Difference in F-Actin Depolymerization Induced by Toxin B from the Clostridium difficile Strain VPI 10463 and Toxin B from the Variant Clostridium difficile Serotype F Strain 1470

Abstract

Clostridium difficile toxin A (TcdA) and toxin B (TcdB) are the causative agent of the C. difficile-associated diarrhea (CDAD) and its severe form, the pseudomembranous colitis (PMC). TcdB from the C. difficile strain VPI10463 mono-glucosylates (thereby inactivates) the small GTPases Rho, Rac, and Cdc42, while Toxin B from the variant C. difficile strain serotype F 1470 (TcdBF) specifically mono-glucosylates Rac but not Rho(A/B/C). TcdBF is related to lethal toxin from C. sordellii (TcsL) that glucosylates Rac1 but not Rho(A/B/C). In this study, the effects of Rho-inactivating toxins on the concentrations of cellular F-actin were investigated using the rhodamine-phalloidin-based F-actin ELISA. TcdB induces F-actin depolymerization comparable to the RhoA-inactivating exoenzyme C3 from C. limosum (C3-lim). In contrast, the Rac-glucosylating toxins TcdBF and TcsL did not cause F-actin depolymerization. These observations led to the conclusion that F-actin depolymerization depends on the toxin’s capability of glucosylating RhoA. Furthermore, the integrity of focal adhesions (FAs) was analyzed using paxillin and p21-activated kinase (PAK) as FA marker proteins. Paxillin dephosphorylation was observed upon treatment of cells with TcdB, TcdBF, or C3-lim. In conclusion, the Rho-inactivating toxins induce loss of cell shape by either F-actin depolymerization (upon RhoA inactivation) or the disassembly of FAs (upon Rac1 inactivation).

Rho-A prenylation and signaling link epithelial homeostasis to intestinal inflammation

“…The potential relevance of these findings for humans was underlined by the observation that significant downregulation of GGTase-I levels and cytosolic Rho-A accumulation was seen in the gut epithelium of IBD patients with active disease. Thus, in addition to the role of Rho-A signaling in cytoskeleton regulation (43), maintenance of cell shape (44), and cell migration (45), Rho-A emerges as a crucial regulator of gut epithelial homeostasis, and this function critically depends on cellular availability of prenylated Rho-A. Our findings indicated that Rho-A signaling is involved in epithelial cell shedding during inflammation.…”

Haemorrhagic toxin and lethal toxin fromClostridium sordelliistrain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins

“…). These observations are consistent with earlier findings showing that in early phases of intoxication the cytopathic effects of the LCGTs) are based on disassembly of focal adhesions and adherens junctions rather than on actin depolymerization (Geny et al ., ; Popoff and Geny, ; May et al ., ). This view is further supported by in vivo data showing that alteration of E‐cadherin intercellular junction between lung vascular endothelial cells is an early and predominant TcsL effect (Geny et al ., ).…”

“…Each toxin type recognizes a distinct set of Rho/Ras protein substrates. In addition, toxin variants such as TcsL from C. sordellii VPI9048 strain and TcdB from C. difficile serotype F have been described, which differ in substrate profile and cytotoxic/cytopathic effects (Chaves‐Olarte et al ., ; Huelsenbeck et al ., ; Rupnik, ; Popoff and Geny, ; May et al ., ). TcdB from C. difficile epidemic strains like R20291 has been found to be more cytotoxic and more lethal for mice than TcdB‐10463, which has been attributed to sequence differences in the C‐terminal binding domain (Stabler et al ., ; Lanis et al ., ).…”

Large clostridial glycosylating toxins modifying small GTPases

“…The presence of nonglycosylatable Rac1 is sufficient to prevent the cytopathic effect of the LCGTs [67] and the Rac1-GTPase plays a key role in the regulation of the organization of the actin cytoskeleton [89]. Rac glucosylation by LCGTs results in rapid dephosphorylation of the Rac effector protein PAK (among many other kinases) and subsequently of paxillin, a focal adhesion-regulating protein [74,88,90]. Furthermore, Rac glucosylation by TcsL alters phosphoinositide metabolism, leading to focal disorganization of adhesion and adherens junction complexes [88].…”