Abstract
BackgroundCell-surface receptors play essential roles in anthrax toxin action by providing the toxin with a high-affinity anchor and self-assembly site on the plasma membrane, mediating the toxin entry into cells through endocytosis, and shifting the pH threshold for prepore-to-pore conversion of anthrax toxin protective antigen (PA) to a more acidic pH, thereby inhibiting premature pore formation. Each of the two known anthrax toxin receptors, ANTXR1 and ANTXR2, has an ectodomain comprised of an N-terminal von Willebrand factor A domain (VWA), which binds PA, and an uncharacterized immunoglobulin-like domain (Ig) that connects VWA to the membrane-spanning domain. Potential roles of the receptor Ig domain in anthrax toxin action have not been investigated heretofore.Methodology/Principal FindingsWe expressed and purified the ANTXR2 ectodomain (R2-VWA-Ig) in E. coli and showed that it contains three disulfide bonds: one in R2-VWA and two in R2-Ig. Reduction of the ectodomain inhibited functioning of the pore, as measured by K+ release from liposomes or Chinese hamster ovary cells or by PA-mediated translocation of a model substrate across the plasma membrane. However, reduction did not affect binding of the ectodomain to PA or the transition of ectodomain-bound PA prepore to the pore conformation. The inhibitory effect depended specifically on reduction of the disulfides within R2-Ig.Conclusions/SignificanceWe conclude that disulfide integrity within R2-Ig is essential for proper functioning of receptor-bound PA pore. This finding provides a novel venue to investigate the mechanism of anthrax toxin action and suggests new strategies for inhibiting toxin action.
Highlights
A common way for pathogenic bacteria to defend themselves against the host’s immune system is to deliver a toxin into the cytoplasm of host cells and disrupt key steps of metabolism
ANTXR2 provides the toxin with a highaffinity anchor and self-assembly site on the plasma membrane and guides its entry into cells; and further the receptor functions as a molecular clamp that shifts the pH threshold of prepore-to-pore conversion to a more acidic pH, thereby preventing premature pore formation before the toxin reaches a suitable compartment
This finding suggests that anthrax toxin action is sensitive to redox conditions and raises the possibility that toxin action may be modulated by reductants to which it is exposed during endocytosis and intracellular trafficking
Summary
A common way for pathogenic bacteria to defend themselves against the host’s immune system is to deliver a toxin into the cytoplasm of host cells and disrupt key steps of metabolism. PA (83 kDa) binds to cell-surface receptors and is cleaved by furin or a furin-like protease to generate an active, 63-kDa form (PA63) [2]. The toxin-receptor complexes are internalized by receptor-mediated endocytosis, and the prepore moiety undergoes an acidic pH-dependent conformational rearrangement within the endosome to form a cation-selective, transmembrane pore [5]. Cell-surface receptors play essential roles in anthrax toxin action by providing the toxin with a high-affinity anchor and self-assembly site on the plasma membrane, mediating the toxin entry into cells through endocytosis, and shifting the pH threshold for prepore-to-pore conversion of anthrax toxin protective antigen (PA) to a more acidic pH, thereby inhibiting premature pore formation. Potential roles of the receptor Ig domain in anthrax toxin action have not been investigated heretofore
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