Publications by authors named "Holger Barth"

122 Publications

Cytotoxic Effects of Recombinant StxA2-His in the Absence of Its Corresponding B-Subunit.

Toxins (Basel) 2021 04 26;13(5). Epub 2021 Apr 26.

Department of Food Microbiology and Hygiene, Institute of Food Science and Biotechnology, Garbenstraße 28, University of Hohenheim, 70599 Stuttgart, Germany.

AB protein toxins are produced by certain bacterial pathogens and are composed of an enzymatically active A-subunit and a B-subunit pentamer, the latter being responsible for cell receptor recognition, cellular uptake, and transport of the A-subunit into the cytosol of eukaryotic target cells. Two members of the AB toxin family were described in Shiga toxin-producing (STEC), namely Shiga toxin (Stx) and subtilase cytotoxin (SubAB). The functional paradigm of AB toxins includes the B-subunit being mandatory for the uptake of the toxin into its target cells. Recent studies have shown that this paradigm cannot be maintained for SubAB, since SubA alone was demonstrated to intoxicate human epithelial cells in vitro. In the current study, we raised the hypothesis that this may also be true for the A-subunit of the most clinically relevant Stx-variant, Stx2a. After separate expression and purification, the recombinant Stx2a subunits StxA2a-His and StxB2a-His were applied either alone or in combination in a 1:5 molar ratio to Vero B4, HeLa, and HCT-116 cells. For all cell lines, a cytotoxic effect of StxA2a-His alone was detected. Competition experiments with Stx and SubAB subunits in combination revealed that the intoxication of StxA2a-His was reduced by addition of SubB1-His. This study showed that the enzymatic subunit StxA2a alone was active on different cells and might therefore play a yet unknown role in STEC disease development.
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http://dx.doi.org/10.3390/toxins13050307DOI Listing
April 2021

Nanodiamond Theranostic for Light-Controlled Intracellular Heating and Nanoscale Temperature Sensing.

Nano Lett 2021 May 21;21(9):3780-3788. Epub 2021 Apr 21.

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128 Mainz, Germany.

Temperature is an essential parameter in all biological systems, but information about the actual temperature in living cells is limited. Especially, in photothermal therapy, local intracellular temperature changes induce cell death but the local temperature gradients are not known. Highly sensitive nanothermometers would be required to measure and report local temperature changes independent of the intracellular environment, including pH or ions. Fluorescent nanodiamonds (ND) enable temperature sensing at the nanoscale independent of external conditions. Herein, we prepare ND nanothermometers coated with a nanogel shell and the photothermal agent indocyanine green serves as a heat generator and sensor. Upon irradiation, programmed cell death was induced in cancer cells with high spatial control. In parallel, the increase in local temperature was recorded by the ND nanothermometers. This approach represents a great step forward to record local temperature changes in different cellular environments inside cells and correlate these with thermal biology.
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http://dx.doi.org/10.1021/acs.nanolett.1c00043DOI Listing
May 2021

The cytotoxic effect of Clostridioides difficile pore-forming toxin CDTb.

Biochim Biophys Acta Biomembr 2021 Jun 6;1863(6):183603. Epub 2021 Mar 6.

Institute of Pharmacology and Toxicology, Ulm University Medical Center, Ulm, Germany.

Clostridioides (C.) difficile is clinically highly relevant and produces several AB-type protein toxins, which are the causative agents for C. difficile-associated diarrhea and pseudomembranous colitis. Treatment with antibiotics can lead to C. difficile overgrowth in the gut of patients due to the disturbed microbiota. C. difficile releases large Rho/Ras-GTPase glucosylating toxins TcdA and TcdB, which are considered as the major virulence factors for C. difficile-associated diseases. In addition to TcdA and TcdB, C. difficile strains isolated from severe cases of colitis produce a third toxin called CDT. CDT is a member of the family of clostridial binary actin ADP-ribosylating toxins and consists of two separate protein components. The B-component, CDTb, binds to the receptor and forms a complex with and facilitates transport and translocation of the enzymatically active A-component, CDTa, into the cytosol of target cells by forming trans-membrane pores through which CDTa translocates. In the cytosol, CDTa ADP-ribosylates G-actin causing depolymerization of the actin cytoskeleton and, eventually, cell death. In the present study, we report that CDTb exhibits a cytotoxic effect in the absence of CDTa. We show that CDTb causes cell rounding and impairs cell viability and the epithelial integrity of CaCo-2 monolayers in the absence of CDTa. CDTb-induced cell rounding depended on the presence of LSR, the specific cellular receptor of CDT. The isolated receptor-binding domain of CDTb was not sufficient to cause cell rounding. CDTb-induced cell rounding was inhibited by enzymatically inactive CDTa or a pore-blocker, implying that CDTb pores in cytoplasmic membranes contribute to cytotoxicity.
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http://dx.doi.org/10.1016/j.bbamem.2021.183603DOI Listing
June 2021

Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo.

Sci Rep 2021 Mar 8;11(1):5429. Epub 2021 Mar 8.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.

Whooping cough is caused by Bordetella pertussis that releases pertussis toxin (PT) which comprises enzyme A-subunit PTS1 and binding/transport B-subunit. After receptor-mediated endocytosis, PT reaches the endoplasmic reticulum from where unfolded PTS1 is transported to the cytosol. PTS1 ADP-ribosylates G-protein α-subunits resulting in increased cAMP signaling. Here, a role of target cell chaperones Hsp90, Hsp70, cyclophilins and FK506-binding proteins for cytosolic PTS1-uptake is demonstrated. PTS1 specifically and directly interacts with chaperones in vitro and in cells. Specific pharmacological chaperone inhibition protects CHO-K1, human primary airway basal cells and a fully differentiated airway epithelium from PT-intoxication by reducing intracellular PTS1-amounts without affecting cell binding or enzyme activity. PT is internalized by human airway epithelium secretory but not ciliated cells and leads to increase of apical surface liquid. Cyclophilin-inhibitors reduced leukocytosis in infant mouse model of pertussis, indicating their promising potential for developing novel therapeutic strategies against whooping cough.
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http://dx.doi.org/10.1038/s41598-021-84817-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940712PMC
March 2021

The enzyme subunit SubA of Shiga toxin-producing E. coli strains demonstrates comparable intracellular transport and cytotoxic activity as the holotoxin SubAB in HeLa and HCT116 cells in vitro.

Arch Toxicol 2021 03 23;95(3):975-983. Epub 2021 Jan 23.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Albert-Einstein-Allee 11, 89081, Ulm, Germany.

The subtilase cytotoxin (SubAB) is secreted by certain Shiga toxin-producing Escherichia coli (STEC) strains and is composed of the enzymatically active subunit SubA and the pentameric binding/transport subunit SubB. We previously demonstrated that SubA (10 µg/ml), in the absence of SubB, binds and intoxicates the human cervix cancer-derived epithelial cell line HeLa. However, the cellular and molecular mechanisms underlying the cytotoxic activity of SubA in the absence of SubB remained unclear. In the present study, the cytotoxic effects mediated by SubA alone were investigated in more detail in HeLa cells and the human colon cancer cell line HCT116. We found that in the absence of SubB, SubA (10 µg/ml) is internalized into the endoplasmic reticulum (ER), where it cleaves the chaperone GRP78, an already known substrate for SubA after its canonical uptake into cells via SubB. The autonomous cellular uptake of SubA and subsequent cleavage of GRP78 in cells is prevented by treatment of cells with 10 µM brefeldin A, which inhibits the transport of protein toxins into the ER. In addition, by analyzing the SubA mutant SubA, we identified the C-terminal SEEL motif as an ER-targeting signal. Conclusively, our results strongly suggest that SubA alone shares the same intracellular transport route and cytotoxic activity as the SubAB holotoxin.
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http://dx.doi.org/10.1007/s00204-020-02965-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904543PMC
March 2021

Intoxication of mammalian cells with binary clostridial enterotoxins is inhibited by the combination of pharmacological chaperone inhibitors.

Naunyn Schmiedebergs Arch Pharmacol 2021 05 7;394(5):941-954. Epub 2020 Dec 7.

Institute of Pharmacology and Toxicology, Ulm University Medical Center, 89081, Ulm, Germany.

Binary enterotoxins Clostridioides difficile CDT toxin, Clostridium botulinum C2 toxin, and Clostridium perfringens iota toxin consist of two separate protein components. The B-components facilitate receptor-mediated uptake into mammalian cells and form pores into endosomal membranes through which the enzymatic active A-components translocate into the cytosol. Here, the A-components ADP-ribosylate G-actin which leads to F-actin depolymerization followed by rounding of cells which causes clinical symptoms. The protein folding helper enzymes Hsp90, Hsp70, and peptidyl-prolyl cis/trans isomerases of the cyclophilin (Cyp) and FK506 binding protein (FKBP) families are required for translocation of A-components of CDT, C2, and iota toxins from endosomes to the cytosol. Here, we demonstrated that simultaneous inhibition of these folding helpers by specific pharmacological inhibitors protects mammalian, including human, cells from intoxication with CDT, C2, and iota toxins, and that the inhibitor combination displayed an enhanced effect compared to application of the individual inhibitors. Moreover, combination of inhibitors allowed a concentration reduction of the individual compounds as well as decreasing of the incubation time with inhibitors to achieve a protective effect. These results potentially have implications for possible future therapeutic applications to relieve clinical symptoms caused by bacterial toxins that depend on Hsp90, Hsp70, Cyps, and FKBPs for their membrane translocation into the cytosol of target cells.
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http://dx.doi.org/10.1007/s00210-020-02029-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102464PMC
May 2021

Croconaine-Based Polymer Particles as Contrast Agents for Photoacoustic Imaging.

Macromol Rapid Commun 2020 Nov 12;41(22):e2000418. Epub 2020 Oct 12.

DWI-Leibniz Institute for Interactive Materials, Forckenbeckstraße 50, Aachen, 52074, Germany.

In the development and optimization of imaging methods, photoacoustic imaging (PAI) has become a powerful tool for preclinical biomedical diagnosis and detection of cancer. PAI probes can improve contrast and help identify pathogenic tissue. Such contrast agents must meet several requirements: they need to be biocompatible, and absorb strongly in the near-infrared (NIR) range, while relaxing the photoexcited state thermally and not radiatively. In this work, polymer nanoparticles are produced with croconaine as a monomer unit. Small molecular croconaine dyes are known to act as efficient pigments, which do not show photoluminescence. Here, for the first time croconaine copolymer nanoparticles are produced from croconic acid and a range of aromatic diamines. Following a dispersion polymerization protocol, this approach yields monodisperse particles of adjustable size. All synthesized polymers exhibit broad absorption within the NIR spectrum and therefore represent suitable candidates as contrast agents for PAI. The optical properties of these polymer particles are discussed with respect to the relation between particle size and outstanding photoacoustic performance. Biocompatibility of the polymer particles is demonstrated in cell viability experiments.
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http://dx.doi.org/10.1002/marc.202000418DOI Listing
November 2020

Human α-Defensin-5 Efficiently Neutralizes Toxins TcdA, TcdB, and CDT.

Front Pharmacol 2020 12;11:1204. Epub 2020 Aug 12.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.

Infections with the pathogenic bacterium are coming more into focus, in particular in hospitalized patients after antibiotic treatment. produces the exotoxins TcdA and TcdB. Since some years, hypervirulent strains are described, which produce in addition the binary actin ADP-ribosylating toxin CDT. These strains are associated with more severe clinical presentations and increased morbidity and frequency. Once in the cytosol of their target cells, the catalytic domains of TcdA and TcdB glucosylate and thereby inactivate small Rho-GTPases whereas the enzyme subunit of CDT ADP-ribosylates G-actin. Thus, enzymatic activity of the toxins leads to destruction of the cytoskeleton and breakdown of the epidermal gut barrier integrity. This causes clinical symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis. Therefore, pharmacological inhibition of the secreted toxins is of peculiar medical interest. Here, we investigated the neutralizing effect of the human antimicrobial peptide α-defensin-5 toward TcdA, TcdB, and CDT in human cells. The toxin-neutralizing effects of α-defensin-5 toward TcdA, TcdB, and CDT as well as their medically relevant combination were demonstrated by analyzing toxins-induced changes in cell morphology, intracellular substrate modification, and decrease of trans-epithelial electrical resistance. For TcdA, the underlying mode of inhibition is most likely based on the formation of inactive toxin-defensin-aggregates whereas for CDT, the binding- and transport-component might be influenced. The application of α-defensin-5 delayed intoxication of cells in a time- and concentration-dependent manner. Due to its effect on the toxins, α-defensin-5 should be considered as a candidate to treat severe -associated diseases.
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http://dx.doi.org/10.3389/fphar.2020.01204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435013PMC
August 2020

Clostridial C3 Toxins Enter and Intoxicate Human Dendritic Cells.

Toxins (Basel) 2020 09 1;12(9). Epub 2020 Sep 1.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany.

C3 protein toxins produced by and are mono-ADP-ribosyltransferases, which specifically modify the GTPases Rho A/B/C in the cytosol of monocytic cells, thereby inhibiting Rho-mediated signal transduction in monocytes, macrophages, and osteoclasts. C3 toxins are selectively taken up into the cytosol of monocytic cells by endocytosis and translocate from acidic endosomes into the cytosol. The C3-catalyzed ADP-ribosylation of Rho proteins inhibits essential functions of these immune cells, such as migration and phagocytosis. Here, we demonstrate that C3 toxins enter and intoxicate dendritic cells in a time- and concentration-dependent manner. Both immature and mature human dendritic cells efficiently internalize C3 exoenzymes. These findings could also be extended to the chimeric fusion toxin C2IN-C3lim. Moreover, stimulated emission depletion (STED) microscopy revealed the localization of the internalized C3 protein in endosomes and emphasized its potential use as a carrier to deliver foreign proteins into dendritic cells. In contrast, the enzyme C2I from the binary C2 toxin was not taken up into dendritic cells, indicating the specific uptake of C3 toxins. Taken together, we identified human dendritic cells as novel target cells for clostridial C3 toxins and demonstrated the specific uptake of these toxins via endosomal vesicles.
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http://dx.doi.org/10.3390/toxins12090563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551598PMC
September 2020

PA Delivers the Tumor Metastasis Suppressor Protein NDPK-A/NME1 into Breast Cancer Cells.

Int J Mol Sci 2020 May 6;21(9). Epub 2020 May 6.

Experimental Pharmacology, European Center for Angioscience, Mannheim Medical Faculty, Heidelberg University, 68167 Mannheim, Germany.

Some highly metastatic types of breast cancer show decreased intracellular levels of the tumor suppressor protein NME1, also known as nm23-H1 or nucleoside diphosphate kinase A (NDPK-A), which decreases cancer cell motility and metastasis. Since its activity is directly correlated with the overall outcome in patients, increasing the cytosolic levels of NDPK-A/NME1 in such cancer cells should represent an attractive starting point for novel therapeutic approaches to reduce tumor cell motility and decrease metastasis. Here, we established the protein toxins' transport component PA as transporter for the delivery of His-tagged human NDPK-A into the cytosol of cultured cells including human MDA-MB-231 breast cancer cells. The specifically delivered His-tagged NDPK-A was detected in MDA-MB-231 cells via Western blotting and immunofluorescence microscopy. The PA-mediated delivery of His-NDPK-A resulted in reduced migration of MDA-MB-231 cells, as determined by a wound-healing assay. In conclusion, PA serves for the transport of the tumor metastasis suppressor NDPK-A/NME1 into the cytosol of human breast cancer cells in vitro, which reduced the migratory activity of these cells. This approach might lead to development of novel therapeutic options.
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http://dx.doi.org/10.3390/ijms21093295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246847PMC
May 2020

Super-resolution microscopy unveils transmembrane domain-mediated internalization of cross-reacting material 197 into diphtheria toxin-resistant mouse J774A.1 cells and primary rat fibroblasts in vitro.

Arch Toxicol 2020 05 8;94(5):1753-1761. Epub 2020 Apr 8.

Institute of Biophysics, Ulm University, Albert-Einstein-Allee 11, 89081, Ulm, Germany.

Diphtheria toxin (DT) efficiently inhibits protein synthesis in human cells, resulting in severe disease diphtheria. The sensitivity towards DT varies between mammalian species. Mice and rats are resistant to DT. However, the reason underlying this insensitivity is controversially discussed and not well understood. Therefore, we investigated the steps of DT uptake, i.e. receptor binding and internalization into mouse J774A.1 macrophages and primary rat fibroblasts. We exploited the non-toxic DT-mutant cross-reacting material 197 (CRM197) and three additional receptor binding-deficient mutants (250 nM each) to investigate binding to cell surface and internalization into murine cells via flow cytometry and stimulated emission depletion (STED) super-resolution optical microscopy. Dual-color STED imaging unveiled CRM197 interacting with the murine precursor of the heparin-binding epidermal growth factor-like growth factor (HB-EGF). Moreover, we identified CRM197's transmembrane domain as an additional HB-EGF binding site, which is also involved in the receptor-mediated internalization into murine cells. However, we do not find evidence for translocation of the catalytically active subunit (DTA) into the cytosol when 250 nM DT were applied. In conclusion, we provide evidence that the resistance of murine cells to DT is caused by an insufficiency of DTA to escape from endosomes and reach the cytosol. Possibly, a higher affinity interaction of DT and the HB-EGF is required for translocation, which highlights the role of the receptor in the endosomes during the translocation step. We extend the current knowledge about cellular uptake of the medically relevant DT and CRM197.
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http://dx.doi.org/10.1007/s00204-020-02731-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261736PMC
May 2020

Human peptide α-defensin-1 interferes with Clostridioides difficile toxins TcdA, TcdB, and CDT.

FASEB J 2020 05 19;34(5):6244-6261. Epub 2020 Mar 19.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.

The human pathogenic bacterium Clostridioides difficile produces two exotoxins TcdA and TcdB, which inactivate Rho GTPases thereby causing C. difficile-associated diseases (CDAD) including life-threatening pseudomembranous colitis. Hypervirulent strains produce additionally the binary actin ADP-ribosylating toxin CDT. These strains are hallmarked by more severe forms of CDAD and increased frequency and severity. Once in the cytosol, the toxins act as enzymes resulting in the typical clinical symptoms. Therefore, targeting and inactivation of the released toxins are of peculiar interest. Prompted by earlier findings that human α-defensin-1 neutralizes TcdB, we investigated the effects of the defensin on all three C. difficile toxins. Inhibition of TcdA, TcdB, and CDT was demonstrated by analyzing toxin-induced changes in cell morphology, substrate modification, and decrease in transepithelial electrical resistance. Application of α-defensin-1 protected cells and human intestinal organoids from the cytotoxic effects of TcdA, TcdB, CDT, and their combination which is attributed to a direct interaction between the toxins and α-defensin-1. In mice, the application of α-defensin-1 reduced the TcdA-induced damage of intestinal loops in vivo. In conclusion, human α-defensin-1 is a specific and potent inhibitor of the C. difficile toxins and a promising agent to develop novel therapeutic options against C. difficile infections.
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http://dx.doi.org/10.1096/fj.201902816RDOI Listing
May 2020

Regulation of endo-lysosomal pathway and autophagic flux by broad-spectrum antipathogen inhibitor ABMA.

FEBS J 2020 08 21;287(15):3184-3199. Epub 2020 Jan 21.

Université Paris-Saclay, CEA, INRAE, Médicaments et Technologies pour la Santé (MTS), SIMoS, Gif-sur-Yvette, 91191, France.

The endo-lysosome system is involved in endocytosis, protein sorting, and degradation as well as autophagy. Numerous toxins and pathogens exploit this system to enter host cells and exert their deleterious effects. Modulation of host endo-lysosome pathway may restrict multiple toxins intoxication as well as pathogen infection. ABMA, selected from a high-throughput screening against the cytotoxicity of ricin toxin, exhibits a broad-spectrum antitoxin and antipathogen activity. Here, we show that ABMA selectively retains endocytosed protein and toxin to late endosomes and thus delaying their intracellular trafficking. It also impairs the autophagic flux by excessive fusion of late endosomes and autophagosomes. Its exclusive action on late endosomes and corresponding consequences on the endo-lysosomal pathway and autophagic flux are distinct from known inhibitors such as bafilomycin A1, EGA, or chloroquine. Hence, besides being a broad-spectrum inhibitor of endocytosed toxins and pathogens, ABMA may serve as a molecular tool to dissect endo-lysosome system-related cellular physiology and mechanisms of pathogenesis.
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http://dx.doi.org/10.1111/febs.15201DOI Listing
August 2020

Variants of Subtilase Cytotoxin Subunits Show Differences in Complex Formation In Vitro.

Toxins (Basel) 2019 12 3;11(12). Epub 2019 Dec 3.

Department of Food Microbiology and Hygiene, Institute of Food Science and Biotechnology, Garbenstraße 28, University of Hohenheim, 70599 Stuttgart, Germany.

The subtilase cytotoxin (SubAB) of Shiga toxin-producing (STEC) is a member of the AB toxin family. In the current study, we analyzed the formation of active homo- and hetero-complexes of SubAB variants in vitro to characterize the mode of assembly of the subunits. Recombinant SubA1-His, SubB1-His, SubA2-2-His, and SubB2-2-His subunits, and His-tag-free SubA2-2 were separately expressed, purified, and biochemically characterized by circular dichroism (CD) spectroscopy, size-exclusion chromatography (SEC), and analytical ultracentrifugation (aUC). To confirm their biological activity, cytotoxicity assays were performed with HeLa cells. The formation of AB complexes was investigated with aUC and isothermal titration calorimetry (ITC). Binding of SubAB2-2-His to HeLa cells was characterized with flow cytometry (FACS). Cytotoxicity experiments revealed that the analyzed recombinant subtilase subunits were biochemically functional and capable of intoxicating HeLa cells. Inhibition of cytotoxicity by Brefeldin A demonstrated that the cleavage is specific. All His-tagged subunits, as well as the non-tagged SubA2-2 subunit, showed the expected secondary structural compositions and oligomerization. Whereas SubAB1-His complexes could be reconstituted in solution, and revealed a value of 3.9 ± 0.8 μmol/L in the lower micromolar range, only transient interactions were observed for the subunits of SubAB2-2-His in solution, which did not result in any binding constant when analyzed with ITC. Additional studies on the binding characteristics of SubAB2-2-His on HeLa cells revealed that the formation of transient complexes improved binding to the target cells. Conclusively, we hypothesize that SubAB variants exhibit different characteristics in their binding behavior to their target cells.
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http://dx.doi.org/10.3390/toxins11120703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6950094PMC
December 2019

The Antibiotic Bacitracin Protects Human Intestinal Epithelial Cells and Stem Cell-Derived Intestinal Organoids from Toxin TcdB.

Stem Cells Int 2019 5;2019:4149762. Epub 2019 Aug 5.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany.

Bacitracin is an established antibiotic for local application and inhibits the cell wall synthesis of Gram-positive bacteria. Recently, we discovered a completely different mode of action of bacitracin and reported that this drug protects human cells from intoxication by a variety of medically relevant bacterial protein toxins including CDT, the binary actin ADP-ribosylating toxin of () . Bacitracin prevents the transport of CDT into the cytosol of target cells, most likely by inhibiting the transport function of the binding subunit of this toxin. Here, we tested the effect of bacitracin towards TcdB, a major virulence factor of contributing to severe -associated diseases (CDAD) including pseudomembranous colitis. Bacitracin protected stem cell-derived human intestinal organoids as well as human gut epithelial cells from intoxication with TcdB. Moreover, it prevented the TcdB-induced disruption of epithelia formed by gut epithelium cells and maintained the barrier function as detected by measuring transepithelial electrical resistance (TEER). In the presence of bacitracin, TcdB was not able reach its substrate Rac1 in the cytosol of human epithelial cells, most likely because its pH-dependent transport across cell membranes into the cytosol is decreased by bacitracin. In conclusion, in addition to its direct antibiotic activity against and its inhibitory effect towards the toxin CDT, bacitracin neutralizes the exotoxin TcdB of this important pathogenic bacterium.
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http://dx.doi.org/10.1155/2019/4149762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6701344PMC
August 2019

Supramolecular Toxin Complexes for Targeted Pharmacological Modulation of Polymorphonuclear Leukocyte Functions.

Adv Healthc Mater 2019 09 18;8(17):e1900665. Epub 2019 Jul 18.

Institute of Pharmacology and Toxicology - Ulm University Medical Center, Albert-Einstein-Allee 11, 89081, Ulm, Germany.

The targeted pharmacological modulation of polymorphonuclear leukocytes (PMNs) is of major medical interest. These innate immune cells play a central role in the defense against pathogenic microorganisms. However, their excessive chemotactic recruitment into tissues after traumatic injury is detrimental due to local and systemic inflammation. Rho-GTPases, being the master regulators of the actin cytoskeleton, regulate migration and chemotaxis of PMNs, are attractive pharmacological targets. Herein, supramolecular protein complexes are assembled in a "mix-and-match" approach containing the specific Rho-inhibiting clostridial C3 enzyme and three PMN-binding peptides using an avidin platform. Selective delivery of the C3 Rho-inhibitor with these complexes into the cytosol of human neutrophil-like NB-4 cells and primary human PMNs ex vivo is demonstrated, where they catalyze the adenosine diphosphate (ADP) ribosylation of Rho and induce a characteristic change in cell morphology. Notably, the complexes do not deliver C3 enzyme into human lung epithelial cells, A549 lung cancer cells, and immortalized human alveolar epithelial cells (hAELVi), demonstrating their cell type-selectivity. The supramolecular complexes represent attractive molecular tools to decipher the role of PMNs in infection and inflammation or for the development of novel therapeutic approaches for diseases that are associated with hyperactivity and reactivity of PMNs such as post-traumatic injury.
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http://dx.doi.org/10.1002/adhm.201900665DOI Listing
September 2019

Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication.

FASEB J 2019 04 30;33(4):5755-5771. Epub 2019 Jan 30.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, Ulm, Germany.

The antibiotic bacitracin (Bac) inhibits cell wall synthesis of gram-positive bacteria. Here, we discovered a totally different activity of Bac: the neutralization of bacterial exotoxins. Bac prevented intoxication of mammalian cells with the binary enterotoxins Clostridium botulinum C2, C. perfringens ι, C. difficile transferase (CDT), and Bacillus anthracis lethal toxin. The transport (B) subunits of these toxins deliver their respective enzyme (A) subunits into cells. Following endocytosis, the B subunits form pores in membranes of endosomes, which mediate translocation of the A subunits into the cytosol. Bac inhibited formation of such B pores in lipid bilayers in vitro and in living cells, thereby preventing translocation of the A subunit into the cytosol. Bac preserved the epithelial integrity of toxin-treated CaCo-2 monolayers, a model for the human gut epithelium. In conclusion, Bac should be discussed as a therapeutic option against infections with medically relevant toxin-producing bacteria, including C. difficile and B. anthracis, because it inhibits bacterial growth and neutralizes the secreted toxins.-Schnell, L., Felix, I., Müller, B., Sadi, M., von Bank, F., Papatheodorou, P., Popoff, M. R., Aktories, K., Waltenberger, E., Benz, R., Weichbrodt, C., Fauler, M., Frick, M., Barth, H. Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication.
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http://dx.doi.org/10.1096/fj.201802453RDOI Listing
April 2019

Prognostic impact of polypharmacy and drug interactions in patients with advanced cancer.

Cancer Chemother Pharmacol 2019 04 25;83(4):763-774. Epub 2019 Jan 25.

Department of Medical Oncology and Hematology, Cantonal Hospital, St. Gallen, Switzerland.

The risk of potential drug-drug interactions (PDI) is poorly studied in oncology. We included 105 patients with advanced non-small-cell lung cancer (NSCLC), 100 patients with advanced breast cancer (BC) and 100 patients of the palliative care unit (PCU) receiving systemic palliative treatment between 2010 and 2015. All patients suffered from advanced incurable cancer and received basic palliative care. PDI were assessed using the hospINDEX of all drugs approved in Switzerland in combination with a specific drug interaction software. Primary study objective was to assess the prognostic impact of PDI per patient cohort using Kaplan-Meier statistics. The median number of comedications was 5 (range 0-15). Major-risk PDI were detected in 74 patients (24.3%). The number of comedications was significantly associated with PDI (p < 0.0001). Major-risk PDI increased from 14% in patients with < 4 comedications to 24% in patients with 4-7 comedications, 40% with 8-11 comedications and 67% in patients with > 11 comedications. Median overall survival (OS) was 8.6 months in NSCLC, 33 months in BC and 1.2 months in PCU patients. PDI were significantly associated with inferior OS in BC (HR = 1.32, 95% CI 1.01-1.74, p = 0.049), but not in NSCLC (HR = 1.11, 95% CI 0.84-1.47, p = 0.45) or PCU (HR = 1.12, 95% CI 0.86-1.45, p = 0.41). PDI remained significantly associated with OS in BC (HR = 1.32, p = 0.049) in the adjusted model. In conclusion, PDI are frequent in patients with advanced cancer and increased caution with polypharmacy is warranted when treating such patients.
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http://dx.doi.org/10.1007/s00280-019-03783-9DOI Listing
April 2019

Cyclic Changes in the Amide Bands Within Escherichia coli Biofilms Monitored Using Real-Time Infrared Attenuated Total Reflection Spectroscopy (IR-ATR).

Appl Spectrosc 2019 Apr 20;73(4):424-432. Epub 2019 Feb 20.

3 Institute of Analytical and Bioanalytical Chemistry, Ulm University, Ulm, Germany.

Contrary to the planktonic state of bacteria, their biofilm form represents severe complications in areas such as human medicine or food industry due to the increasing resistance against harsh conditions and treatment. In the present study, infrared attenuated total reflection (IR-ATR) spectroscopy has been applied as an analytic tool studying Escherichia coli ( E. coli) biofilm formation close to real time. We report on IR spectroscopic investigations on the biofilm formation via ATR waveguides probing the biofilm in the spectral window of 1800-900 cm at dynamic flow conditions, which facilitated monitoring the growth dynamics during several days. Key IR bands are in the range 1700-1590 cm (amide I), 1580-1490 cm (amide II), and 1141-1006 cm extracellular polymeric substances (EPS), which were evaluated as a function of time. Cyclic fluctuations of the amide I and amide II bands and a continuous increase of the EPS band were related to the starvation of bottom-layered bacteria caused by the nutrient gradient. Potential death of bacteria may then result in cannibalistic behavior known for E. coli colonies. Observing this behavior via IR spectroscopy allows revealing these cyclical changes in bottom-layered bacteria within the biofilm under continuous nutrient flow, in molecular detail, and during extended periods for the first time.
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http://dx.doi.org/10.1177/0003702819829081DOI Listing
April 2019

Combined Pharmacological Inhibition of Cyclophilins, FK506-Binding Proteins, Hsp90, and Hsp70 Protects Cells From C2 Toxin.

Front Pharmacol 2018 13;9:1287. Epub 2018 Nov 13.

Institute of Pharmacology and Toxicology, Ulm University Medical Center, Ulm, Germany.

The C2 toxin is an exotoxin causing severe enterotoxic symptoms. The C2 toxin consists of the binding/translocation component C2II, and the enzymatic active component C2I. After proteolytic activation, C2IIa forms heptamers that bind C2I. The C2IIa/C2I complex is taken up into mammalian target cells via receptor-mediated endocytosis. Acidification of endosomes leads to conformational changes in both components. C2IIa heptamers form a pore into the endosomal membrane, and C2I becomes unfolded and translocates through the narrow C2IIa pores into the cytosol of the cell. Here, C2I covalently transfers an ADP-ribose moiety from its co-substrate NAD onto G-actin, which leads to depolymerization of F-actin resulting in rounding up of adherent cells. Translocation of C2I into the cytosol depends on the activity of the chaperones Hsp90 and Hsp70 and peptidyl-prolyl isomerases of the cyclophilin (Cyp) and FK506-binding protein (FKBP) families. Here, we demonstrated that C2I is detected in close proximity with Hsp90, Cyp40, and FKBP51 in cells, indicating their interaction. This interaction was dependent on the concentration of C2 toxin and detected in mammalian Vero and human HeLa cells. Moreover, the present study reveals that combination of radicicol, VER-155008, cyclosporine A, and FK506, which are specific pharmacological inhibitors of Hsp90, Hsp70, Cyps, and FKBPs, respectively, resulted in a stronger inhibition of intoxication of cells with C2 toxin compared to application of the single inhibitors. Thus, the combination of inhibitors showed enhanced protection of cells against the cytotoxic effects of C2 toxin. Cell viability was not significantly impaired by application of the inhibitor combination. Moreover, we confirmed that the combination of radicicol, VER-155008, CsA, and FK506 in particular inhibit the membrane translocation step of C2I into the cytosol whereas receptor binding and enzyme activity of the toxin were not affected. Our findings further characterize the mode of action of Hsp90, Hsp70, Cyps, and FKBPs during membrane translocation of bacterial toxins and furthermore supply starting points for developing of novel therapeutic strategies against diseases caused by bacterial toxins that depend on Hsp90, Hsp70, Cyps, and FKBPs.
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http://dx.doi.org/10.3389/fphar.2018.01287DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243138PMC
November 2018

Autonomous Ultrafast Self-Healing Hydrogels by pH-Responsive Functional Nanofiber Gelators as Cell Matrices.

Adv Mater 2019 Jan 9;31(2):e1805044. Epub 2018 Nov 9.

Max Planck Institute for Polymer Research, 55128, Mainz, Germany.

The synthesis of hybrid hydrogels by pH-controlled structural transition with exceptional rheological properties as cellular matrix is reported. "Depsi" peptide sequences are grafted onto a polypeptide backbone that undergo a pH-induced intramolecular O-N-acyl migration at physiological conditions affording peptide nanofibers (PNFs) as supramolecular gelators. The polypeptide-PNF hydrogels are mechanically remarkably robust. They reveal exciting thixotropic behavior with immediate in situ recovery after exposure to various high strains over long periods and self-repair of defects by instantaneous reassembly. High cytocompatibility, convenient functionalization by coassembly, and controlled enzymatic degradation but stability in 2D and 3D cell culture as demonstrated by the encapsulation of primary human umbilical vein endothelial cells and neuronal cells open many attractive opportunities for 3D tissue engineering and other biomedical applications.
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http://dx.doi.org/10.1002/adma.201805044DOI Listing
January 2019

Primary resistance of human patients to botulinum neurotoxins A and B.

Ann Clin Transl Neurol 2018 Aug 17;5(8):971-975. Epub 2018 Jul 17.

Department of Biomedical Sciences University of Padova Via Ugo Bassi 58/B 35131 Padova Italy.

Botulinum neurotoxin serotypes A and B are successfully used to treat a variety of human diseases characterized by hyperactive peripheral nerve terminals. However, a number of patients are primary resistant to these pharmaceuticals, without having antitoxin-neutralizing antibodies. A straightforward explanation of this phenomenon posits that mutations of the toxin sites of interaction with their receptors or protein substrates prevent their neuroparalytic action. After a careful investigation of available human genomic databases, we conclude that it is very unlikely that humans are resistant to these two therapeutic neurotoxins because of mutations that would affect their binding or intracellular proteolytic actions.
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http://dx.doi.org/10.1002/acn3.586DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6093845PMC
August 2018

Boosting Antitumor Drug Efficacy with Chemically Engineered Multidomain Proteins.

Adv Sci (Weinh) 2018 Aug 14;5(8):1701036. Epub 2018 Jun 14.

Max-Planck Institute for Polymer Research Ackermannweg 10 55128 Mainz Germany.

A facile chemical approach integrating supramolecular chemistry, site-selective protein chemistry, and molecular biology is described to engineer synthetic multidomain protein therapeutics that sensitize cancer cells selectively to significantly enhance antitumor efficacy of existing chemotherapeutics. The desired bioactive entities are assembled via supramolecular interactions at the nanoscale into structurally ordered multiprotein complexes comprising a) multiple copies of the chemically modified cyclic peptide hormone somatostatin for selective targeting and internalization into human A549 lung cancer cells expressing SST-2 receptors and b) a new cysteine mutant of the C3bot1 (C3) enzyme from , a Rho protein inhibitor that affects and influences intracellular Rho-mediated processes like endothelial cell migration and blood vessel formation. The multidomain protein complex, SST3-Avi-C3, retargets C3 enzyme into non-small cell lung A549 cancer cells and exhibits exceptional tumor inhibition at a concentration ≈100-fold lower than the clinically approved antibody bevacizumab (Avastin) in vivo. Notably, SST3-Avi-C3 increases tumor sensitivity to a conventional chemotherapeutic (doxorubicin) in vivo. These findings show that the integrated approach holds vast promise to expand the current repertoire of multidomain protein complexes and can pave the way to important new developments in the area of targeted and combination cancer therapy.
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http://dx.doi.org/10.1002/advs.201701036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6097141PMC
August 2018

Toxins of Locus of Enterocyte Effacement-Negative Shiga Toxin-Producing .

Toxins (Basel) 2018 06 14;10(6). Epub 2018 Jun 14.

Department of Food Microbiology and Hygiene, Institute of Food Science and Biotechnology, Garbenstrasse 28, University of Hohenheim, 70599 Stuttgart, Germany.

Studies on Shiga toxin-producing (STEC) typically examine and classify the virulence gene profiles based on genomic analyses. Among the screened strains, a subgroup of STEC which lacks the locus of enterocyte effacement (LEE) has frequently been identified. This raises the question about the level of pathogenicity of such strains. This review focuses on the advantages and disadvantages of the standard screening procedures in virulence profiling and summarizes the current knowledge concerning the function and regulation of toxins encoded by LEE-negative STEC. Although LEE-negative STEC usually come across as food isolates, which rarely cause infections in humans, some serotypes have been implicated in human diseases. In particular, the LEE-negative O104:H7 German outbreak strain from 2011 and the Australian O113:H21 strain isolated from a HUS patient attracted attention. Moreover, the LEE-negative STEC O113:H21 strain TS18/08 that was isolated from minced meat is remarkable in that it not only encodes multiple toxins, but in fact expresses three different toxins simultaneously. Their characterization contributes to understanding the virulence of the LEE-negative STEC.
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http://dx.doi.org/10.3390/toxins10060241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6024878PMC
June 2018

Human Serum Albumin Is an Essential Component of the Host Defense Mechanism Against Clostridium difficile Intoxication.

J Infect Dis 2018 09;218(9):1424-1435

2nd Infectious Diseases Division, National Institute for Infectious Diseases "L. Spallanzani", Roma, Italy.

Background: The pathogenic effects of Clostridium difficile are primarily attributable to the production of the large protein toxins (C difficile toxins [Tcd]) A (TcdA) and B (TcdB). These toxins monoglucosylate Rho GTPases in the cytosol of host cells, causing destruction of the actin cytoskeleton with cytotoxic effects. Low human serum albumin (HSA) levels indicate a higher risk of acquiring and developing a severe C difficile infection (CDI) and are associated with recurrent and fatal disease.

Methods: We used a combined approach based on docking simulation and biochemical analyses that were performed in vitro on purified proteins and in human epithelial colorectal adenocarcinoma cells (Caco-2), and in vivo on stem cell-derived human intestinal organoids and zebrafish embryos.

Results: Our results show that HSA specifically binds via its domain II to TcdA and TcdB and thereby induces their autoproteolytic cleavage at physiological concentrations. This process impairs toxin internalization into the host cells and reduces the toxin-dependent glucosylation of Rho proteins.

Conclusions: Our data provide evidence for a specific HSA-dependent self-defense mechanism against C difficile toxins and provide an explanation for the clinical correlation between CDI severity and hypoalbuminemia.
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http://dx.doi.org/10.1093/infdis/jiy338DOI Listing
September 2018

Pharmacological Cyclophilin Inhibitors Prevent Intoxication of Mammalian Cells with Toxin.

Toxins (Basel) 2018 05 1;10(5). Epub 2018 May 1.

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany.

The toxin (PT) is one important virulence factor causing the severe childhood disease whooping cough which still accounted for approximately 63,000 deaths worldwide in children in 2013. PT consists of PTS1, the enzymatically active (A) subunit and a non-covalently linked pentameric binding/transport (B) subunit. After endocytosis, PT takes a retrograde route to the endoplasmic reticulum (ER), where PTS1 is released into the cytosol. In the cytosol, PTS1 ADP-ribosylates inhibitory alpha subunits of trimeric GTP-binding proteins (Giα) leading to increased cAMP levels and disturbed signalling. Here, we show that the cyclophilin (Cyp) isoforms CypA and Cyp40 directly interact with PTS1 in vitro and that Cyp inhibitors cyclosporine A (CsA) and its tailored non-immunosuppressive derivative VK112 both inhibit intoxication of CHO-K1 cells with PT, as analysed in a morphology-based assay. Moreover, in cells treated with PT in the presence of CsA, the amount of ADP-ribosylated Giα was significantly reduced and less PTS1 was detected in the cytosol compared to cells treated with PT only. The results suggest that the uptake of PTS1 into the cytosol requires Cyps. Therefore, CsA/VK112 represent promising candidates for novel therapeutic strategies acting on the toxin level to prevent the severe, life-threatening symptoms caused by PT.
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http://dx.doi.org/10.3390/toxins10050181DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983237PMC
May 2018

Human alpha-defensin-1 protects cells from intoxication with Clostridium perfringens iota toxin.

Pathog Dis 2018 03;76(2)

Institute of Pharmacology and Toxicology, University of Ulm Medical Center, 89081 Ulm, Germany.

Iota toxin is produced by Clostridium perfringens type E strains and associated with diarrhea in cattle and lambs. This binary protein toxin comprises the enzyme component iota a (Ia), which ADP-ribosylates G-actin, and the separate transport component iota b (Ib), which delivers Ia into the cytosol of target cells. Ib binds to cell receptors and forms biologically active toxin complexes with Ia, which cause rounding of adherent cells due to the destruction of the actin cytoskeleton. Here, we report that the human peptide α-defensin-1 protects cultured cells including human colon cells from intoxication with iota toxin. In contrast, the related ß-defensin-1 had no effect, indicating a specific mode of action. The α-defensin-1 did not inhibit ADP-ribosylation of actin by Ia in vitro. Pretreatment of Ib with α-defensin-1 prior to addition of Ia prevented intoxication. Additionally, α-defensin-1 protected cells from cytotoxic effects mediated by Ib in the absence of Ia, implicating that α-defensin-1 interacts with Ib to prevent the formation of biologically active iota toxin on cells. In conclusion, the findings contribute to a better understanding of the functions of α-defensin-1 and suggest that this human peptide might be an attractive starting point to develop novel pharmacological options to treat/prevent diseases associated with iota toxin-producing Clostridium perfringens strains.
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http://dx.doi.org/10.1093/femspd/fty022DOI Listing
March 2018

Cellular Uptake and Mode-of-Action of Clostridium difficile Toxins.

Adv Exp Med Biol 2018 ;1050:77-96

Institute of Experimental and Clinical Pharmacology and Toxicology, Albert Ludwig University of Freiburg, Freiburg im Breisgau, Germany.

Research on the human gut pathogen Clostridium difficile and its toxins has gained much attention, particularly as a consequence of the increasing threat to human health presented by emerging hypervirulent strains. Toxin A (TcdA) and B (TcdB) are the two major virulence determinants of C. difficile. Both are single-chain proteins with a similar multidomain architecture. Certain hypervirulent C. difficile strains also produce a third toxin, namely binary toxin CDT (Clostridium difficile transferase). As C. difficile toxins are the causative agents of C. difficile-associated diseases (CDAD), such as antibiotics-associated diarrhea and pseudomembranous colitis, considerable efforts have been expended to unravel their molecular mode-of-action and the cellular mechanisms responsible for their uptake. Notably, a high proportion of studies on C. difficile toxins were performed in European laboratories. In this chapter we will highlight important recent advances in C. difficile toxins research.
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http://dx.doi.org/10.1007/978-3-319-72799-8_6DOI Listing
June 2018

High Conservation of Tetanus and Botulinum Neurotoxins Cleavage Sites on Human SNARE Proteins Suggests That These Pathogens Exerted Little or No Evolutionary Pressure on Humans.

Toxins (Basel) 2017 12 19;9(12). Epub 2017 Dec 19.

Department of Biomedical Sciences, University of Padova, Via Ugo Bassi 58/B, 35131 Padova, Italy.

The Genome Aggregation Database presently contains >120,000 human genomes. We searched in this database for the presence of mutations at the sites of tetanus (TeNT) and botulinum neurotoxins (BoNTs) cleavages of the three SNARE proteins: VAMP, SNAP-25 and Syntaxin. These mutations could account for some of the BoNT/A resistant patients. At the same time, this approach was aimed at testing the possibility that TeNT and BoNT may have acted as selective agents in the development of resistance to tetanus or botulism. We found that mutations of the SNARE proteins are very rare and concentrated outside the SNARE motif required for the formation of the SNARE complex involved in neuroexocytosis. No changes were found at the BoNT cleavage sites of VAMP and syntaxins and only one very rare mutation was found in the essential C-terminus region of SNAP-25, where Arg198 was replaced with a Cys residue. This is the P1' cleavage site for BoNT/A and the P1 cleavage site for BoNT/C. We found that the Arg198Cys mutation renders SNAP-25 resistant to BoNT/A. Nonetheless, its low frequency (1.8 × 10) indicates that mutations of SNAP-25 at the BoNT/A cleavage site are unlikely to account for the existence of BoNT/A resistant patients. More in general, the present findings indicate that tetanus and botulinum neurotoxins have not acted as selective agents during human evolution as it appears to have been the case for tetanus in rats and chicken.
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http://dx.doi.org/10.3390/toxins9120404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744124PMC
December 2017

Directing intracellular supramolecular assembly with N-heteroaromatic quaterthiophene analogues.

Nat Commun 2017 11 29;8(1):1850. Epub 2017 Nov 29.

Max Planck Institute for Polymer Research, Ackermannweg 10, 55128, Mainz, Germany.

Self-assembly in situ, where synthetic molecules are programmed to organize in a specific and complex environment i.e., within living cells, can be a unique strategy to influence cellular functions. Here we present a small series of rationally designed oligothiophene analogues that specifically target, locate and dynamically self-report their supramolecular behavior within the confinement of a cell. Through the recognition of the terminal alkyl substituent and the amphiphilic pyridine motif, we show that the cell provides different complementary pathways for self-assembly that can be traced easily with fluorescence microscopy as their molecular organization emits in distinct fluorescent bands. Importantly, the control and induction of both forms are achieved by time, temperature and the use of the intracellular transport inhibitor, bafilomycin A1. We showcase the importance of both intrinsic (cell) and extrinsic (stimulus) factors for self-organization and the potential of such a platform toward developing synthetic functional components within living cells.
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http://dx.doi.org/10.1038/s41467-017-02020-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707410PMC
November 2017