ReviewMembrane translocation by anthrax toxin
Introduction
For many disease-causing bacteria a key strategy for survival within mammalian hosts is to deliver selected enzymes (effector proteins) into the cytosol of host cells, primarily with the aim of killing or disabling key cellular elements of the immune system. Delivery of an effector protein requires specialized machinery to enable it to cross a membrane at some level within the host cell, and much attention has been devoted to defining such machinery and understanding how it functions. Some of the simplest and most extensively studied systems for protein translocation are found in intracellularly acting bacterial toxins. Our current understanding of translocation by anthrax toxin, which has proven to be one of the most tractable toxins for studying this process, is summarized here. Readers may also wish to consult other recent reviews relevant to this topic (Collier and Young, 2003, Finkelstein, 2009, Puhar and Montecucco, 2007, Young and Collier, 2007).
Anthrax toxin is an ensemble of three large, multidomain proteins, which are secreted from Bacillus anthracis as monomers and self-assemble on receptor-bearing cells into a series of toxic, hetero-oligomeric complexes (Pimental et al., 2004, Smith, 2000). Two of the proteins are enzymic intracellular effectors: Lethal Factor (LF, 90 kDa), a Zn++-dependent protease (Duesbery et al., 1998, Vitale et al., 1998), and Edema Factor (EF, 89 kDa), a Ca++- and calmodulin-dependent adenylyl cyclase (Leppla, 1982). The third is a receptor binding and pore-forming protein, called Protective Antigen (PA, 83 kDa), which transports EF and LF from the extracellular milieu to the cytosolic compartment of mammalian cells. EF and LF can be transported to the cytosol by PA and act independently of one other, a fact that has given rise to the terms Edema Toxin, EdTx, and Lethal Toxin, LeTx, for the binary combinations, EF + PA and LF + PA, respectively (Ezzell et al., 1984, Friedlander, 1986). However, all three components of the toxin are produced during B. anthracis infections, and can combine to form ternary complexes as well as binary complexes during self-assembly at the cell surface (Pimental et al., 2004). In addition, any given host cell that is affected by EF is almost certainly affected by LF, and vice versa; and there is evidence of synergy between these two effector proteins (Cui et al., 2007, Rossi Paccani et al., 2007, Tournier et al., 2005). Thus, while the terms Edema Toxin and Lethal Toxin are useful in analyzing and describing experimental findings, it is also appropriate to think of the ensemble of PA, EF, and LF as a single system.
Section snippets
Activation, acidic pH, and pore formation
Leppla and co-workers showed that PA must be proteolytically activated in order to interact with LF and EF (Singh et al., 1989). The activation involves cleavage of the native protein into N-terminal 20-kDa and C-terminal 63-kDa fragments (PA20 and PA63, respectively) and may be effected in vivo by cell-associated furin-family proteases (Klimpel et al., 1992) or by proteases in the blood of animals (Ezzell and Abshire, 1992, Moayeri et al., 2007). For research purposes trypsin is commonly used
Systems for study
A cell-based assay developed by Olsnes and co-workers for probing the translocation of diphtheria toxin across the plasma membrane was adapted to anthrax toxin (Falnes et al., 1994, Wesche et al., 1998). In that assay, radiolabeled translocation ligands were bound to proteolytically activated PA at the surface of CHO or L6 cells, and translocation was induced by lowering the pH of the medium. The cells were then treated with Pronase E to degrade exposed label at the cell surface, and
Acknowledgements
Work in the author’s laboratory on anthrax toxin has been supported by NIH Grant AI022021. Some of the proteins used were produced by the Biomolecule Production Core under the New England Regional Center of Excellence under Grant AI057159. The author holds equity in PharmAthene, Inc.
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