Difference in Mono-O-Glucosylation of Ras Subtype GTPases Between Toxin A and Toxin B From Clostridioides difficile Strain 10463 and Lethal Toxin From Clostridium sordellii Strain 6018

Abstract

Clostridioides difficile toxin A (TcdA) and Toxin B (TcdB) trigger inflammasome activation with caspase-1 activation in cultured cells, which in turn induce the release of IL-6, IFN-γ, and IL-8. Release of these proinflammatory responses is positively regulated by Ras-GTPases, which leads to the hypothesis that Ras glucosylation by glucosylating toxins results in (at least) reduced proinflammatory responses. Against this background, data on toxin-catalyzed Ras glucosylation are required to estimate of pro-inflammatory effect of the glucosylating toxins. In this study, a quantitative evaluation of the GTPase substrate profiles glucosylated in human colonic (Caco-2) cells treated with either TcdA, TcdB, or the related Clostridium sordellii lethal toxin (TcsL) was performed using multiple reaction monitoring (MRM) mass spectrometry. (H/K/N)Ras are presented to be glucosylated by TcsL and TcdA but by neither TcdB isoform tested. Furthermore, the glucosylation of (H/K/N)Ras was detected in TcdA-(not TcdB)-treated cells, as analyzed exploiting immunoblot analysis using the Ras glucosylation-sensitive 27H5 antibody. Furthermore, [14C]glucosylation of substrate GTPase was found to be increased in a cell-free system complemented with Caco-2 lysates. Under these conditions, (H/K/N)Ras glucosylation by TcdA was detected. In contrast, TcdB-catalyzed (H/K/N)Ras glucosylation was detected by neither MRM analysis, immunoblot analysis nor [14C]glucosylation in a cell-free system. The observation that TcdA (not TcdB) glucosylates Ras subtype GTPases correlates with the fact that TcdB (not TcdA) is primarily responsible for inflammatory responses in CDI. Finally, TcsL more efficaciously glucosylated Ras subtype GTPase as compared with TcdA, reinforcing the paradigm that TcsL is the prototype of a Ras glucosylating toxin.

Subtyping analysis reveals new variants and accelerated evolution of Clostridioides difficile toxin B

“…There is currently no standard nomination for TcdB variants, miscellaneous names referred to same toxin variants were sometimes assigned in different studies. For example, TcdB variant initially identified from NAP1/BI/027 strains was later called TcdB 027 28,29 , TcdB HV 22,30 , TcdB NAP1 15 , TcdB2 31 , TcdB R20291 32,33 , or TcdB-R20291 34 . In 2005, Rupnik et al 35 proposed nomenclature of C. difficile toxin variants by adding the bacterial strain in which the toxin was originally found.…”

Clostridioides difficile Toxin A-Induced Wnt/β-Catenin Pathway Inhibition Is Mediated by Rac1 Glucosylation

“…In addition, we cannot exclude the involvement of additional mechanisms to modulate the activity of Rac1 in the present study, considering that specific modification at a single threonine residue in the small GTPases leads these important key players of several signaling pathways to their functional inactivation. TcdA and TcdB glucosylate Rac1/Cdc42 at threonine-35 and RhoA, decreasing their active levels into the cells ( Halabi-Cabezon et al, 2008 ; Schoentaube et al, 2009 ; Genth et al, 2018 ).…”

“…In addition, whereas TcdB has been shown to inhibit the activity of the Wnt/β-catenin signaling pathway by binding to the FZD-7 receptor expressed by cells in the colonic epithelium ( Tao et al, 2016 ), thereby preventing its activation by Wnt3a, TcdA has not shown affinity for FZD2, FZD3, or FZD-7 ( Gupta et al, 2017 ). Like TcdA, TcdB induces inactivation of Rho GTPases including Rac-1 ( Halabi-Cabezon et al, 2008 ; Schoentaube et al, 2009 ; Genth et al, 2018 ), which should cause Wnt/β-catenin signaling pathway inhibition. However, the investigation on TcdB effect in this pathway was not the aim of this study.…”

Global and Targeted Profiling of Gtp‐binding Proteins in Biological Samples by Mass Spectrometry

“…In total, 134 (for tryptic digestion) and 96 (for chymotryptic digestion) transitions from eight small GTPases were monitored in two scheduled LC–MRM runs (Junemann et al, 2017). Together, the substrate profiles of the three LCTs (TcdA, TcdB, and TcsL) in Caco‐2 cells were interrogated, and it was found that (H‐/K‐/N‐)Ras and Rap(1/2) could be glucosylated by TcsL and TcdA, but not TcdB (Genth et al, 2018).…”