Exosome-associated Shiga toxin 2 is released from cells and causes severe toxicity in mice

Miho Watanabe-Takahashi,Kiyotaka Nishikawa,Jun Motoyama,Kentaro Shimasaki,Jyoji Yamate,Yasuhiro Natori,Ryohei Yanoshita,Hirofumi Ukai,Masaya Ikegawa,Waka Sato,Fumi Kano,Shinji Yamasaki,Masayuki Murata,Atsuo Miyazawa,Yuri Nishino,Noriko Toyama-Sorimachi,Jumpei Omi,Miwa Tamura-Nakano

doi:10.1038/s41598-018-29128-9

Abstract

Shiga toxin (Stx), a major virulence factor of enterohemorrhagic Escherichia coli (EHEC), is classified into two subgroups, Stx1 and Stx2. Clinical data clearly indicate that Stx2 is associated with more severe toxicity than Stx1, but the molecular mechanism underlying this difference is not fully understood. Here, we found that after being incorporated into target cells, Stx2, can be transported by recycling endosomes, as well as via the regular retrograde transport pathway. However, transport via recycling endosome did not occur with Stx1. We also found that Stx2 is actively released from cells in a receptor-recognizing B-subunit dependent manner. Part of the released Stx2 is associated with microvesicles, including exosome markers (referred to as exo-Stx2), whose origin is in the multivesicular bodies that formed from late/recycling endosomes. Finally, intravenous administration of exo-Stx2 to mice causes more lethality and tissue damage, especially severe renal dysfunction and tubular epithelial cell damage, compared to a free form of Stx2. Thus, the formation of exo-Stx2 might contribute to the severity of Stx2 in vivo, suggesting new therapeutic strategies against EHEC infections.

Highlights

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli (EHEC) that causes bloody diarrhea and hemorrhagic colitis and exposure can result in fatal systemic complications, such as hemolytic uremic syndrome (HUS) and acute encephalopathy[1,2,3,4]
We found that Stx2a, but not Stx1a, is actively released after incorporation into target cells, in manner dependent on the B-subunit
Our observations suggest a novel molecular mechanism, in which a unique structure containing activated Stx2a is formed, which is not formed by Stx1a, and this unique form contributes to the severe toxicity of Stx2a in vivo

Summary

Introduction

Shiga toxin (Stx) is a major virulence factor of enterohemorrhagic Escherichia coli (EHEC) that causes bloody diarrhea and hemorrhagic colitis and exposure can result in fatal systemic complications, such as hemolytic uremic syndrome (HUS) and acute encephalopathy[1,2,3,4]. After binding to Gb3, Stx is internalized into cells through endocytosis to form early endosomes and transported to the Golgi in a retrograde manner, and to the endoplasmic reticulum (ER), from where the A-subunit is released into the cytosol to inhibit protein synthesis[18,19,20,21] In this retrograde transport process, furin, a membrane-anchored protease that is primarily present in the trans-Golgi network (TGN), cleaves the A-subunit into an enzymatically active A1-fragment (27.5 kDa) and a carboxy terminal A2-fragment (4.5 kDa) that are linked by a disulfide bond and bind to the B-subunit, to be fully activated[22,23]. By using genome-wide siRNA screening, UNC50, a membrane protein present in the Golgi, was identified to regulate the trafficking of Stx[2] B-subunit, but not Stx[1] B-subunit, from early endosomes to the Golgi[27] Despite these observations, it is unclear whether differences in the intracellular transport of these toxins are related to the marked difference in toxin severity in vivo. Microparticles can be produced by Stx[1] or by EHEC O157:H7-derived lipopolysaccharide (LPS) when incubated with whole blood cells[34], suggesting that other structures or machineries might contribute to the difference in toxicity of Stx[2] in vivo

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Results

Conclusion