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Abstract
Lethal Toxin from Clostridium sordellii (TcsL), which is casually involved in the toxic shock syndrome and in gas gangrene, enters its target cells by receptor-mediated endocytosis. Inside the cell, TcsL mono-O-glucosylates and thereby inactivates Rac/Cdc42 and Ras subtype GTPases, resulting in actin reorganization and an activation of p38 MAP kinase. While a role of p38 MAP kinase in TcsL-induced cell death is well established, data on a role of p38 MAP kinase in TcsL-induced actin reorganization are not available. In this study, TcsL-induced Rac/Cdc42 glucosylation and actin reorganization are differentially analyzed in p38alpha−/− MSCV empty vector MEFs and the corresponding cell line with reconstituted p38alpha expression (p38alpha−/− MSCV p38alpha MEFs). Genetic deletion of p38alpha results in reduced susceptibility of cells to TcsL-induced Rac/Cdc42 glucosylation and actin reorganization. Furthermore, SB203580, a pyridinyl imidazole inhibitor of p38alpha/beta MAP kinase, also protects cells from TcsL-induced effects in both p38−/− MSCV empty vector MEFs and in p38alpha−/− MSCV p38alpha MEFs, suggesting that inhibition of p38beta contributes to the protective effect of SB203580. In contrast, the effects of the related C. difficile Toxin B are responsive neither to SB203580 treatment nor to p38alpha deletion. In conclusion, the protective effects of SB203580 and of p38alpha deletion are likely not based on inhibition of the toxins’ glucosyltransferase activity rather than on inhibited endocytic uptake of specifically TcsL into target cells.
Highlights
Toxin-producing strains of C. difficile and C. sordellii cause intestinal infections, includingC. difficile-associated diarrhea (CDAD) in humans and horses, and C. sordellii-induced hemorrhagic enteritis and enterotoxemia in cattle, sheep, and other ruminants [1,2,3,4]
Toxin from Clostridium sordellii (TcsL)-induced cell rounding was analyzed in p38alpha −/− MSCV p38alpha MEFs treated with SB203580, a pyridinyl imidazole inhibitor of p38alpha/beta MAP kinase [27]
SB203080 concentration-dependently reduced TcsL-induced cell rounding, with a SB203580 concentration of 10 μM being sufficient for almost complete prevention of TcsL-induced cell rounding (Figure 2A,B)
Summary
Toxin-producing strains of C. difficile and C. sordellii cause intestinal infections, includingC. difficile-associated diarrhea (CDAD) in humans and horses, and C. sordellii-induced hemorrhagic enteritis and enterotoxemia in cattle, sheep, and other ruminants [1,2,3,4]. The major virulence factors involved in these infections are toxin A (TcdA) and toxin B (TcdB) of C. difficile, and lethal toxin (TcsL) and hemorrhagic toxin (TcsH) from C. sordellii. These single chained toxins exhibit an AB-like toxin structure with the C-terminal delivery domain mediating cell entry of the N-terminal glucosyltransferase domain by receptor-mediated endocytosis [5,6]. The endocytosed glucosyltransferase domain associates with membrane phosphatidylserine facilitating mono-O-glucosylation of small GTPases of the Rho and Ras subfamilies in a monovalent and divalent metal ion-dependent manner [7,8,9].
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