Internalization of Clostridium botulinum C2 Toxin Is Regulated by Cathepsin B Released from Lysosomes.

Masahiro Nagahama,Keiko Kobayashi,Sadayuki Ochi,Masaya Takehara

doi:10.3390/toxins13040272

Masahiro Nagahama, Keiko Kobayashi + Show 2 more

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https://doi.org/10.3390/toxins13040272

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Abstract

Clostridium botulinum C2 toxin is a clostridial binary toxin consisting of actin ADP-ribosyltransferase (C2I) and C2II binding components. Activated C2II (C2IIa) binds to cellular receptors and forms oligomer in membrane rafts. C2IIa oligomer assembles with C2I and contributes to the transport of C2I into the cytoplasm of host cells. C2IIa induces Ca2+-induced lysosomal exocytosis, extracellular release of the acid sphingomyelinase (ASMase), and membrane invagination and endocytosis through generating ceramides in the membrane by ASMase. Here, we reveal that C2 toxin requires the lysosomal enzyme cathepsin B (CTSB) during endocytosis. Lysosomes are a rich source of proteases, containing cysteine protease CTSB and cathepsin L (CTSL), and aspartyl protease cathepsin D (CTSD). Cysteine protease inhibitor E64 blocked C2 toxin-induced cell rounding, but aspartyl protease inhibitor pepstatin-A did not. E64 inhibited the C2IIa-promoted extracellular ASMase activity, indicating that the protease contributes to the activation of ASMase. C2IIa induced the extracellular release of CTSB and CTSL, but not CTSD. CTSB knockdown by siRNA suppressed C2 toxin-caused cytotoxicity, but not siCTSL. These findings demonstrate that CTSB is important for effective cellular entry of C2 toxin into cells through increasing ASMase activity.

Highlights

Clostridium botulinum C2 toxin is a member of the family of clostridial binary actin-ADP-ribosylating toxins
When cells were treated with C2IIa, βHex activity in culture medium increased in a time-dependent manner
C2IIa-induced βHex release decreased in the presence of lysosomal exocytosis inhibitor bromoenol lactone (BEL), indicating that lysosomal exocytosis is promoted by C2IIa

Summary

Introduction

Clostridium botulinum C2 toxin is a member of the family of clostridial binary actin-ADP-ribosylating toxins. Internalization of C2I to host cells is performed with the proteolytically cleaved form of C2II (C2IIa). C2I catalyzes monoADP-ribosylation of actin monomer (G-actin) at Arg177, leading to filamentous actin (F-actin) depolymerization and cell morphological change (rounding) [1,3]. Other members of this toxin family are Clostridium perfringens iota-toxin, Clostridium spiroforme toxin, and‘Clostridium difficile ADP-ribosyltransferase [1,2,3,4]. The C2I/C2IIa complex is delivered to early and recycling endosomes through the host endosomal trafficking pathway [3,6,7]. A small amount of C2I and C2IIa is transported together back to the cell membranes by recycling endosomes, whereas other components are delivered to late endosomes/lysosomes for degradation [7]

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