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Nicholas J Mantis - One of the best experts on this subject based on the ideXlab platform.

  • sites of vulnerability on ricin b chain revealed through epitope mapping of Toxin neutralizing monoclonal antibodies
    PLOS ONE, 2020
    Co-Authors: David J Vance, Amanda Y Poon, Nicholas J Mantis
    Abstract:

    Ricin Toxin's B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin's ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of Toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) Toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular Toxin Transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB's duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as "bait" in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB's two domains may contribute to distinct steps in the intoxication pathway.

  • sites of vulnerability on ricin b chain revealed through epitope mapping of Toxin neutralizing monoclonal antibodies
    bioRxiv, 2020
    Co-Authors: David J Vance, Amanda Y Poon, Nicholas J Mantis
    Abstract:

    Ricin Toxins B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes efficient uptake and intracellular trafficking of ricins ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (, {beta}, {gamma}). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1 and 2{gamma}) and function non-cooperatively. Previous studies have revealed two distinct classes of Toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit ([~]90%) Toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular Toxin Transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTBs duplicative structure. To circumvent this problem, full-length RTB or the two individual domains, RTB-D1 and RTB-D2, were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as "bait" in mAb capture assays. The results indicated that SylH3 captured RTB-D1, while 24B11 captured RTB-D2. Analysis of additional Toxin-neutralizing and non-neutralizing mAbs along with single chain antibodies (VHHs) known to compete with SylH3 or 24B11 confirmed these domain assignments. These results not only indicate that so-called type I and type II mAbs segregate on the basis of domain specificity, but suggest that RTBs two domains may contribute to distinct steps in the intoxication pathway.

  • Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin's Enzymatic Subunit Interfere with Intracellular Toxin Transport
    Scientific Reports, 2016
    Co-Authors: A. Yermakova, Joanne M. O'hara, Tove Irene Klokk, Richard Cole, Kirsten Sandvig, Nicholas J Mantis
    Abstract:

    Ricin is a member of the A-B family of bacterial and plant Toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin's enzymatic subunit (RTA), interfere with Toxin Transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin's egress from EEA-1(+) and Rab7(+) vesicles and enhanced Toxin accumulation in LAMP-1(+) vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde Transport. We conclude that interference with Toxin retrograde Transport may be a hallmark of Toxin-neutralizing antibodies directed against disparate epitopes on RTA.

  • Immunoglobulin A antibodies against ricin A and B subunits protect epithelial cells from ricin intoxication
    Infection and Immunity, 2006
    Co-Authors: Nicholas J Mantis, Carolyn R. Mcguinness, Oluwakemi Sonuyi, Gary Edwards, Stephanie A Farrant
    Abstract:

    Epithelial cells of the respiratory and gastrointestinal tracts are extremely vulnerable to the cytotoxic effects of ricin, a Shiga-like Toxin with ribosome-inactivating properties. While mucosal immunity to ricin correlates with secretory immunoglobulin A (IgA) antibody levels in vivo, the potential of IgA to protect epithelial cells from ricin in vitro has not been examined due to the unavailability of well-defined antiToxin IgA antibodies. Here we report the characterization of four monoclonal IgA antibodies (IgA MAbs) produced from the Peyer's patches and mesenteric lymph nodes of BALB/c mice immunized intragastrically with ricin toxoid. Two IgA MAbs (33G2 and 35H6) were active against ricin's lectin subunit (RTB), and two (23D7 and 25A4) reacted with the Toxin's enzymatic subunit (RTA). All four IgA MAbs neutralized ricin in a Vero cell cytotoxicity assay, blocked Toxin-induced interleukin-8 release by the human monocyte/macrophage cell line 28SC, and protected polarized epithelial cell monolayers from ricin-mediated protein synthesis inhibition. 33G2 and 35H6 reduced ricin binding to the luminal surfaces of human intestinal epithelial cells to undetectable levels in tissue section overlay assays, whereas 23D7 had no effect on Toxin attachment. 23D7 and 25A4 did, however, reduce ricin transcytosis across MDCK II cell monolayers, possibly by interfering with intracellular Toxin Transport. We conclude that IgA antibodies against RTA and RTB can protect mucosal epithelial cells from ricin intoxication.

Thomas Brüser - One of the best experts on this subject based on the ideXlab platform.

  • evidence for an adaptation of a phage derived holin endolysin system to Toxin Transport in clostridioides difficile
    Frontiers in Microbiology, 2018
    Co-Authors: Denise Mehnerbreitfeld, Ralf Gerhard, Claudia Rathmann, Thomas Riedel, Ingo Just, Jörg Overmann, Thomas Brüser
    Abstract:

    The pathogenicity locus (PaLoc) of Clostridioides difficile usually comprises five genes (tcdR, tcdB, tcdE, tcdA, tcdC). While the proteins TcdA and TcdB represent the main Toxins of this pathogen, TcdR and TcdC are involved in the regulation of their production. TcdE is a holin family protein, members of which are usually involved in the Transport of cell wall-degrading enzymes (endolysins) for phage-induced lysis. In the past, TcdE has been shown to contribute to the release of TcdA and TcdB, but it is unclear whether it mediates a specific Transport or rather a lysis of cells. TcdE of C. difficile strains analyzed so far can be produced in three isoforms that are initiated from distinct N-terminal ATG codons. When produced in Escherichia coli, we found that the longest TcdE isoform had a moderate effect on cell growth, whereas the shortest isoform strongly induced lysis. The effect of the longest isoform was inhibitory for cell lysis, implying a regulatory function of the N-terminal 24 residues. We analyzed the PaLoc sequence of 44 C. difficile isolates and found that four of these apparently encode only the short TcdE isoforms, and the most closely related holins from C. difficile phages only possess one of these initiation codons, indicating that an N-terminal extension of TcdE evolved in C. difficile. All PaLoc sequences comprised also a conserved gene encoding a short fragment of an endolysin remnant of a phage holin/endolysin pair. We could produce this peptide, which we named TcdL, and demonstrated by bacterial two-hybrid analysis a self-interaction and an interaction with TcdB that might serve to mediate TcdE-dependent Transport.

  • Presentation_1_Evidence for an Adaptation of a Phage-Derived Holin/Endolysin System to Toxin Transport in Clostridioides difficile.pdf
    2018
    Co-Authors: Denise Mehner-breitfeld, Ralf Gerhard, Claudia Rathmann, Thomas Riedel, Ingo Just, Jörg Overmann, Thomas Brüser
    Abstract:

    The pathogenicity locus (PaLoc) of Clostridioides difficile usually comprises five genes (tcdR, tcdB, tcdE, tcdA, tcdC). While the proteins TcdA and TcdB represent the main Toxins of this pathogen, TcdR and TcdC are involved in the regulation of their production. TcdE is a holin family protein, members of which are usually involved in the Transport of cell wall-degrading enzymes (endolysins) for phage-induced lysis. In the past, TcdE has been shown to contribute to the release of TcdA and TcdB, but it is unclear whether it mediates a specific Transport or rather a lysis of cells. TcdE of C. difficile strains analyzed so far can be produced in three isoforms that are initiated from distinct N-terminal ATG codons. When produced in Escherichia coli, we found that the longest TcdE isoform had a moderate effect on cell growth, whereas the shortest isoform strongly induced lysis. The effect of the longest isoform was inhibitory for cell lysis, implying a regulatory function of the N-terminal 24 residues. We analyzed the PaLoc sequence of 44 C. difficile isolates and found that four of these apparently encode only the short TcdE isoforms, and the most closely related holins from C. difficile phages only possess one of these initiation codons, indicating that an N-terminal extension of TcdE evolved in C. difficile. All PaLoc sequences comprised also a conserved gene encoding a short fragment of an endolysin remnant of a phage holin/endolysin pair. We could produce this peptide, which we named TcdL, and demonstrated by bacterial two-hybrid analysis a self-interaction and an interaction with TcdB that might serve to mediate TcdE-dependent Transport.

Manfred J. Schmitt - One of the best experts on this subject based on the ideXlab platform.

  • adapting yeast as model to study ricin Toxin a uptake and trafficking
    Toxins, 2011
    Co-Authors: Björn Becker, Manfred J. Schmitt
    Abstract:

    The plant A/B Toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin Toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a Toxin (RTAHDEL) showed a similar cytotoxic effect on yeast as a corresponding RTAKDEL variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in Toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B Toxin Transport from the plasma membrane through the endosomal compartment to the ER.

  • Adapting Yeast as Model to Study Ricin Toxin A Uptake and Trafficking
    MDPI AG, 2011
    Co-Authors: Björn Becker, Manfred J. Schmitt
    Abstract:

    The plant A/B Toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin Toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a Toxin (RTAHDEL) showed a similar cytotoxic effect on yeast as a corresponding RTAKDEL variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in Toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B Toxin Transport from the plasma membrane through the endosomal compartment to the ER

  • Adapting Yeast as Model to Study Ricin Toxin A Uptake and Trafficking ‡
    2011
    Co-Authors: Björn Becker, Manfred J. Schmitt
    Abstract:

    Abstract: The plant A/B Toxin ricin represents a heterodimeric glycoprotein belonging to the family of ribosome inactivating proteins, RIPs. Its toxicity towards eukaryotic cells results from the depurination of 28S rRNA due to the N-glycosidic activity of ricin Toxin A chain, RTA. Since the extention of RTA by a mammalian-specific endoplasmic reticulum (ER) retention signal (KDEL) significantly increases RTA in vivo toxicity against mammalian cells, we here analyzed the phenotypic effect of RTA carrying the yeast-specific ER retention motif HDEL. Interestingly, such a Toxin (RTA HDEL) showed a similar cytotoxic effect on yeast as a corresponding RTA KDEL variant on HeLa cells. Furthermore, we established a powerful yeast bioassay for RTA in vivo uptake and trafficking which is based on the measurement of dissolved oxygen in Toxin-treated spheroplast cultures of S. cerevisiae. We show that yeast spheroplasts are highly sensitive against external applied RTA and further demonstrate that its toxicity is greatly enhanced by replacing the C-terminal KDEL motif by HDEL. Based on the RTA resistant phenotype seen in yeast knock-out mutants defective in early steps of endocytosis (∆end3) and/or in RTA depurination activity on 28S rRNA (∆rpl12B) we feel that the yeast-based bioassay described in this study is a powerful tool to dissect intracellular A/B Toxin Transport from the plasma membrane through the endosomal compartment to the ER

  • endocytotic uptake and retrograde Transport of a virally encoded killer Toxin in yeast
    Molecular Microbiology, 2000
    Co-Authors: Katrin Eisfeld, Frank Riffer, Johannes Mentges, Manfred J. Schmitt
    Abstract:

    We demonstrate that a virally encoded yeast 'killer' Toxin is entering its eukaryotic target cell by endocytosis, subsequently travelling the yeast secretory pathway in reverse to exhibit its lethal effect. The K28 killer Toxin is a secreted alpha/beta heterodimer that kills sensitive yeasts in a receptor-mediated fashion by blocking DNA synthesis in the nucleus. In vivo processing of the Toxin precursor results in a protein whose beta-C-terminus carries the endoplasmic reticulum (ER) retention signal HDEL, which, as we show here, is essential for retrograde Toxin Transport. Yeast end3/4 mutants as well as cells lacking the HDEL receptor (Deltaerd2) or mutants defective in Golgi-to-ER protein recycling (erd1) are Toxin resistant because the Toxin can no longer enter and/or retrograde pass the cell. Site-directed mutagenesis further indicated that the Toxin's beta-HDEL motif ensures retrograde Transport, although in a Toxin-secreting yeast the beta-C-terminus is initially masked by an R residue (beta-HDELR) until Kex1p cleavage uncovers the Toxin's targeting signal in a late Golgi compartment. Prevention of Kex1p processing results in high-level secretion of a biologically inactive protein incapable of re-entering the secretory pathway. Finally, we present evidence that ER-to-cytosol Toxin export is mediated by the Sec61p translocon and requires functional copies of the lumenal ER chaperones Kar2p and Cne1p.

Grant J Jensen - One of the best experts on this subject based on the ideXlab platform.

  • programmed secretion arrest and receptor triggered Toxin export during antibacterial contact dependent growth inhibition
    Cell, 2018
    Co-Authors: Zachary C Ruhe, Josephine Y Nguyen, David A Low, Poorna Subramanian, Kiho Song, Taylor A Stevens, Grant J Jensen
    Abstract:

    Contact-dependent growth inhibition (CDI) entails receptor-mediated delivery of CdiA-derived Toxins into Gram-negative target bacteria. Using electron cryotomography, we show that each CdiA effector protein forms a filament extending ∼33 nm from the cell surface. Remarkably, the extracellular filament represents only the N-terminal half of the effector. A programmed secretion arrest sequesters the C-terminal half of CdiA, including the Toxin domain, in the periplasm prior to target-cell recognition. Upon binding receptor, CdiA secretion resumes, and the periplasmic FHA-2 domain is transferred to the target-cell outer membrane. The C-terminal Toxin region of CdiA then penetrates into the target-cell periplasm, where it is cleaved for subsequent translocation into the cytoplasm. Our findings suggest that the FHA-2 domain assembles into a transmembrane conduit for Toxin Transport into the periplasm of target bacteria. We propose that receptor-triggered secretion ensures that FHA-2 export is closely coordinated with integration into the target-cell outer membrane.

David A Low - One of the best experts on this subject based on the ideXlab platform.

  • programmed secretion arrest and receptor triggered Toxin export during antibacterial contact dependent growth inhibition
    Cell, 2018
    Co-Authors: Zachary C Ruhe, Josephine Y Nguyen, David A Low, Poorna Subramanian, Kiho Song, Taylor A Stevens, Grant J Jensen
    Abstract:

    Contact-dependent growth inhibition (CDI) entails receptor-mediated delivery of CdiA-derived Toxins into Gram-negative target bacteria. Using electron cryotomography, we show that each CdiA effector protein forms a filament extending ∼33 nm from the cell surface. Remarkably, the extracellular filament represents only the N-terminal half of the effector. A programmed secretion arrest sequesters the C-terminal half of CdiA, including the Toxin domain, in the periplasm prior to target-cell recognition. Upon binding receptor, CdiA secretion resumes, and the periplasmic FHA-2 domain is transferred to the target-cell outer membrane. The C-terminal Toxin region of CdiA then penetrates into the target-cell periplasm, where it is cleaved for subsequent translocation into the cytoplasm. Our findings suggest that the FHA-2 domain assembles into a transmembrane conduit for Toxin Transport into the periplasm of target bacteria. We propose that receptor-triggered secretion ensures that FHA-2 export is closely coordinated with integration into the target-cell outer membrane.

  • contact dependent growth inhibition Toxins exploit multiple independent cell entry pathways
    Proceedings of the National Academy of Sciences of the United States of America, 2015
    Co-Authors: Julia L E Willett, Grant C Gucinski, Jackson P Fatherree, David A Low
    Abstract:

    Contact-dependent growth inhibition (CDI) systems function to deliver Toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a Toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI Toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control Toxin Transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for Toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal "translocation domains." These results highlight the plasticity of CDI Toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.