###### Abstract  
Pathogenic _Bacillus_ and Clostridal (i.e., _Clostridium_ and _Clostridioides_) bacteria express a diverse repertoire of effector proteins to promote disease. This includes production of binary toxins, which enter host epithelial cells and seriously damage the intestinal tracts of insects, animals, and humans. In particular, binary toxins form an AB-type complex composed of a catalytic subunit that is toxic (A) and an oligomeric cell-binding and delivery subunit (B), where upon delivery of A into the cytoplasm of the host cell it catalytically ADP-ribosylates actin and rapidly induces host cell death. In this review, binary toxins expressed by _Bacillus thuringiensis_, _Clostridioides difficile_, and _Clostridium perfringens_ will be discussed, with particular focus placed upon the structural elucidations of their respective B subunits and how these findings help to deconvolute how toxic enzyme delivery into target host cells is achieved by these deadly bacteria. binary toxins; pore-forming proteins; insecticidal; biotechnology; oligomerization; molecular mechanism +
Footnote †: journal: _MCD_  
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_botulinum_ C2 toxin (C2; C2I and C2 II), and vegetative insecticidal proteins 1 and 2 (Vpb1 and Vpa2) from _Bacillus thuringiensis_[2; 4; 5]. In addition, anthrax toxin is historically discussed in the context of binary toxin structure and function [2; 6]. However, it is important to note that anthrax toxin is technically not a binary toxin as it is an assembly of three independently secreted proteins, which include two enzymatic subunits, lethal factor (LF) and edema factor (EF), and a cell-binding/translocase subunit known as the protective antigen (PA) [7]. The similarities between anthrax toxin and Clostridal binary toxins led to speculation that the B component of binary toxins, such as CDTb, bound to three enzymatic partners, an assumption later disproven by cryo-EM studies that revealed CDTa and CDTb form a one-to-one bipartite structure [8]. The structural and functional similarities of the PA to the B subunits of toxins within the binary toxin family make it a necessary and foundational inclusion in the discussion of them. Recent advances are reviewed here, including structures and functional relationships involving bacterial binary and binary-like toxins from pathogenic members of the _Bacillus_, _Clostidium_, and _Clostridioides_ genera. How these discoveries reinforced and/or challenged several ongoing molecular mechanisms proposed for inducing toxicity are also discussed. Figure 1: Pathways and states to consider for binary toxin receptor binding, toxin uptake, and cellular toxicity. A and B subunits are secreted independently by vegetative bacteria. One extracellular pathway  
indicated by blue arrows considers that the B subunit is proteolytically activated and heptamerizes prior to receptor binding [9; 10]. Shown by red arrows is another relevant pathway to consider, illustrating that the monomeric B subunit precursor binds to the host cell receptor first, and then it is proteolytically activated, which triggers the clustering of the receptor and facilitates B subunit heptamerization [9; 10]. A dashed purple arrow includes a pathway that considers the highly stable CDT di-heptamer structures that were determined at atomic resolution in symmetric and asymmetric states [11]. Likewise, dissociation of di-heptamers into heptamers was found to occur upon decreasing Ca\({}^{2+}\) ion concentrations associated with receptor-mediated endocytosis [9; 10; 12; 13]. Thus, depletion of Ca\({}^{2+}\) ions and/or endosomal acidification need to be considered during endosomal delivery of binary toxins, particularly since CDTb-dependent pore formation in the membrane was observed upon lowering Ca\({}^{2+}\) levels to levels found inside of an endosome [11; 12; 13].