Harnessing the Membrane Translocation Properties of AB Toxins for Therapeutic Applications

Abstract

Over the last few decades, proteins and peptides have become increasingly more common as FDA-approved drugs, despite their inefficient delivery due to their inability to cross the plasma membrane. In this context, bacterial two-component systems, termed AB toxins, use various protein-based membrane translocation mechanisms to deliver toxins into cells, and these mechanisms could provide new insights into the development of bio-based drug delivery systems. These toxins have great potential as therapies both because of their intrinsic properties as well as the modular characteristics of both subunits, which make them highly amenable to conjugation with various drug classes. This review focuses on the therapeutical approaches involving the internalization mechanisms of three representative AB toxins: botulinum toxin type A, anthrax toxin, and cholera toxin. We showcase several specific examples of the use of these toxins to develop new therapeutic strategies for numerous diseases and explain what makes these toxins promising tools in the development of drugs and drug delivery systems.

Keywords: anthrax toxin; botulinum toxin; cholera toxin; drug delivery; endocytosis; membrane translocation; therapeutic applications.

Figure 1

Internalization mechanisms of botulinum toxin type A, anthrax toxin, and cholera toxin. (A) Botulinum toxin binds to polysialogangliosides (PSGs) and then to synaptic vesicle protein 2 (SV2), which leads to the internalization of the toxin in small synaptic vesicles. The low pH induces a structural change of botulinum toxin heavy chain (HC) that leads to the unfolding of the light chain (LC) and its translocation through the membrane. Once in the cytosol, the disulfide bond between the HC and LC is reduced, and the LC refolds. LC cleaves SNAP-25 and impairs synaptic vesicle fusion. (B) Anthrax toxin binds to its receptors, CMG2 or TEM8, and is cleaved by a furin-family protease. In this form, PA oligomerizes and clusters in lipid rafts at the plasma membrane. The oligomeric form of PA recruits LF or EF. The receptor-PA complex is endocytosed and is targeted to early endosomes. While some PA pores start to form at the limiting membrane of endosomes (a), some are sorted in intraluminal vesicles (ILVs) and targeted to lysosomes (b). On the way to lysosomes, the PA oligomers undergo pH-dependent PA pore formation in the membrane of ILVs. The pores allow the translocation of unfolded LF through the membrane. These vesicles fuse with the limiting membrane of late endosomes and release their content in the cytosol, where LF cleaves MAPKKs and EF converts ATP into cAMP. (C) The cholera toxin B subunit binds in a pentameric form to the membrane on GM1 in lipid raft domains of the plasma membrane. CTA2 interacts with the pentamer and links the catalytically active CTA1 subunit via a disulfide bond. Once endocytosed in endosomes, the toxin is transported to the trans-Golgi network (TGN) and then to the endoplasmic reticulum (ER) using retro-translocation. The reductive environment of the ER frees CTA1 by breaking the disulfide bond, which is then translocated through the ER membrane using ERAD-associated mechanisms. In the cytosol, CTA1 constitutively activates Gαs, increasing cAMP levels.

Figure 2

Schematic representation of the different constructs described in this study and brief description of their properties. The three original toxins at the top of their respective compartments are highlighted. A and B domains of each toxin’s subunits are represented in red and green, respectively. The text on the right briefly depict either the internalization process of the original toxin or the therapeutic properties of the chimeric constructs.