Abstract
The Clostridium botulinum C3 exoenzyme selectively ADP-ribosylates low molecular weight GTP-binding proteins RhoA, B and C. This covalent modification inhibits Rho signaling activity, resulting in distinct actin cytoskeleton changes. Although C3 exoenzyme has no binding, the translocation domain assures that C3 enters cells and acts intracellularly. C3 uptake is thought to occur due to the high concentration of the C3 enzyme. However, recent work indicates that C3 is selectively endocytosed, suggesting a specific endocytotic pathway, which is not yet understood. In this study, we show that the C3 exoenzyme binds to cell surfaces and is internalized in a time-dependent manner. We show that the intermediate filament, vimentin, is involved in C3 uptake, as indicated by the inhibition of C3 internalization by acrylamide, a known vimentin disruption agent. Inhibition of C3 internalization was not observed by chemical inhibitors, like bafilomycin A, methyl-β-cyclodextrin, nocodazole or latrunculin B. Furthermore, the internalization of C3 exoenzyme was markedly inhibited in dynasore-treated HT22 cells. Our results indicate that C3 internalization depends on vimentin and does not depend strictly on both clathrin and caveolae.
Highlights
C3 exoenzyme (C3) is produced by certain strains of different bacteria, such as Clostridia, Bacilli and Staphylococci [1,2,3,4]
Our results indicate that C3 internalization depends on vimentin and does not depend strictly on both clathrin and caveolae
Our results show that internalization of the C3 exoenzyme is dynamin-dependent, and the intermediate filament, vimentin, is involved in C3 binding and uptake
Summary
C3 exoenzyme (C3) is produced by certain strains of different bacteria, such as Clostridia, Bacilli and Staphylococci [1,2,3,4]. Dynamin mediates the pinching off of the budding coated vesicle from the membrane [17] This endocytotic pathway is reported for diphtheria toxin [18,19]. Caveolae-mediated endocytosis is one of the better characterized forms of clathrin-independent endocytosis [20] Bacterial toxins, such as cholera toxin, are associated with caveolae and can be internalized by caveolae/raft-dependent endocytosis [21,22]. Clostridial binary toxins, such as Clostridium botulinum C2, use a clathrin-and caveolae-independent pathway, but this endocytosis is dependent on dynamin and Rho-GDI [23]. These observations suggest that many bacterial toxins use established endocytotic pathways for internalization into the target cells. Our results show that internalization of the C3 exoenzyme is dynamin-dependent, and the intermediate filament, vimentin, is involved in C3 binding and uptake
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