Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis y Simon Cocklin 1 , Monika Jost 2 , Noreen M Robertson 1 , Stephen D Weeks 1 , Hans-Walter Weber 2 , Emily Young 1 , Samar Seal 3 , Can Zhang 4 , Elise Mosser 3 , Patrick J Loll 1 , Aleister J Saunders 4 , Richard F Rest 3 and Irwin M Chaiken 1 * 1 Department of Biochemistry and A. J. Drexel Institute of Basic and Applied Protein Science, Drexel University College of Medicine, 11102 New College Building, MS# 497, 245 N. 15th Street, Philadelphia, Pennsylvania 19102, USA 2 Department of Radiation Oncology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, Pennsylvania 19102, USA 3 Department of Microbiology and Immunology, Drexel University College of Medicine, 2900 Queen Lane, Philadelphia, Pennsylvania 19129, USA 4 Department of Bioscience and Biotechnology, 3141 Chestnut Street, Drexel University, Philadelphia, Pennsylvania 19104, USA Bacillus anthracis 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—binding properties of this protein. Recombinant anthrolysin O (rALO 35–512 ) and two N-terminally truncated versions of ALO (rALO 390–512 and rALO 403–512 ) from B. anthracis were overproduced in Escherichia coli 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-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO 35–512 , but not rALO 390–512 or rALO 403–512 , resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated 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 35–512 and rALO 403–512 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 35–512 and rALO 403–512 , whereas other sterols tested did not support binding. The rALO 403–512 —membrane interaction demonstrated an equilibrium dissociation constant (K D ) in the low nanomolar range, whereas rALO 35–512 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-binding proteins and peptides. Copyright# 2006 John Wiley & Sons, Ltd. Keywords: surface plasmon resonance; membrane; cholesterol dependent cytolysin; pore formation; kinetic assay Received 28 October 2005; revised 10 March 2006; accepted 12 March 2006 INTRODUCTION Anthrax is an acute infectious disease, caused by the spore- forming bacterium Bacillus anthracis, that has reemerged as a threat due to the fact that Bacillus spores can be produced and distributed as biological weapons of warfare or terrorism (Quintiliani and Quintiliani, 2003; Fennelly et al., 2004). Throughout the course of infection, B. anthracis is capable of avoiding a variety of antibacterial host factors while proceeding through different compartments and environ- mental conditions in the host organism (Little and Ivins, 1999). Bacillus species secrete large amounts of proteins that are important for their adaptation and survival. Among these secreted proteins in B. anthracis are the main virulence factors lethal toxin and edema toxin. In addition, the genome of B. anthracis contains regions that code for putative proteins similar to known membrane damaging proteins. Although B. anthracis is generally thought of as non- hemolytic, a recent study by Shannon et al. (2003) revealed that B. anthracis produces biologically relevant amounts of a hemolysin in culture supernatants, whose expression is dependent on growth conditions, growth phase, and the type JOURNAL OF MOLECULAR RECOGNITION J. Mol. Recognit. (in press) Published online in Wiley InterScience (www.interscience.wiley.com) DOI:10.1002/jmr.784 *Correspondence to: I. M. Chaiken, Drexel University College of Medicine, 11313 New College Building, MS #497, 245 N. 15th St., Philadelphia, PA 19102. E-mail: imc23@drexel.edu Contract/grant sponsors: Pennsylvania Department of Health; National Science Foundation STTR Phase II grant. y This paper is published as part of a special issue entitled ‘‘Bioaffinity 2005, Uppsala, August 14–18, Sweden’’. Copyright # 2006 John Wiley & Sons, Ltd.