# Targeting the Early Endosome-to-Golgi Transport of Shiga Toxins as a Therapeutic Strategy
## 3 Early Endosome-to-Golgi Transport of STx2
### Similarities and Differences in the Transport of STx2B and STxB/STx1B  
Similar to STxB/STx1B, retrograde trafficking of STx2B requires dynamin, epsinR, retromer component Vps26, and the SNARE protein syntaxin 5 (Table 2) [14]. However, early endosome-to-Golgi traffic of STx2 has slower kinetics than that of STxB/STx1B, and STx2B takes a longer time to reach the Golgi than STxB/STx1B [14]. A striking difference between the toxins is that early endosome-to-Golgi transport of STx2B is GPP130-independent [10]. Unlike STxB/STx1B, STx2B does not bind GPP130 [10]. In STxB/STx1B, residues H78 and N79 in a surface-exposed loop ($4-$5 loop) are required for binding GPP130 (Figure 3). Alanine mutations at these sites abolish GPP130 binding and block the early endosome-to-Golgi transport of STx1B [10]. However, the corresponding residues in STx2B are E76 and S77 (Figure 3), respectively, explaining the lack of interaction between STx2B and GPP130 [10]. Nevertheless, the spatial orientation of the $4-$5 loop is nearly identical between STxB/STx1B and STx2B (Figure 3), and alanine mutations of E76 and S77 in STx2B also block the early endosome-to-Golgi transport of STx2B [14]. Hence, STxB/STx1B and STx2B use a conserved structural motif for early endosome-to-Golgi transport, raising the possibility that STx2B may also employ a yet unidentified endosomal receptor to traffic from early endosomes to the Golgi. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Factor** & **Function** & **Reference** \\ \hline Dynamin & Membrane scission & [14] \\ EpsinR & Clathrin adaptor & [14] \\ Vps26 & Retromer component & [14] \\ Syntaxin 5 & SNARE complex component & [14] \\ UNC50 & GBF1 interactor, recruits GBF1 to the Golgi & [58] \\ Rab2a & Small GTPase & [20] \\ STAM & ESCRT (endosomal sorting complexes required for transport)-0 complex component & [20] \\ FUT1 & Fuosylation enzyme & [20] \\ Vps39 & HOPS (homotypic fusion and vacuole protein sorting) complex component & [20] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Factors required for the endosome-to-Golgi transport of STx2B. Late Endosome-Lysosome Fusion Modulates Early Endosome-to-Golgi Trafficking of STx2B--Insights Obtained from a Genome-Wide siRNA Screen  
In an effort to comprehensively understand the commonalities and differences in the mechanisms of retrograde trafficking of STxB/STx1B and STx2B, as well as identify druggable host processes required for toxin trafficking, a viability-based genome-wide siRNA screen was performed in 2017 [58]. The primary screen identified 564, 535, and 196 genes (or hits) that, respectively, are required for toxicity induced by STx1-only, STx2-only, and both toxins [58]. Subsequent efforts focused on 35 hits required for STx2-only toxicity and known or predicted to generate proteins that localize to endosomes/Golgi, because these factors were most likely to specifically modulate the endosome-to-Golgi trafficking of STx2. A secondary screen revealed that 12 of these 35 hits reproducibly protect against STx2-toxicity when depleted (UNC50, B4 GALT5, FUT1, KDR/VEGFR2, SNX14, STAM, TPCN1, SNX1, ASTN2, ATP11A, AP1 AR, and Rab2a) (Table 2) [58]. Subsequent studies revealed that depletion of UNC50, Rab2a, STAM, or FUT1 blocks endosome-to-Golgi transport of STx2B and, notably, also impacts lysosomes and/or the autophagy pathway (Table 2) [20; 58]. Three other factors in this list, TPCN1, SNX14, and KDR/VEGFR2, are also known modulators of lysosomal function and/or autophagy [59; 60; 61]. The above findings raised the interesting hypothesis that biogenesis or function of lysosomes and/or autophagy is essential for the trafficking and toxicity of STx2. This hypothesis was tested by leveraging several facts. Firstly, in the endolysosomal pathway, early endosomes mature into late endosomes that fuse with lysosomes, whereas, in the autophagic pathway, lysosomes fuse with autophagosomes [62; 63; 64; 65; 66].