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Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo.

Bischof LJ, Kao CY, Los FC, Gonzalez MR, Shen Z, Briggs SP, van der Goot FG, Aroian RV - PLoS Pathog. (2008)

Bottom Line: We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR.The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities.These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, United States of America.

ABSTRACT
Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

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Loss of specific UPR pathways cause hypersensitivity to PFT but not other toxins or a pathogenic bacteria.(A) Comparison of ER stress response mutants to wild-type N2 on 25% Cry5B-expressing E. coli plates indicate ire-1(v33) and xbp-1(zc12) are hypersensitive to Cry5B intoxication. Two representative worms are shown for each strain 48 hours after feeding either on E. coli without Cry5B or on E. coli of which 25% expressed Cry5B. Scale bar is 0.2 mm. (B) A lethal concentration assay was performed using purified Cry5B toxin to quantitatively compare sensitivities of wild-type N2 and the ER stress mutants. Lethality was determined after 8 days. This semi-log graph represents three independent experiments, and each data point is the mean and standard deviations of the experiments. (C) A Cry5B developmental inhibition assay was performed beginning with synchronized worms at the first larval stage. Worms were grown on plates containing different percentages of Cry5B-expressing E. coli (% Cry5B as indicated under the figure), and the percent of worms reaching the L4 stage or adulthood 72 hours later is indicated. ire-1(v33) was included only on the plates with 0% Cry5B. Data are presented as mean and standard deviation. (D) A lethal concentration assay comparing sensitivity to CuSO4 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 8 days of CuSO4 exposure, the same time frame as the Cry5B lethality assay. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (E) A lethal concentration assay comparing sensitivity to H2O2 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 4 hours of H2O2 exposure. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (F) A lifespan assay was used to compare the ER stress mutants to slow killing by P. aeruginosa PA14. This graph represents combined data from three experiments.
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ppat-1000176-g002: Loss of specific UPR pathways cause hypersensitivity to PFT but not other toxins or a pathogenic bacteria.(A) Comparison of ER stress response mutants to wild-type N2 on 25% Cry5B-expressing E. coli plates indicate ire-1(v33) and xbp-1(zc12) are hypersensitive to Cry5B intoxication. Two representative worms are shown for each strain 48 hours after feeding either on E. coli without Cry5B or on E. coli of which 25% expressed Cry5B. Scale bar is 0.2 mm. (B) A lethal concentration assay was performed using purified Cry5B toxin to quantitatively compare sensitivities of wild-type N2 and the ER stress mutants. Lethality was determined after 8 days. This semi-log graph represents three independent experiments, and each data point is the mean and standard deviations of the experiments. (C) A Cry5B developmental inhibition assay was performed beginning with synchronized worms at the first larval stage. Worms were grown on plates containing different percentages of Cry5B-expressing E. coli (% Cry5B as indicated under the figure), and the percent of worms reaching the L4 stage or adulthood 72 hours later is indicated. ire-1(v33) was included only on the plates with 0% Cry5B. Data are presented as mean and standard deviation. (D) A lethal concentration assay comparing sensitivity to CuSO4 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 8 days of CuSO4 exposure, the same time frame as the Cry5B lethality assay. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (E) A lethal concentration assay comparing sensitivity to H2O2 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 4 hours of H2O2 exposure. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (F) A lifespan assay was used to compare the ER stress mutants to slow killing by P. aeruginosa PA14. This graph represents combined data from three experiments.

Mentions: To determine whether the ER stress response played a role in the defense of C. elegans against the PFT, C. elegans mutants in the ER stress response pathway were qualitatively compared to wild-type N2 animals in their susceptibilities to Cry5B. The mutants that were tested included those encoding the three ER stress transducer genes, atf-6(ok551), pek-1(ok275), and ire-1(v33), as well as xbp-1(zc12); these mutations are predicted or known to be loss of function mutations in their respective genes [11],[12],[13]. In the absence of Cry5B, the wild type and mutant worms are healthy adults with similar appearance, except ire-1(v33), which is clearly smaller than the other strains (Figure 2A). In the presence of low-moderate levels of the PFT Cry5B, wild-type worms are slightly intoxicated compared to those found on control no-toxin plates, as evidenced by their smaller sizes and paler appearances (Figure 2A). To the same extent seen with wild-type worms, atf-6(ok551) and pek-1(ok275) mutant animals are also slightly intoxicated on low-moderate levels of the PFT Cry5B, indicating lack of either of these genes does not result in overt hypersensitivity or hyper-resistance to Cry5B (Figure 2A). However under the same conditions, the ire-1(v33) and xbp-1(zc12) mutant worms are more severely intoxicated than wild-type worms as they are relatively smaller and considerably paler compared to their corresponding no toxin controls. The hypersensitivity to Cry5B resulting from lack of ire-1 and xbp-1 was also seen using RNA interference (RNAi; data not shown), confirming the phenotype is caused by loss of function in these genes. We call this hypersensitivity phenotype “Hpo” for hypersensitive to pore-forming toxin.


Activation of the unfolded protein response is required for defenses against bacterial pore-forming toxin in vivo.

Bischof LJ, Kao CY, Los FC, Gonzalez MR, Shen Z, Briggs SP, van der Goot FG, Aroian RV - PLoS Pathog. (2008)

Loss of specific UPR pathways cause hypersensitivity to PFT but not other toxins or a pathogenic bacteria.(A) Comparison of ER stress response mutants to wild-type N2 on 25% Cry5B-expressing E. coli plates indicate ire-1(v33) and xbp-1(zc12) are hypersensitive to Cry5B intoxication. Two representative worms are shown for each strain 48 hours after feeding either on E. coli without Cry5B or on E. coli of which 25% expressed Cry5B. Scale bar is 0.2 mm. (B) A lethal concentration assay was performed using purified Cry5B toxin to quantitatively compare sensitivities of wild-type N2 and the ER stress mutants. Lethality was determined after 8 days. This semi-log graph represents three independent experiments, and each data point is the mean and standard deviations of the experiments. (C) A Cry5B developmental inhibition assay was performed beginning with synchronized worms at the first larval stage. Worms were grown on plates containing different percentages of Cry5B-expressing E. coli (% Cry5B as indicated under the figure), and the percent of worms reaching the L4 stage or adulthood 72 hours later is indicated. ire-1(v33) was included only on the plates with 0% Cry5B. Data are presented as mean and standard deviation. (D) A lethal concentration assay comparing sensitivity to CuSO4 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 8 days of CuSO4 exposure, the same time frame as the Cry5B lethality assay. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (E) A lethal concentration assay comparing sensitivity to H2O2 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 4 hours of H2O2 exposure. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (F) A lifespan assay was used to compare the ER stress mutants to slow killing by P. aeruginosa PA14. This graph represents combined data from three experiments.
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ppat-1000176-g002: Loss of specific UPR pathways cause hypersensitivity to PFT but not other toxins or a pathogenic bacteria.(A) Comparison of ER stress response mutants to wild-type N2 on 25% Cry5B-expressing E. coli plates indicate ire-1(v33) and xbp-1(zc12) are hypersensitive to Cry5B intoxication. Two representative worms are shown for each strain 48 hours after feeding either on E. coli without Cry5B or on E. coli of which 25% expressed Cry5B. Scale bar is 0.2 mm. (B) A lethal concentration assay was performed using purified Cry5B toxin to quantitatively compare sensitivities of wild-type N2 and the ER stress mutants. Lethality was determined after 8 days. This semi-log graph represents three independent experiments, and each data point is the mean and standard deviations of the experiments. (C) A Cry5B developmental inhibition assay was performed beginning with synchronized worms at the first larval stage. Worms were grown on plates containing different percentages of Cry5B-expressing E. coli (% Cry5B as indicated under the figure), and the percent of worms reaching the L4 stage or adulthood 72 hours later is indicated. ire-1(v33) was included only on the plates with 0% Cry5B. Data are presented as mean and standard deviation. (D) A lethal concentration assay comparing sensitivity to CuSO4 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 8 days of CuSO4 exposure, the same time frame as the Cry5B lethality assay. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (E) A lethal concentration assay comparing sensitivity to H2O2 revealed xbp-1(zc12) is not hypersensitive compared to wild-type N2. Lethality was determined after 4 hours of H2O2 exposure. Data, plotted semi-log, are the mean and standard deviation of three independent experiments. (F) A lifespan assay was used to compare the ER stress mutants to slow killing by P. aeruginosa PA14. This graph represents combined data from three experiments.
Mentions: To determine whether the ER stress response played a role in the defense of C. elegans against the PFT, C. elegans mutants in the ER stress response pathway were qualitatively compared to wild-type N2 animals in their susceptibilities to Cry5B. The mutants that were tested included those encoding the three ER stress transducer genes, atf-6(ok551), pek-1(ok275), and ire-1(v33), as well as xbp-1(zc12); these mutations are predicted or known to be loss of function mutations in their respective genes [11],[12],[13]. In the absence of Cry5B, the wild type and mutant worms are healthy adults with similar appearance, except ire-1(v33), which is clearly smaller than the other strains (Figure 2A). In the presence of low-moderate levels of the PFT Cry5B, wild-type worms are slightly intoxicated compared to those found on control no-toxin plates, as evidenced by their smaller sizes and paler appearances (Figure 2A). To the same extent seen with wild-type worms, atf-6(ok551) and pek-1(ok275) mutant animals are also slightly intoxicated on low-moderate levels of the PFT Cry5B, indicating lack of either of these genes does not result in overt hypersensitivity or hyper-resistance to Cry5B (Figure 2A). However under the same conditions, the ire-1(v33) and xbp-1(zc12) mutant worms are more severely intoxicated than wild-type worms as they are relatively smaller and considerably paler compared to their corresponding no toxin controls. The hypersensitivity to Cry5B resulting from lack of ire-1 and xbp-1 was also seen using RNA interference (RNAi; data not shown), confirming the phenotype is caused by loss of function in these genes. We call this hypersensitivity phenotype “Hpo” for hypersensitive to pore-forming toxin.

Bottom Line: We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR.The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities.These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

View Article: PubMed Central - PubMed

Affiliation: Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, California, United States of America.

ABSTRACT
Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of the UPR is protective in vivo against PFTs since animals that lack either the ire-1-xbp-1 or the atf-6 arms of the UPR are more sensitive to PFT than wild-type animals. The UPR acts directly in the cells targeted by the PFT. Loss of the UPR leads to a normal response against unrelated toxins or a pathogenic bacterium, indicating its PFT-protective role is specific. The p38 mitogen-activated protein (MAPK) kinase pathway has been previously shown to be important for cellular defenses against PFTs. We find here that the UPR is one of the key downstream targets of the p38 MAPK pathway in response to PFT since loss of a functional p38 MAPK pathway leads to a failure of PFT to properly activate the ire-1-xbp-1 arm of the UPR. The UPR-mediated activation and response to PFTs is distinct from the canonical UPR-mediated response to unfolded proteins both in terms of its activation and functional sensitivities. These data demonstrate that the UPR, a fundamental intracellular pathway, can operate in intrinsic cellular defenses against bacterial attack.

Show MeSH
Related in: MedlinePlus