10th German Pharm-Tox Summit / 91. Jahrestagung der Deutschen Gesellschaft für Experimentelle und Klinische Pharmakologie und Toxikologie (DGPT)

Prof. Dr.

Chair

Prof.

Human fibrinogen inhibits Clostridium botulinum C2 toxin

Biogenic Toxins

TOX04

AbstractBacterial AB-type protein toxins intoxicate human and animal cells in the absence of the toxin-producing bacteria, thereby causing several severe diseases in humans and animals. Clostridium botulinum C2 toxin (C2) is a binary actin ADP-ribosylating toxin consisting of the binding/translocation subunit C2II and the separate enzyme subunit C2I [1]. C2II requires proteolytic activation to generate the biological active form C2IIa which then forms a heptameric ring-like structure and binds to the cell surface. C2I binds to C2IIa, and the toxin-receptor complex is internalized via receptor-mediated endocytosis. Acidification of the endosome results in the insertion of the C2IIa pore into the endosomal membrane to translocate C2I into the cytosol [2]. There, C2I ADP-ribosylates G-actin [3] resulting in cell rounding and barrier disruption.In conclusion, we discovered a novel function of the human blood coagulation factor fibrinogen as a potent inhibitor of C2 toxin, suggesting a novel role of fibrinogen in innate immunity.ResultsHere we discovered that fibrinogen protects human HeLa and CaCo-2 cells from intoxication with C2 and prevents C2 toxin-associated apoptosis and barrier disruption. However, fibrinogen had no protective effect against related bacterial toxins including anthrax, CDT, and iota. Fibrinogen had no effect on intoxication of cells with Clostridioides difficile TcdB but prevented the transport of its glycosyltransferase domain via C2IIa into cells, indicating that the inhibitory effect is highly specific for C2IIa. Consistently, fibrinogen prevented the binding of C2IIa to cells as shown by flow cytometry while leaving the enzyme activity unaffected. ELISA and SPR analysis revealed that fibrinogen specifically binds to C2IIa in vitro but not to the binding subunits of related binary anthrax or iota toxins. In addition, human blood plasma containing fibrinogen interacted with C2IIa, whereas serum without fibrinogen or plasma after fibrinogen depletion by thrombin cleavage did not.[1] K. Aktories and H. Barth (2004), Int. J. Med. Microbiol.[2] H. Barth et al. (2000), J. Biol. Chem., 18704–18711[3] K. Aktories et al. (1986), Nature, 322, 6077

Toxicology

Short talk

TcdA and TcdB from Clostridioides difficile are inhibited by the antimicrobial peptide Angie 5

The spore-forming, toxin-producing nosocomial human gut pathogen Clostridioides (C.) difficile is the causative agent of gastrointestinal infections, leading to mild to severe diarrhea. More life-threatening conditions, such as pseudomembranous colitis, colonic perforation or toxic megacolon are attributed to severe C. difficile infections (CDI). The two AB-type protein toxins, toxin A (TcdA) and toxin B (TcdB) are the main virulence factors of C. difficile and responsible for CDI symptoms. Both toxins are large, single-chain proteins with multiple domains, and members of the clostridial glucosylating toxins family. After binding of the toxins to target cells, receptor-mediated endocytosis occurs. Next, acidification of respective endosomes triggers the insertion and pore formation of the toxins into the endosomal membrane. Through this pore, their toxic glucosyltransferase domain (GTD) is delivered into the cytosol, where it glucosylates its target proteins, small GTPases of the Rho and/or Ras family. This leads, amongst others, to the collapse of the actin cytoskeleton, and ultimately, to cell death. Here, we show that the in silico predicted antimicrobial peptides, termed Angies as they derive from the human endogenous protein angiogenin, are inhibitors of TcdA and TcdB. Among the six different Angie peptides designed and tested, Angie 5 showed the strongest inhibitory capacity in HeLa and Vero cells, as well as in the physiologically more relevant human colon carcinoma cell line CaCo-2. This was demonstrated by monitoring the toxin-induced cell rounding due to actin depolymerization in the cells, using fluorescence microscopy, among other assays. Furthermore, Angie 5 induced a delay in the specific TcdA/TcdB-mediated glucosylation of its substrate protein Rac1, as shown by Western blot. These results contribute to the basis for the future development of antimicrobial peptide-based anti-toxin strategies against C. difficile-associated diseases (CDADs).Noschka, R., Gerbl, F., Löffler, F., Kubis, J., Rodríguez, A. A., Mayer, D., … Stenger, S. (2021). Unbiased Identification of Angiogenin as an Endogenous Antimicrobial Protein With Activity Against Virulent Mycobacterium tuberculosis. Frontiers in Microbiology, 11, 618278. https://doi.org/10.3389/fmicb.2020.618278

Synthesis, characterization and biomedical application of Shiga-Toxins by cell-free protein synthesis

Ribosome-inactivating proteins (RIPs) are found amongst bacteria and plants. RIPs are known to act enzymatically upon the eukaryotic 60S ribosomal subunit or translation elongation factor 2. Thus, RIPs are potent inhibitors of eukaryotic protein translation. This led to great scientific interest due to their potential harmful nature to human health but also pose a possibility in biomedical research as anti-cancer pharmaceuticals. Since the synthesis of RIPs is rather limited, their functional characterization is challenging. Here, we present cell-free protein synthesis (CFPS) as an alternative for the synthesis, characterization and application of RIPs. CFPS is especially valuable for the synthesis of toxins as a lysate rather than viable cells is used as the toxic nature of the protein cannot harm the cell"s viability. CFPS based on eukaryotic lysates was used to synthesize Shiga-toxins including the AB5 holotoxins as well as the single subunits. We show that the synthesis of the individual subunits as well as the holotoxin was possible. The activity of the Shiga holotoxin could be shown in cell-based assays on HeLa and U87-MG cells at concentrations of 0.1 nM. In some cases, the mode of action of toxins is difficult to investigate. Hence, the Shiga A-subunit was modified by the introduction of non-canonical amino acids in order to fluorescently label the toxin. This technique can be used to track the toxin within the cell. We show that diverse labeling strategies could be established to fluorescently label the A-subunit. At last, in a biomedical interest, the A-subunit of the Shiga-toxin was fused to an interleukin 13 (IL13) in order to address glioblastoma cells as a targeted toxin. Further analyses of the targeted toxin on HeLa, U87-MG and SH-SY5Y were performed. These data showed that the cells expressing the IL13 receptor were directly addressed by the targeted toxin and the target cells were apoptotic at concentrations of 1 nM. The receptor binding was further confirmed by ELISA binding assays. Taken together these data show the versatile use of CFPS for the analysis and modification of RIPs.

Exploiting Host Cell NEDDylation Inhibitors for Combating Bacterial Toxins

NEDDylation is a reversible post-translational modification (PTM) that attaches the ubiquitin-like molecule NEDD8 to lysine residues on protein substrates, impacting their stability, degradation, and interactions (Li et al., 2020). Recently, it was found that NEDDylation affected virus entry and infection. For instance, incubation of cells with the NEDDylation inhibitor MLN4924 prevented endocytic uptake of the herpes simplex virus (HSV) (Lee et al., 2022).AB-type bacterial toxins share similar cell entry mechanisms with viruses. Therefore, we aimed to examine the potential involvement of NEDDylation in the cellular internalization of the diphtheria toxin (DT), a prototypical, well-established AB-type toxin model. Surprisingly, we found that intoxication with DT was delayed in HeLa cells pretreated with the MLN4924 compound. Further mechanistic analysis revealed that MLN4924 neither affected binding or activation of DT at the cell surface nor interfered with endosomal acidification, a critical step during the endocytic uptake of AB-type toxins. Moreover, we could demonstrate the inhibitory potential of MLN4924 also against two other bacterial toxins, namely Clostridioides difficile toxin B (TcdB) and Clostridium botulinum C2 toxin. Importantly, arctigenin, which represents another NEDDylation inhibitor acting at a later step of the NEDDylation cascade than MLN4924, was also capable of inhibiting DT. Notably, combining both NEDDylation inhibitors synergistically increased their inhibitory potential against DT.In conclusion, our pivotal study provides first evidence of the involvement of NEDDylation during the cellular uptake and/or intoxication process of bacterial AB-type toxins, thus offering a foundation for further research into these molecular mechanisms and for the development of anti-toxin strategies.References: Li, J., Zou, J., Littlejohn, R., Liu, J., & Su, H. (2020). Neddylation, an Emerging Mechanism Regulating Cardiac Development and Function. Frontiers in physiology, 11, 612927. https://doi.org/10.3389/fphys.2020.612927 Lee, B. H., Tebaldi, G., Pritchard, S. M., & Nicola, A. V. (2022). Host Cell Neddylation Facilitates Alphaherpesvirus Entry in a Virus-Specific and Cell-Dependent Manner. Microbiology spectrum, 10(5), e0311422. https://doi.org/10.1128/spectrum.03114-22

Short talks 1
Biogenic Toxins

Appointment

Chair

Programme

Short talks 1Biogenic Toxins﻿

Appointment

Chair

Programme

Short talks 1
Biogenic Toxins