The N-Terminal Part of the Enzyme Component (C2I) of the Binary Clostridium botulinum C2 Toxin Interacts with the Binding Component C2II and Functions as a Carrier System for a Rho ADP-Ribosylating C3-Like Fusion Toxin

Abstract

The binary actin–ADP-ribosylating Clostridium botulinum C2 toxin consists of the enzyme component C2I and the binding component C2II, which are separate proteins. The active component C2I enters cells through C2II by receptor-mediated endocytosis and membrane translocation. The N-terminal part of C2I (C2IN), which consists of 225 amino acid residues but lacks ADP-ribosyltransferase activity, was identified as the C2II contact site. A fusion protein (C2IN-C3) of C2IN and the full-length C3-like ADP-ribosyltransferase from Clostridium limosum was constructed. The fusion protein C2IN-C3 ADP-ribosylated Rho but not actin in CHO cell lysates. Together with C2II, C2IN-C3 induced complete rounding up of CHO and HeLa cells after incubation for 3 h. No cell rounding was observed without C2II or with the original C3-like transferase from C. limosum. The data indicate that the N-terminal 225 amino acid residues of C2I are sufficient to cause the cellular uptake of C. limosum transferase via the binding component of C2II, thereby increasing the cytotoxicity of the C3-like exoenzyme several hundred-fold.

Cyclophilin A facilitates translocation of theClostridium botulinumC2 toxin across membranes of acidified endosomes into the cytosol of mammalian cells

“…Because the N-terminal domain of C2I (C2IN) is sufficient to mediate C2IIa-facilitated membrane translocation of C2I (Barth et al, 1998), we tested whether uptake of the recombinant fusion toxin C2IN-C3lim is also inhibited by CsA. The Clostridium limosum ADP-ribosyltransferase C3lim alone is not taken up by HeLa cells and therefore this protein represents an ideal reporter enzyme to detect translocation of C2IN into the cytosol.…”

“…The Clostridium limosum ADP-ribosyltransferase C3lim alone is not taken up by HeLa cells and therefore this protein represents an ideal reporter enzyme to detect translocation of C2IN into the cytosol. C2IN-C3 is internalized via the C2 toxin system into the host cell cytosol, where the C3 ADP-ribosyltransferase activity selectively modifies Rho-GTPases (Barth et al, 1998). HeLa cells were pretreated with CsA and subsequently the cells were incubated together with C2IN-C3 (100 ng ml -1 ) plus C2IIa (200 ng ml -1 ) in the presence or absence of CsA.…”

Cell Entry of C3 Exoenzyme from Clostridium botulinum

“…In the last twenty years, several molecular and cell biology standard methods have been applied to study the internalization and effects of C3 (Just et al 1992;Aullo et al 1993;Olson et al 1998;Barth et al 1998;Marvaud et al 2002;Park et al 2003;Fahrer et al 2010;Lillich et al 2012;Rohrbeck et al 2015). Yet there are substantial gaps in our knowledge about uptake and molecular effects of C3.…”

“…To accelerate C3 uptake and thus cytoskeletal reorganization, C3 has been introduced in eukaryotic cells by microinjection (Ridley and Hall 1992;Olson et al 1998), electroporation (Tokman et al 1997), or permeabilization with digitonin or streptolysin O (Mackay et al 1997;Fensome et al 1998;Koch et al 1993). Moreover, chimeric fusion toxins, in which C3 was fused to the binding domain of the C2 toxin (Barth et al 1998) or diphtheria toxin (Aullo et al 1993), were used. Furthermore, a membrane-permeating form of C3 was created by fusing Tat (trans-activating transcription factor) transduction domain of human immunodeficiency virus (Frankel and Pabo 1988) to the C3 amino terminus (Tan et al 2007).…”

Bacterial protein toxins targeting Rho GTPases

“…C3 exoenzyme was fused to the C2II-binding part of C2I, thereby replacing the actin speci¢c ADP-ribosyltransferase with the Rho-speci¢c C3. The treatment of cultured cells with C3 fusion toxin along with C2II results in a C3-speci¢c phenotype at more than a 300fold lower concentration of the fusion toxin than with C3 [16].…”