title: The staphylococcal leukocidin bicomponent toxin forms large ionic channels.
abstract: The genes encoding the F and S components of a leukocidin, LukF (HlgB) and LukS (HlgC), a pore-forming binary toxin, were amplified from the Smith 5R strain of Staphylococcus aureus both with and without sequences encoding 3'-hexahistidine tags. The His-tagged components were expressed in Escherichia coli and purified under nondenaturing conditions. In addition, the two unmodified proteins and the His-tagged versions were produced in an E. coli cell-free in vitro transcription and translation system. An SDS-stable oligomer of approximately 200 kDa appeared when both components were cotranslated in the presence of rabbit erythrocyte membranes. Hemolytic activity of the combined components against rabbit erythrocytes was measured for both in vitro- and in vivo-produced polypeptides, yielding similar HC(50) values of approximately 0.14 microg/mL. The pore-forming properties of the recombinant leukocidin were also investigated with planar lipid bilayers of diphytanoylphosphatidylcholine. Although leukocidins and staphylococcal alpha-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS [1 M KCl and 5 mM HEPES (pH 7.4)], which is more than 3 times greater than that of alpha-hemolysin measured under the same conditions. Therefore, if the leukocidin pore were a cylinder, its diameter would be almost twice that of alpha-hemolysin. In addition, the leukocidin pore is weakly cation selective and exhibits gating at low positive potentials, while alpha-hemolysin is weakly anion selective and gates only at high potentials. Taken together, these data suggest that the structure of the oligomeric pore formed by the leukocidin examined here has diverged significantly from that of alpha-hemolysin.
authors.affiliation: Department of Medical Biochemistry and Genetics, The Texas A&M University System Health Science Center, College Station, Texas 77843-1114, USA.
authors.forename: G
authors.initials: G
authors.lastname: Miles
authors.forename: S
authors.initials: S
authors.lastname: Cheley
authors.forename: O
authors.initials: O
authors.lastname: Braha
authors.forename: H
authors.initials: H
authors.lastname: Bayley
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D002458:Cell Fractionation; D003001:Cloning, Molecular; D004910:Erythrocyte Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007473:Ion Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007956:Leukocidins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008051:Lipid Bilayers / Q000378:metabolism; D008564:Membrane Potentials / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007473:Ion Channels; D007956:Leukocidins; D008051:Lipid Bilayers; D011994:Recombinant Proteins; C108483:leukocidin S-component protein, Staphylococcus; C074300:LukF protein, Staphylococcus aureus