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Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

Eshraghi A, Dixon SD, Tamilselvam B, Kim EJ, Gargi A, Kulik JC, Damoiseaux R, Blanke SR, Bradley KA - PLoS Pathog. (2014)

Bottom Line: Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs.In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication.These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

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Hrd1 is required for CDT intoxication.(a) Co-immunoprecipitation of Derl2 and Hrd1. Derl2 was immunoprecipitated as in figure 2i and samples were analyzed for Hrd1 by western blot. (b) CRISPR mediated deletion of Hrd1 (ΔHrd1) results in decreased expression as judged by western blot of Hrd1 from α-Hrd1 immunoprecipitated protein from normalized cell lysates. (c) Co-immunoprecipitation of Derl2 with Hrd1. Hrd1 was immunoprecipitated and samples were analyzed for Derl2 by western blot. (d–g) Wild type 293 and ΔHrd1 cells were intoxicated with Aa-CDT (d), Hd-CDT (e), Ec-CDT (f) and Cj-CDT (g) similar to figure 1. Percent viability is normalized to unintoxicated controls and error bars indicate standard error. (h–j) Retrograde trafficking of Hd-CDT in ΔHrd1 cells is blocked at the endoplasmic reticulum. pDsRed2-ER (red) transfected 293 cells and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with DAPI (nuclei, blue) and α-Hd-CdtB (green) antibody. White scale bars indicate 5 µm. (i,j) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus (i), or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER (j). Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments.
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ppat-1004295-g003: Hrd1 is required for CDT intoxication.(a) Co-immunoprecipitation of Derl2 and Hrd1. Derl2 was immunoprecipitated as in figure 2i and samples were analyzed for Hrd1 by western blot. (b) CRISPR mediated deletion of Hrd1 (ΔHrd1) results in decreased expression as judged by western blot of Hrd1 from α-Hrd1 immunoprecipitated protein from normalized cell lysates. (c) Co-immunoprecipitation of Derl2 with Hrd1. Hrd1 was immunoprecipitated and samples were analyzed for Derl2 by western blot. (d–g) Wild type 293 and ΔHrd1 cells were intoxicated with Aa-CDT (d), Hd-CDT (e), Ec-CDT (f) and Cj-CDT (g) similar to figure 1. Percent viability is normalized to unintoxicated controls and error bars indicate standard error. (h–j) Retrograde trafficking of Hd-CDT in ΔHrd1 cells is blocked at the endoplasmic reticulum. pDsRed2-ER (red) transfected 293 cells and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with DAPI (nuclei, blue) and α-Hd-CdtB (green) antibody. White scale bars indicate 5 µm. (i,j) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus (i), or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER (j). Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments.

Mentions: Derl2 is part of the Hrd1-containing “retrotranslocon”, a protein complex that mediates retrotranslocation of ERAD substrates [47]. Indeed, Hrd1 was co-immunoprecipitated with Derl2 from wildtype but not Derl2-deficient cells (Fig. 3a). Similarly, Derl2 could be co-immunoprecipitated from wildtype cells, but not from cells in which Hrd1 was targeted by CRISPR (Fig. 3b–3c). Intoxication of Hrd1-deficient cells revealed that this gene, like Derl2, is required for cell killing by multiple CDTs (Fig. 3d–3g, S4). Interestingly, cells lacking Hrd1 displayed full sensitivity to intoxication by Cj-CDT (Fig. 3g). Similar to Derl2 deficient cells, deletion of Hrd1 resulted in retention of Hd-CDT in the ER 240 minutes post-intoxication (Fig. 3h–3j). These data suggest that the Derl2 and Hrd1-containing retrotranslocon is required for intoxication by multiple CDTs, implicating a role for the ERAD pathway in cellular entry for a subset of this family of toxins.


Cytolethal distending toxins require components of the ER-associated degradation pathway for host cell entry.

Eshraghi A, Dixon SD, Tamilselvam B, Kim EJ, Gargi A, Kulik JC, Damoiseaux R, Blanke SR, Bradley KA - PLoS Pathog. (2014)

Hrd1 is required for CDT intoxication.(a) Co-immunoprecipitation of Derl2 and Hrd1. Derl2 was immunoprecipitated as in figure 2i and samples were analyzed for Hrd1 by western blot. (b) CRISPR mediated deletion of Hrd1 (ΔHrd1) results in decreased expression as judged by western blot of Hrd1 from α-Hrd1 immunoprecipitated protein from normalized cell lysates. (c) Co-immunoprecipitation of Derl2 with Hrd1. Hrd1 was immunoprecipitated and samples were analyzed for Derl2 by western blot. (d–g) Wild type 293 and ΔHrd1 cells were intoxicated with Aa-CDT (d), Hd-CDT (e), Ec-CDT (f) and Cj-CDT (g) similar to figure 1. Percent viability is normalized to unintoxicated controls and error bars indicate standard error. (h–j) Retrograde trafficking of Hd-CDT in ΔHrd1 cells is blocked at the endoplasmic reticulum. pDsRed2-ER (red) transfected 293 cells and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with DAPI (nuclei, blue) and α-Hd-CdtB (green) antibody. White scale bars indicate 5 µm. (i,j) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus (i), or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER (j). Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments.
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Show All Figures
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ppat-1004295-g003: Hrd1 is required for CDT intoxication.(a) Co-immunoprecipitation of Derl2 and Hrd1. Derl2 was immunoprecipitated as in figure 2i and samples were analyzed for Hrd1 by western blot. (b) CRISPR mediated deletion of Hrd1 (ΔHrd1) results in decreased expression as judged by western blot of Hrd1 from α-Hrd1 immunoprecipitated protein from normalized cell lysates. (c) Co-immunoprecipitation of Derl2 with Hrd1. Hrd1 was immunoprecipitated and samples were analyzed for Derl2 by western blot. (d–g) Wild type 293 and ΔHrd1 cells were intoxicated with Aa-CDT (d), Hd-CDT (e), Ec-CDT (f) and Cj-CDT (g) similar to figure 1. Percent viability is normalized to unintoxicated controls and error bars indicate standard error. (h–j) Retrograde trafficking of Hd-CDT in ΔHrd1 cells is blocked at the endoplasmic reticulum. pDsRed2-ER (red) transfected 293 cells and ΔHrd1 cells were incubated with Hd-CDT on ice, washed and incubated at 37°C for 240 minutes. Cells were then fixed and stained with DAPI (nuclei, blue) and α-Hd-CdtB (green) antibody. White scale bars indicate 5 µm. (i,j) Quantification of microscopy results comparing the percentage of cells with at least one green puncta localized to the nucleus (i), or Pearson's coefficient values indicating colocalization of the Hd-CdtB signal with the ER (j). Images and quantitation are representative of those collected from a total of 30 randomly chosen cells analyzed during two independent experiments and error bars represent standard deviations. Unless otherwise noted, data are representative of at least three independent experiments.
Mentions: Derl2 is part of the Hrd1-containing “retrotranslocon”, a protein complex that mediates retrotranslocation of ERAD substrates [47]. Indeed, Hrd1 was co-immunoprecipitated with Derl2 from wildtype but not Derl2-deficient cells (Fig. 3a). Similarly, Derl2 could be co-immunoprecipitated from wildtype cells, but not from cells in which Hrd1 was targeted by CRISPR (Fig. 3b–3c). Intoxication of Hrd1-deficient cells revealed that this gene, like Derl2, is required for cell killing by multiple CDTs (Fig. 3d–3g, S4). Interestingly, cells lacking Hrd1 displayed full sensitivity to intoxication by Cj-CDT (Fig. 3g). Similar to Derl2 deficient cells, deletion of Hrd1 resulted in retention of Hd-CDT in the ER 240 minutes post-intoxication (Fig. 3h–3j). These data suggest that the Derl2 and Hrd1-containing retrotranslocon is required for intoxication by multiple CDTs, implicating a role for the ERAD pathway in cellular entry for a subset of this family of toxins.

Bottom Line: Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs.In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication.These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
Intracellular acting protein exotoxins produced by bacteria and plants are important molecular determinants that drive numerous human diseases. A subset of these toxins, the cytolethal distending toxins (CDTs), are encoded by several Gram-negative pathogens and have been proposed to enhance virulence by allowing evasion of the immune system. CDTs are trafficked in a retrograde manner from the cell surface through the Golgi apparatus and into the endoplasmic reticulum (ER) before ultimately reaching the host cell nucleus. However, the mechanism by which CDTs exit the ER is not known. Here we show that three central components of the host ER associated degradation (ERAD) machinery, Derlin-2 (Derl2), the E3 ubiquitin-protein ligase Hrd1, and the AAA ATPase p97, are required for intoxication by some CDTs. Complementation of Derl2-deficient cells with Derl2:Derl1 chimeras identified two previously uncharacterized functional domains in Derl2, the N-terminal 88 amino acids and the second ER-luminal loop, as required for intoxication by the CDT encoded by Haemophilus ducreyi (Hd-CDT). In contrast, two motifs required for Derlin-dependent retrotranslocation of ERAD substrates, a conserved WR motif and an SHP box that mediates interaction with the AAA ATPase p97, were found to be dispensable for Hd-CDT intoxication. Interestingly, this previously undescribed mechanism is shared with the plant toxin ricin. These data reveal a requirement for multiple components of the ERAD pathway for CDT intoxication and provide insight into a Derl2-dependent pathway exploited by retrograde trafficking toxins.

Show MeSH
Related in: MedlinePlus