title: Purified Bacillus anthracis lethal toxin complex formed in vitro and during infection exhibits functional and biological activity.
abstract: Anthrax protective antigen (PA, 83 kDa), a pore-forming protein, upon protease activation to 63 kDa (PA(63)), translocates lethal factor (LF) and edema factor (EF) from endosomes into the cytosol of the cell. The relatively small size of the heptameric PA(63) pore (approximately 12 angstroms) raises questions as to how large molecules such as LF and EF can move through the pore. In addition, the reported high binding affinity between PA and EF/LF suggests that EF/LF may not dissociate but remain complexed with activated PA(63). In this study, we found that purified (PA(63))(7)-LF complex exhibited biological and functional activities similar to the free LF. Purified LF complexed with PA(63) heptamer was able to cleave both a synthetic peptide substrate and endogenous mitogen-activated protein kinase kinase substrates and kill susceptible macrophage cells. Electrophysiological studies of the complex showed strong rectification of the ionic current at positive voltages, an effect similar to that observed if LF is added to the channels formed by heptameric PA(63) pore. Complexes of (PA(63))(7)-LF found in the plasma of infected animals showed functional activity. Identifying active complex in the blood of infected animals has important implications for therapeutic design, especially those directed against PA and LF. Our studies suggest that the individual toxin components and the complex must be considered as critical targets for anthrax therapeutics.
authors.affiliation: Developmental Therapeutics Program, Target Structure-based Drug Discovery Group, NCI SAIC-Frederick, National Institutes of Health, Frederick, Maryland 21702, USA. rekha.panchal@amedd.army.mil
authors.forename: Rekha G
authors.initials: RG
authors.lastname: Panchal
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Halverson
authors.forename: Wilson
authors.initials: W
authors.lastname: Ribot
authors.forename: Douglas
authors.initials: D
authors.lastname: Lane
authors.forename: Tara
authors.initials: T
authors.lastname: Kenny
authors.forename: Teresa G
authors.initials: TG
authors.lastname: Abshire
authors.forename: John W
authors.initials: JW
authors.lastname: Ezzell
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Hoover
authors.forename: Bradford
authors.initials: B
authors.lastname: Powell
authors.forename: Stephen
authors.initials: S
authors.lastname: Little
authors.forename: John J
authors.initials: JJ
authors.lastname: Kasianowicz
authors.forename: Sina
authors.initials: S
authors.lastname: Bavari
mesh_terms: D000818:Animals; D000881:Anthrax / Q000378:metabolism*; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D001408:Bacillus anthracis / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism*; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D004594:Electrophysiology; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers; D020929:Mitogen-Activated Protein Kinase Kinases / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011817:Rabbits; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D008051:Lipid Bilayers; D010455:Peptides; C030325:anthrax toxin; D020929:Mitogen-Activated Protein Kinase Kinases