Abstract
The large clostridial glucosylating toxin B (TcdB) is a major virulence factor of the nosocomial pathogen Clostridioides difficile. TcdB inhibits small GTPases by glucosylation leading to impaired downstream signaling. TcdB also possesses a glucosyltransferase independent effect described as pyknosis. To elucidate the impact of TcdB and its glucosylation-inactive mutant TcdBNXN on the kinome of human cells, SILAC labeled HEp-2 cells were treated with 2 nM TcdB for 8 h. Phosphopeptides were enriched using SCX chromatography, IMAC and TiO2 followed shotgun mass spectrometry analysis. Overall 4,197 phosphopeptides were identified; more than 1,200 phosphosites responded to treatment with TcdB or TcdBNXN. The data suggested that predominantly stress-activated MAPK-dependent signaling pathways were triggered by toxin B treatment.
Highlights
Clostridioides difficile is one of the most common human nosocomial pathogens and causes antibiotic-associated diarrhea leading sometimes to severe pseudomembranous colitis (Loo et al, 2005; Voth and Ballard, 2005)
HEp-2 cells were used to elucidate the impact of TcdB and TcdBNXN on the phosphorylation status of cellular proteins designated as phosphoproteome
The glucosyltransferase-deficient mutant TcdBNXN only revealed the pyknotic phenotype for almost all cells
Summary
Clostridioides difficile is one of the most common human nosocomial pathogens and causes antibiotic-associated diarrhea leading sometimes to severe pseudomembranous colitis (Loo et al, 2005; Voth and Ballard, 2005). In case of TcdB Rho, Rac, and Cdc are glucosylated causing a perturbed downstream signaling of the affected small GTPases (Kuehne et al, 2010; Zeiser et al, 2013; Genth et al, 2014) This leads initially to a rearrangement of the cytoskeleton and cell cycle arrest (cytopathic effect) (Reinert et al, 2005; Halabi-Cabezon et al, 2008) and to apoptosis (cytotoxic effect) (Matarrese et al, 2007). It has been shown that high concentrations of TcdB, but not TcdA, have an additional effect causing necrotic cell death termed pyknosis (Farrow et al, 2013; Wohlan et al, 2014; Chumbler et al, 2016; Frädrich et al, 2016) This is manifested in morphological changes such as chromatin condensation, ballooning of the plasma membrane and loss of membrane integrity.
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