To quantify perinuclear clustering of endosomal markers and lysosomes, the plugin was used on the average projection of acquired Z-stacks

To quantify perinuclear clustering of endosomal markers and lysosomes, the plugin was used on the average projection of acquired Z-stacks. cytosol (5, 6, 7, 8, 9). Direct transport from early endosomes to the Golgi is critical as it allows the toxins to evade late endosomes where proteolytic enzymes are active (5, 6, 7, 8, 9). As STx1 and STx2 must traffic to the cytosol to induce cytotoxicity, blocking toxin transport in general, and at the early endosome-to-Golgi step in particular, has emerged as a encouraging therapeutic strategy (5, 6, 10, 11). As an example, treatment with manganese degrades the endosomal STx1 receptor GPP130 and thereby blocks the early endosome-to-Golgi transport of STx1, diverts STx1 to late endosomes for degradation, and protects cells and mice against lethal STx1 toxicosis (6). However, in order to be therapeutically effective, a toxin transport inhibitor must block STx2 because STx2 is usually 400 times more harmful than STx1 in vivo (12), and in humans, disease severity correlates with STx2 production (13). In spite of the greater disease relevance, molecular mechanisms of STx2 transport, which is GPP130 impartial and manganese insensitive (7), are poorly understood. This space in knowledge has hindered therapeutic development, and currently, there are no toxin transport inhibitors approved for use in humans. Here, we utilize data from a genome-wide siRNA screen and statement the unexpected finding that early endosome-to-Golgi transport of STx2 requires efficient fusion of late endosomes with lysosomes. Inhibition of late endosomeClysosome fusion alters endosomal recruitment of retromer, which is required for the early endosome-to-Golgi transport of STx2 (9), providing a possible explanation for the effects on toxin trafficking. Through a subsequent screen FLT3-IN-4 of clinically approved drugs that target lysosomes, we identify tamoxifen (TAM) to be a potent inhibitor of the early endosome-to-Golgi transport and toxicity of STx2 and STx1. Further, we show that TAM functions as a poor base to increase endolysosomal pH, which alters endosomal dynamics and impacts endosomal recruitment of retromer. Finally, we show that TAM increases the survival of mice exposed to lethal STx2 or STx1. These findings identify a previously unknown role of late endosomeClysosome fusion in cargo transport at the early endosome/Golgi interface. Moreover, our work suggests that it may be possible to repurpose TAM for treating STEC infections. Results Biogenesis or function of lysosomes and/or autophagy is required for STx2 transport and toxicity To elucidate the mechanisms of STx2 trafficking, we recently performed a viability-based genome-wide siRNA screen and recognized 12 endosome/Golgi-localized host proteins that, when depleted, reproducibly guarded against STx2-induced cell death (8). Surprisingly, 6 of 12 recognized hits (Rab2a, FUT1, STAM, TPCN1, SNX14, and VEGFR2) regulate lysosome biogenesis/function and/or autophagy (Table 1). Based on this, here we hypothesized that biogenesis or function of lysosomes and/or Rabbit Polyclonal to CCDC102B the FLT3-IN-4 autophagy pathway is required for the trafficking and toxicity of STx2, and targeting FLT3-IN-4 lysosomes/autophagy may provide a therapeutically viable means to block STx2 trafficking. Table 1. Role of TPCN1, Rab2a, SNX14, STAM, VEGFR2, and FUT1 in lysosome function and/or autophagy. clone, two individual stop codons were launched in cells experienced a higher number of LC3-positive punctae than WT cells FLT3-IN-4 (Fig 1C and D), indicating that autophagy and/or lysosome function was compromised. Toxin transport assays revealed that, consistent with our previous studies (8, 9), in WT cells, STx2 B-subunit (STx2B) bound the cell surface and trafficked to the Golgi within 60 min (Fig 1E and F). In cells, STx2B also bound the cell surface, but at 60 min, a pool of the toxin failed to traffic to the Golgi and instead was degraded (Fig FLT3-IN-4 1E and F). At earlier time points, in cells, STx2B was detected in Rab5-positive punctae (Fig 1G), indicating that internalization to early endosomes was not affected. Degradation of STx2B in cells was blocked by pretreatment with leupeptin/pepstatin or expression of dominant unfavorable Rab7 (Fig 1HCK), suggesting that this toxin was degraded in late endosomes/lysosomes. Toxin degradation in cells, in spite of possible changes in lysosomal function, was not amazing because soluble cargo are effectively degraded in prelysosomal late endosomes, where proteolytic enzymes are active (32). The block in transport was rescued by overexpression of WT, but not dominant unfavorable or constitutively active, Rab2a (Fig 1L and M). Identical results were obtained using a second clone in which the CRISPR/Cas9 system introduced a stop codon in one allele and an inactivating point mutation in the.