Cross-linked forms of the isolated N-terminal domain of the lethal factor are potent inhibitors of anthrax toxin.
The proteins that comprise anthrax toxin self-assemble at the mammalian cell surface into a series of toxic complexes, each containing a heptameric form of protective antigen (PA) plus up to a total of three molecules of the enzymatic moieties of the toxin (lethal factor [LF] and edema factor [EF]). These complexes are trafficked to the endosome, where the PA heptamer forms a pore in the membrane under the influence of low pH, and bound LF and EF unfold and translocate through the pore to the cytosol. To explore the hypothesis that the PA pore can translocate multiple, cross-linked polypeptides simultaneously, we cross-linked LF(N), the N-terminal domain of LF, via an introduced cysteine at its N or C terminus and characterized the products. Both dimers and trimers of LF(N) retained the ability to bind to PA pores and block ion conductance, but they were unable to translocate across the membrane, even at high voltages or with a transmembrane pH gradient. The multimers were remarkably potent inhibitors of toxin action in mammalian cells (20- to 50-fold more potent than monomeric LF(N)) and in a zebrafish model system. These findings show that the PA pore cannot translocate multimeric, cross-linked polypeptides and demonstrate a new approach to generating potent inhibitors of anthrax toxin.
Juris
Stephen J
SJ
Harvard Medical School, Department of Microbiology and Molecular Genetics, 200 Longwood Avenue, Boston, MA 02115, USA.
Melnyk
Roman A
RA
Bolcome
Robert E
RE
Chan
Joanne
J
Collier
R John
RJ
D000818:Animals; D000881:Anthrax / Q000517:prevention & control; D000942:Antigens, Bacterial / Q000378:metabolism; D000992:Antitoxins / Q000737:chemistry* / Q000494:pharmacology*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005393:Fish Diseases / Q000517:prevention & control; D017434:Protein Structure, Tertiary; D015027:Zebrafish
D000942:Antigens, Bacterial; D000992:Antitoxins; D001427:Bacterial Toxins; C030325:anthrax toxin