The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

Stieglitz, Florian; Gerhard, Ralf; Pich, Andreas

doi:10.3389/fmicb.2021.725612

Cited by 13 publications

(11 citation statements)

References 36 publications

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“…BioID2 assays and mass spectrometry were performed essentially as previously reported (63, 77). Detailed information is described in SI Appendix, Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Ena/VASP clustering at microspike tips involves Lamellipodin but not I-BAR proteins, and absolutely requires unconventional Myosin-X

Pokrant¹,

Hein²,

Körber³

et al. 2022

Preprint

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Sheet-like membrane protrusions at the leading edge, termed lamellipodia, drive cell migration using active actin polymerization. Microspikes comprise actin-filament bundles embedded within lamellipodia, but their relevance for lamellipodium function has remained elusive. Microspike formation requires the specific activity of clustered Ena/VASP proteins at their tips to enable processive actin assembly in the presence of capping protein, but the factors and mechanisms mediating Ena/VASP clustering are poorly understood. Systematic analyses of B16-F1 melanoma mutants lacking potential candidate proteins revealed that neither inverse BAR-domain proteins, nor lamellipodin or Abi are essential for clustering, although they differentially contribute to lamellipodial VASP accumulation. In contrast, unconventional myosin-X (MyoX) identified here as proximal to VASP was obligatory for Ena/VASP clustering and microspike formation. Notably, MyoX removal caused marked defects in protrusion and migration. Strikingly, Ena/VASP-deficiency uncoupled MyoX cluster dynamics from actin assembly in lamellipodia, establishing their tight functional association in microspike formation and lamellipodia stabilization.

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“…BioID2 assays and mass spectrometry were performed essentially as previously reported (63, 77). Detailed information is described in SI Appendix, Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Ena/VASP clustering at microspike tips involves Lamellipodin but not I-BAR proteins, and absolutely requires unconventional Myosin-X

Pokrant¹,

Hein²,

Körber³

et al. 2022

Preprint

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“…Cell Culture was proceeded as previously described [ 89 ]. HEp-2 cells were maintained in a 75 cm 2 flask in humidified atmosphere at 37 °C and 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Tryptic digestion of proteins proceeded as previously described [ 89 ]. Protein concentrations were calculated using DC protein assay (Bio-Rad, Hercules, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

Clostridium novyi’s Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells

Schweitzer

Genth

Pich

2022

IJMS

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C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.

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“…The specific concentration and purity of toxins was estimated by SDS gel electrophoresis. We have produced CDT as authors of this study have described before in Stieglitz et al (2021) . This report provides evidence by 1,000 CDT-regulated phosphorylation sites that CDT used in this study is LPS-free and did not induce any classical LPS-dependent signaling pathway such as the ERK pathway [ Supplementary Tables 1 and 2 in Stieglitz et al (2021) ].…”

Section: Methodsmentioning

confidence: 99%

Clostridioides difficile Toxin CDT Induces Cytotoxic Responses in Human Mucosal-Associated Invariant T (MAIT) Cells

et al. 2021

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Clostridioides difficile is the major cause of antibiotic-associated colitis (CDAC) with increasing prevalence in morbidity and mortality. Severity of CDAC has been attributed to hypervirulent C. difficile strains, which in addition to toxin A and B (TcdA, TcdB) produce the binary toxin C. difficile transferase (CDT). However, the link between these toxins and host immune responses as potential drivers of immunopathology are still incompletely understood. Here, we provide first experimental evidence that C. difficile toxins efficiently activate human mucosal-associated invariant T (MAIT) cells. Among the tested toxins, CDT and more specifically, the substrate binding and pore-forming subunit CDTb provoked significant MAIT cell activation resulting in selective MAIT cell degranulation of the lytic granule components perforin and granzyme B. CDT-induced MAIT cell responses required accessory immune cells, and we suggest monocytes as a potential CDT target cell population. Within the peripheral blood mononuclear cell fraction, we found increased IL-18 levels following CDT stimulation and MAIT cell response was indeed partly dependent on this cytokine. Surprisingly, CDT-induced MAIT cell activation was found to be partially MR1-dependent, although bacterial-derived metabolite antigens were absent. However, the role of antigen presentation in this process was not analyzed here and needs to be validated in future studies. Thus, MR1-dependent induction of MAIT cell cytotoxicity might be instrumental for hypervirulent C. difficile to overcome cellular barriers and may contribute to pathophysiology of CDAC.

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The Binary Toxin of Clostridioides difficile Alters the Proteome and Phosphoproteome of HEp-2 Cells

Cited by 13 publications

References 36 publications

Ena/VASP clustering at microspike tips involves Lamellipodin but not I-BAR proteins, and absolutely requires unconventional Myosin-X

Ena/VASP clustering at microspike tips involves Lamellipodin but not I-BAR proteins, and absolutely requires unconventional Myosin-X

Clostridium novyi’s Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells

Clostridioides difficile Toxin CDT Induces Cytotoxic Responses in Human Mucosal-Associated Invariant T (MAIT) Cells

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