO<jats:sub>35\u2013512<\/jats:sub> and rALO<jats:sub>403\u2013512<\/jats:sub> demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO<jats:sub>35\u2013512<\/jats:sub> and rALO<jats:sub>403\u2013512<\/jats:sub>, whereas other sterols tested did not support binding. The rALO<jats:sub>403\u2013512<\/jats:sub>\u2014membrane interaction demonstrated an equilibrium dissociation constant (<jats:italic>K<\/jats:italic><jats:sub>D<\/jats:sub>) in the low nanomolar range, whereas rALO<jats:sub>35\u2013512<\/jats:sub> exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane\u2010binding proteins and peptides. Copyright\u00a9 2006 John Wiley &amp; Sons, Ltd.<\/jats:p>","DOI":"10.1002\/jmr.784","type":"journal-article","created":{"date-parts":[[2006,6,15]],"date-time":"2006-06-15T12:42:38Z","timestamp":1150375358000},"page":"354-362","source":"Crossref","is-referenced-by-count":15,"title":["Real\u2010time monitoring of the membrane\u2010binding and insertion properties of the cholesterol\u2010dependent cytolysin anthrolysin O from <i>Bacillus anthracis<\/i>"],"prefix":"10.1002","volume":"19","author":[{"given":"Simon","family":"Cocklin","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monika","family":"Jost","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Noreen M","family":"Robertson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Stephen D","family":"Weeks","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hans\u2010Walter","family":"Weber","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Emily","family":"Young","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samar","family":"Seal","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Can","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elise","family":"Mosser","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick J","family":"Loll","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleister J","family":"Saunders","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard F","family":"Rest","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Irwin M","family":"Chaiken","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2006,6,15]]},"reference":[{"key":"e_1_2_1_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ab.2005.06.013"},{"key":"e_1_2_1_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2004.04.118"},{"key":"e_1_2_1_4_1","doi-asserted-by":"publisher","DOI":"10.1016\/B978-0-12-152818-8.50022-0"},{"key":"e_1_2_1_5_1","doi-asserted-by":"publisher","DOI":"10.3109\/10409238309102790"},{"key":"e_1_2_1_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/0005-2736(80)90475-7"},{"key":"e_1_2_1_7_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0005-2736(98)00091-1"},{"key":"e_1_2_1_8_1","doi-asserted-by":"publisher","DOI":"10.1006\/abio.1999.4389"},{"key":"e_1_2_1_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2005.02.003"},{"key":"e_1_2_1_10_1","doi-asserted-by":"publisher","DOI":"10.3201\/eid1006.020738"},{"key":"e_1_2_1_11_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2033520100"},{"key":"e_1_2_1_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/nsmb862"},{"key":"e_1_2_1_13_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00018-002-8471-1"},{"key":"e_1_2_1_14_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.str.2005.04.019"},{"key":"e_1_2_1_15_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M303151200"},{"key":"e_1_2_1_16_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M009865200"},{"key":"e_1_2_1_17_1","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111039200"},{"key":"e_1_2_1_18_1","doi-asserted-by":"publisher","DOI":"10.1038\/nature03923"},{"key":"e_1_2_1_19_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF02532453"},{"key":"e_1_2_1_20_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1286-4579(99)80004-5"},{"key":"e_1_2_1_21_1","doi-asserted-by":"publisher","DOI":"10.1023\/B:JSFG.0000029237.70316.52"},{"key":"e_1_2_1_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-3495(95)80014-7"},{"key":"e_1_2_1_23_1","doi-asserted-by":"publisher","DOI":"10.1021\/bi00368a032"},{"key":"e_1_2_1_24_1","doi-asserted-by":"publisher","DOI":"10.1084\/jem.20041215"},{"key":"e_1_2_1_25_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0403229101"},{"key":"e_1_2_1_26_1","doi-asserted-by":"publisher","DOI":"10.1097\/00063198-200305000-00011"},{"issue":"11","key":"e_1_2_1_27_1","first-page":"823","article-title":"Structural insights into the membrane\u2010anchoring mechanism of a cholesterol\u2010dependent cytolysin","volume":"9","author":"Ramachandran R","year":"2002","journal-title":"Nat. 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