RESUMEN
Clostridium sordellii lethal toxin (TcsL) belongs to the family of clostridial glucosylating toxins. TcsL exhibits glucosyltransferase activity to inactivate Rho and Ras proteins. On cultured cells, TcsL causes actin reorganization ("cytopathic effect") and apoptotic cell death ("cytotoxic effect"). This study is based on the concept that the cytotoxic effects of TcsL depend on the glucosylation of critical substrate proteins rather than on the glucosyltransferase activity per se. The cytotoxic effects of TcsL depend on the glucosyltransferase activity of TcsL, as neither chemically inactivated TcsL nor a glucosyltransferase-deficient mutant version of TcsL caused it. The TcsL homologous toxin B from Clostridium difficile serotype F strain 1470 (TcdBF) also failed to cause cytotoxic effects. Correlation of the toxins' respective protein substrate specificities highlighted (H/K/N)Ras as critical substrate proteins for the cytotoxic effects. (H/K/N)Ras are critical upstream regulators of phosphatidylinositide 3'-OH kinase (PI3K)/Akt survival signaling. Tauroursodeoxycholic acid (TUDCA) classified to activate PI3K/Akt signaling downstream of apoptosis-inducing stimuli prevented the cytotoxic effects of TcsL. In conclusion, (H/K/N)Ras glucosylation and subsequent inhibition of PI3K/Akt signaling are critical for the cytotoxic effects of TcsL.
Asunto(s)
Toxinas Bacterianas/toxicidad , Leucemia Basofílica Aguda/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Animales , Toxinas Bacterianas/química , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Glicosilación/efectos de los fármacos , Glicosiltransferasas/metabolismo , Leucemia Basofílica Aguda/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Ratas , Ácido Tauroquenodesoxicólico/farmacologíaRESUMEN
Small GTPases of the Rho family play versatile roles in the formation and development of axons and dendrites, effects often studied by the Rho-inactivating C3 transferase (C3bot) from Clostridium botulinum. Recently, we reported that transferase-deficient C3bot also exerted axonotrophic activity. Using overlapping peptides from the C3bot sequence, we identified a small peptide of 29 amino acids (covering residues 154-182) from the C-terminal region of C3bot that promotes both axonal and dendritic growth, as well as branching of hippocampal neurons, at submicromolar concentrations. Several C3bot constructs, including the short peptide, enhanced the number of axonal segments from mid- to higher-order segments. C3bot(154-182) also increased the number of synaptophysin-expressing terminals, up-regulated various synaptic proteins, and functionally increased the glutamate uptake. Staining against the vesicular glutamate and GABA transporters further revealed that the effect was attributable to a higher number of glutamatergic and GABAergic inputs on proximal dendrites of enhanced green fluorescent protein (EGFP)-transfected neurons. Using organotypical slice cultures, we also detected trophic effects of C3bot(154-182) on length and density of outgrowing fibers from the entorhinal cortex that were comparable to the effects elicited by full-length C3bot. In addition, an enhanced reinnervation was observed in a hippocampal-entorhinal lesion model. In summary, the neurotrophic effect of C3bot is executed by a C-terminal peptide fragment covering aa 154-182 of C3; it triggers dendritic and axonal growth and branching as well as increased synaptic connectivity. In contrast to full-length C3, this C3 peptide selectively acts on neurons but not on glial cells.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Aminoácidos/farmacología , Axones/fisiología , Toxinas Botulínicas/metabolismo , Dendritas/fisiología , Neuronas/fisiología , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/genética , Aminoácidos/química , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Biomarcadores/metabolismo , Toxinas Botulínicas/química , Toxinas Botulínicas/genética , Células Cultivadas , Clostridium botulinum/genética , Clostridium botulinum/metabolismo , Técnicas de Cocultivo , Dendritas/efectos de los fármacos , Dendritas/genética , Dendritas/metabolismo , Embrión de Mamíferos , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Hipocampo/embriología , Inmunohistoquímica , Ratones , Ratones Endogámicos , Proteínas Asociadas a Microtúbulos/metabolismo , Peso Molecular , Proteínas de Neurofilamentos/química , Proteínas de Neurofilamentos/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Factores de TiempoRESUMEN
Toxin A (TcdA) and Toxin B (TcdB) are the major pathogenicity factors of the Clostridium difficile-associated diarrhoea (CDAD). The single-chained protein toxins enter their target cells by receptor-mediated endocytosis. New data show the critical role of auto-catalytic processing for target cell entry. Inside the cell, the toxins mono-glucosylate and thereby inactivate low molecular mass GTP-binding proteins of the Rho subfamily. Toxin-treated cells respond to RhoA glucosylation with up-regulation and activation of the pro-apoptotic Rho family protein RhoB. These data reinforce the critical role of the glucosyltransferase activity for programmed cell death and show that TcdA and TcdB, generally classified as broad-spectrum inhibitors of Rho proteins, are also capable of activating Rho proteins.
Asunto(s)
Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/farmacología , Toxinas Bacterianas/farmacología , Enterotoxinas/metabolismo , Enterotoxinas/farmacología , Humanos , Modelos Biológicos , Proteínas de Unión al GTP rho/antagonistas & inhibidores , Proteínas de Unión al GTP rho/metabolismoRESUMEN
ADP-ribosylation of Rho(A,B,C) by the family of exoenzyme C3-like transferases induces reorganization of the actin cytoskeleton based on inactivation of RhoA. No data are available on the role of RhoB in C3-treated cells. In murine fibroblasts treated with the cell-permeable exoenzyme C3 from Clostridium limosum (C3), an increase in the level of RhoB was observed. This upregulation of RhoB was based on transcriptional activation, as it was responsive to inhibition by actinomycin D and accompanied by activation of the rhoB promoter. Upregulation of RhoB was not observed in cells treated with either the actin ADP-ribosylating C2 toxin from Clostridium botulinum or latrunculin B, suggesting that inactivation of Rho but not actin reorganization was required for the upregulation of RhoB. This notion was confirmed, as the Rho/Rac/Cdc42-glucosylating toxin B from Clostridium difficile (TcdB) but not the Rac/R-Ras-glucosylating variant toxin B from C. difficile strain 1470 serotype F (TcdBF) induced a strong upregulation of RhoB. Upregulation of RhoB was further observed in response to the Rac/(H-,K-,N-,R-)Ras-glucosylating lethal toxin from Clostridium sordellii. The level of active, GTP-bound RhoB was increased in TcdB-treated cells compared to untreated cells (as determined by Rhotekin pull-down assay). In contrast, no active RhoB was found in C3-treated cells. RhoB-GTP was required for the TcdB-induced apoptosis (cytotoxic effect), as this effect was responsive to inhibition by C3. In conclusion, RhoB was upregulated by Rho-/Ras-inactivating toxins, as a consequence of the inactivation of either Rho(A,B,C) or (H-,K-,N-)Ras. In TcdB-treated cells, RhoB escaped its inactivation and was required for the cytotoxic effect.