Manganese Blocks Intracellular Trafficking of Shiga Toxin and Protects Against Shiga Toxicosis

Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
Science (Impact Factor: 33.61). 01/2012; 335(6066):332-5. DOI: 10.1126/science.1215930
Source: PubMed


Infections with Shiga toxin (STx)–producing bacteria cause more than a million deaths each year and have no definitive treatment.
To exert its cytotoxic effect, STx invades cells through retrograde membrane trafficking, escaping the lysosomal degradative
pathway. We found that the widely available metal manganese (Mn2+) blocked endosome-to-Golgi trafficking of STx and caused its degradation in lysosomes. Mn2+ targeted the cycling Golgi protein GPP130, which STx bound in control cells during sorting into Golgi-directed endosomal
tubules that bypass lysosomes. In tissue culture cells, treatment with Mn2+ yielded a protection factor of 3800 against STx-induced cell death. Furthermore, mice injected with nontoxic doses of Mn2+ were completely resistant to a lethal STx challenge. Thus, Mn2+ may represent a low-cost therapeutic agent for the treatment of STx infections.

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    • "Mukhopadhyay and Linstedt reported that manganese was able to block the intracellular trafficking of Stx1 through the Golgi apparatus of Stx-susceptible HeLa cells engineered to overexpress the glycolipid Gb3[14]; by doing so MnCl2 appeared to block the toxic effects of Stx1. Hope that manganese could be used as a treatment for STEC infection diminished, however, when Gaston et al. and additional work by Mukhopadhyay et al. showed that the protective effects of manganese did not extend to Stx2 [65,66]. "
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    ABSTRACT: Background Zinc supplements can treat or prevent enteric infections and diarrheal disease. Many articles on zinc in bacteria, however, highlight the essential nature of this metal for bacterial growth and virulence, suggesting that zinc should make infections worse, not better. To address this paradox, we tested whether zinc might have protective effects on intestinal epithelium as well as on the pathogen. Results Using polarized monolayers of T84 cells we found that zinc protected against damage induced by hydrogen peroxide, as measured by trans-epithelial electrical resistance. Zinc also reduced peroxide-induced translocation of Shiga toxin (Stx) across T84 monolayers from the apical to basolateral side. Zinc was superior to other divalent metals to (iron, manganese, and nickel) in protecting against peroxide-induced epithelial damage, while copper also showed a protective effect. The SOS bacterial stress response pathway is a powerful regulator of Stx production in STEC. We examined whether zinc’s known inhibitory effects on Stx might be mediated by blocking the SOS response. Zinc reduced expression of recA, a reliable marker of the SOS. Zinc was more potent and more efficacious than other metals tested in inhibiting recA expression induced by hydrogen peroxide, xanthine oxidase, or the antibiotic ciprofloxacin. The close correlation between zinc’s effects on recA/SOS and on Stx suggested that inhibition of the SOS response is one mechanism by which zinc protects against STEC infection. Conclusions Zinc’s ability to protect against enteric bacterial pathogens may be the result of its combined effects on host tissues as well as inhibition of virulence in some pathogens. Research focused solely on the effects of zinc on pathogenic microbes may give an incomplete picture by failing to account for protective effects of zinc on host epithelia.
    Full-text · Article · Jun 2014 · BMC Microbiology
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    • "Treatment of patients with EHEC infection with antibiotics is controversial because there is the risk of the release of large amounts of Stx from dead EHEC leading to life-threatening diseases [4], [6]. Oral administration of Stx-specific IgA is one of the candidate therapies for the treatment of Stx-caused food poisoning. "
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    ABSTRACT: Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a "plantibody". Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb3, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.
    Full-text · Article · Nov 2013 · PLoS ONE
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    • "Within five minutes post-injection of MnCl2 at the reported dose of 50 mg/kg, mice exhibited signs of distress including: hunching, isolation in the corner of cages and temporary tremor. These signs were not reported for BALB/c mice [8]. We thus used half the reported dose of MnCl2 for our in vivo Stx toxicity assays. "
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    ABSTRACT: Shiga toxin (Stx), the main virulence factor of Shiga toxin producing Escherichia coli, is a major public health threat, causing hemorrhagic colitis and hemolytic uremic syndrome. Currently, there are no approved therapeutics for these infections; however manganese has been reported to provide protection from the Stx1 variant isolated from Shigella dysenteriae (Stx1-S) both in vitro and in vivo. We investigated the efficacy of manganese protection from Stx1-S and the more potent Stx2a isoform, using experimental systems well-established for studying Stx: in vitro responses of Vero monkey kidney cells, and in vivo toxicity to CD-1 outbred mice. Manganese treatment at the reported therapeutic concentration was toxic to Vero cells in culture and to CD-1 mice. At lower manganese concentrations that were better tolerated, we observed no protection from Stx1-S or Stx2a toxicity. The ability of manganese to prevent the effects of Stx may be particular to certain cell lines, mouse strains, or may only be manifested at high, potentially toxic manganese concentrations.
    Preview · Article · Jul 2013 · PLoS ONE
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