SummaryThe C3 transferases from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) mono-ADP-ribosylate and thereby inactivate RhoA, -B and -C of eukaryotic cells. Due to their extremely poor cellular uptake, C3 transferases were supposed to be exoenzymes rather than exotoxins, challenging their role in pathogenesis. Here, we report for the first time that low concentrations of both C3lim and C3bot are selectively internalized into macrophages/monocytes in less than 3 h, inducing the reorganization of the actin cytoskeleton by ADP-ribosylation of Rho. We demonstrate that C3 transferases are internalized into the cytosol of macrophages/monocytes via acidified early endosomes. Bafilomycin A1, an inhibitor of endosomal acidification, protected J774A.1 macrophages and human promyelotic leukaemia cells (HL-60) from intoxication by C3. Moreover, confocal laser scanning microscopy revealed colocalization of C3 with early endosomes. An extracellular acidic pulse enabled direct translocation of cell surface-bound C3 across the cytoplasmic membrane to the cytosol. In line with this finding, both C3 proteins exhibited membrane activity in lipid bilayer membranes only under acidic conditions (pH < 5.5). In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis.

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The binary C2 toxin from Clostridium botulinum mono-ADP-ribosylates G-actin in the cytosol of eukaryotic cells. This modification leads to depolymerization of actin filaments accompanied by cell rounding within 3 h of incubation but does not immediately induce cell death. Here we investigated the long-term responses of mammalian cell lines (HeLa and Vero) following C2 toxin treatment. Cells stayed round even though the toxin was removed from the medium after its internalization into the cells. No unmodified actin reappeared in the C2 toxin-treated cells within 48 h. Despite actin being completely ADP-ribosylated after about 7 h, no obvious decrease in the overall amount of actin was observed for at least 48 h. Therefore, ADP-ribosylation was not a signal for an accelerated degradation of actin in the tested cell lines. C2 toxin treatment resulted in delayed apoptotic cell death that became detectable about 15 to 24 h after toxin application in a portion of the cells. Poly(ADP)-ribosyltransferase 1 (PARP-1) was cleaved in C2 toxin-treated cells, an indication of caspase 3 activation and a hallmark of apoptosis. Furthermore, specific caspase inhibitors prevented C2 toxin-induced apoptosis, implying that caspases 8 and 9 were activated in C2 toxin-treated cells. C2I, the ADP-ribosyltransferase component of the C2 toxin, remained active in the cytosol for at least 48 h, and no extensive degradation of C2I was observed. From our data, we conclude that the long-lived nature of C2I in the host cell cytosol was essential for the nonreversible cytotoxic effect of C2 toxin, resulting in delayed apoptosis of the tested mammalian cells.Clostridium botulinum C2 toxin, the prototype of the family of binary actin ADP-ribosylating toxins, mono-ADP-ribosylates G-actin at Arg-177 (1). C2 toxin consists of the enzyme component C2I (431 amino acid residues, 49.3 kDa) and the binding/translocation component C2II (721 amino acid residues, 80.8 kDa). The ADP-ribosyltransferase activity of C2I is located in its C-terminal domain (5), while the enzymatically inactive N-terminal domain (C2IN, amino acid residues 1 to 225) mediates interaction with C2IIa and translocation of C2I into the cytosol (6). Following limited proteolysis, the active C2IIa protein (ϳ60 kDa) forms ring-shaped heptamers that assemble with C2I and mediate binding of the toxin complex to the cellular receptor, a carbohydrate structure (2, 11, 19). During cellular uptake, the heptamers form pores in endosomal membranes, thereby facilitating translocation of C2I into the cytosol (7). Translocation of C2I requires unfolding (14) and subsequent refolding of the protein in the cytosol by the host cell chaperone Hsp90 (13).In the cytosol, C2I catalyzes covalent transfer of the ADPribose moiety from NAD (NAD ϩ ) to Arg-177 of G-actin (1). Mono-ADP-ribosylation of actin induces the depolymerization of F-actin and thereby a complete loss of actin filaments, resulting in rounding of cultured monolayer cells (1). The ADPribosylation at Arg-177 turns G-actin monomers into "cappin...

The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N-and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.Several pathogenic bacteria produce toxins and effector proteins, which attack the cytoskeleton of eukaryotic cells by mono-ADP-ribosylation of actin. In past years, we focused on the mode of action of the Clostridium botulinum C2 toxin as the prototype of binary actin-ADP-ribosylating toxins (1). The enzyme component of the C2 toxin (C2I) 3 ADP-ribosylates G-actin at arginine 177 (2). This leads to depolymerization of actin filaments and finally to cell rounding. The proteolytically activated binding/translocation component (C2IIa) forms heptamers, which assemble with C2I and bind to the cellular receptor (3). Following receptor-mediated endocytosis, C2IIa forms pores in the membrane of acidic endosomes. For translocation of the C2I protein through the lumen of these pores, a partial unfolding of C2I is required (4). The subsequent refolding of C2I in the cytosol is facilitated by the host cell chaperone Hsp90 (5). The interaction of C2I with C2IIa is mediated by the N-terminal domain of the C2I protein (C2IN, amino acid residues 1-225). C2IN, which is enzymatically inactive and does not induce cell rounding when applied in combination with C2IIa to cells, was successfully used as an adaptor for the C2IIa-mediated delivery of different proteins into the cytosol of eukaryotic cells (6).The SpvB protein from Salmonella enterica was identified as a new member of bacterial actin-ADP-ribosylating enzymes...

Depending on the matrix (e.g., water, air) and the classification of a substance (e.g., contaminant), a multitude of limits and guidelines has been defined. The motivation for defining such a limit or guideline can be different. The most important reason is to protect the population from adverse health effects such as acute chronic toxicity or cancer. Another reason could be the protection of ecosystems which could be more vulnerable than humans. Moreover, aesthetic considerations, like the taste and/or odor of drinking water, can result in limitations of chemicals. In the following chapter, definitions of and examples for limits in water, air, or occupational environments are given. These lists are by no means exhaustible.