# Targeting the Early Endosome-to-Golgi Transport of Shiga Toxins as a Therapeutic Strategy
## 1 Introduction  
Infections caused by _Shigella_ bacteria that produce Shiga toxin (STx) and Shiga toxin-producing _Escherichia coli_ that produce Shiga toxin 1 and 2 (STx1 and STx2) are major causes of water- and food-borne disease in the world [1, 2]. _Shigella_ infections predominate in developing countries due to poor sanitation conditions [2]. In contrast, STEC infections are more commonly seen in North America, Europe, and Australia [3]. Infected patients initially develop gastrointestinal disease [1, 4]. In a subset, the released toxins enter the bloodstream and target renal microvascular endothelial and tubular epithelial cells to cause life-threatening hemolytic uremic syndrome [1, 4, 5]. While _Shigella_ infections can be treated with antibiotics, drug resistance is an emerging problem [2]. In the case of STEC-induced disease, there are no definitive treatments, and antibiotic therapy is contraindicated because it may increase toxin release from the bacteria and enhance the risk of severe renal damage [1, 6, 7]. Overall, there is an urgent need to develop effective interventions to counter disease caused by these bacterial toxins.  
STx, STx1, and STx2 belong to the AB\({}_{5}\) class of bacterial exotoxins and are formed by the association of an A-subunit with a pentameric B-subunit [8, 9]. The A-subunit has ribosomal RNA\(N\)-glycosidase activity and induces toxicity by inhibiting protein synthesis in host cells [4; 8; 9]. The pentameric B-subunits mediate retrograde trafficking from the cell surface to the cytosol [4; 8; 9; 10]. STx1 is essentially identical to STx with 100% conservation in the B subunit and a conservative serine-to-threonine substitution in the A subunit [9; 11]. However, STx2 shares only \(\sim\)55% sequence identity with STx and STx1 [9; 11]. Notably, with STEC infections, STx2 is the more disease-relevant toxin; the in vivo median lethal dose (LD\({}_{50}\)) of STx2 is \(\sim\)400-fold lower than that of STx1, and production of STx2 is associated with disease severity [1; 12; 13].  
STx, STx1, and STx2 traffic via an elaborate retrograde route to reach the cytosol of host cells. The steps involved are, sequentially, binding to globotriaosylceramide, the cell-surface glycosphingolipid receptor for the toxins, followed by endocytosis and internalization to early endosomes, direct transit from early endosomes to the Golgi apparatus, and delivery to the endoplasmic reticulum from where the A-subunits are translocated to the cytosol (Figure 1A) [9; 14; 15; 16]. The early endosome-to-Golgi transport step is critical as it allows the toxins to bypass degradative late endosomes and lysosomes (Figure 1A) [9; 14]. As retrograde trafficking is essential for toxicity, there is a high interest in developing inhibitors of toxin transport for therapy [9; 17; 18; 19; 20]. Targeting the early endosome-to-Golgi step has particular appeal, as inhibition of toxin transport at this step is expected to reroute the toxins to degradative late endosomes/lysosomes (Figure 1B). Here, we review current understanding of the molecular mechanisms of the early endosome-to-Golgi trafficking of STx, STx1, and STx2, and we summarize how basic science advances are being utilized for the development of small-molecule inhibitors of toxin transport.