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Enteropathogenic Escherichia coli Uses NleA to Inhibit NLRP3 Inflammasome Activation.

Yen H, Sugimoto N, Tobe T - PLoS Pathog. (2015)

Bottom Line: It was found that the reduction is not because of blocked NF-κB activity; instead, the reduction results from inhibited caspase-1 activation by NleA.We further showed that the effector interacts with regions of NLRP3 containing the PYD and LRR domains.Additionally, NleA was found to associate with non-ubiquitinated and ubiquitinated NLRP3 and to interrupt de-ubiquitination of NLRP3, which is a required process for inflammasome activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, Graduate School of Medicine, Osaka University, Osaka, Japan.

ABSTRACT
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are related strains capable of inducing severe gastrointestinal disease. For optimal infection, these pathogens actively modulate cellular functions through the deployment of effector proteins in a type three secretion system (T3SS)-dependent manner. In response to enteric pathogen invasion, the Nod-like receptor pyrin domain containing (NLRP) inflammasome has been increasingly recognized as an important cytoplasmic sensor against microbial infection by activating caspase-1 and releasing IL-1β. EPEC and EHEC are known to elicit inflammasome activation in macrophages and epithelial cells; however, whether the pathogens actively counteract such innate immune responses is unknown. Using a series of compound effector-gene deletion strains of EPEC, we screened and identified NleA, which could subdue host IL-1β secretion. It was found that the reduction is not because of blocked NF-κB activity; instead, the reduction results from inhibited caspase-1 activation by NleA. Immunostaining of human macrophage-like cells following infection revealed limited formation of inflammasome foci with constituents of total caspase-1, ASC and NLRP3 in the presence of NleA. Pulldown of PMA-induced differentiated THP-1 lysate with purified MBP-NleA reveals that NLRP3 is a target of NleA. The interaction was verified by an immunoprecipitation assay and direct interaction assay in which purified MBP-NleA and GST-NLRP3 were used. We further showed that the effector interacts with regions of NLRP3 containing the PYD and LRR domains. Additionally, NleA was found to associate with non-ubiquitinated and ubiquitinated NLRP3 and to interrupt de-ubiquitination of NLRP3, which is a required process for inflammasome activation. Cumulatively, our findings provide the first example of EPEC-mediated suppression of inflammasome activity in which NieA plays a novel role in controlling the host immune response through targeting of NLRP3.

No MeSH data available.


Related in: MedlinePlus

Identification of NleA and NleE as inhibitors of host IL-1β secretion.Differentiated THP-1 cells were infected for 1 hr followed by addition of gentamicin to terminate extracellular bacteria. Cells were further incubated for 6 hrs, and culture supernatant was collected for ELISA analysis. (A) IL-1β secretion from EPEC-infected cells. Cells were uninfected or infected with WT, ΔescF (T3SS-deficient strain) and TOE-A6 (non-LEE effector-deleted strain), and the amount of IL-1β in the culture supernatant was measured. (B) TOE-A4 and TOE-A5 isogenic mutants lost their inhibitory property on IL-1β secretion. Cells were infected with WT and TOE-A strains (A1-A6), followed by measurement of the amount of IL-1β secreted in culture supernatants. (C and D) Complementation test for TOE-A4 or TOE-A5 identified NleE and NleA as inhibitors. TOE-A4 possessing nleE, or nleB, or espL were used for infection of THP-1 cells along with the parental strain (C). Alternatively, TOE-A5 possessing nleA, or nleF, or nleH2 were used for infection of THP-1 cells along with the parental strain (D). * p < 0.05 by Student’s t-test. Experiments were done in triplicate and repeated independently three times for (A) and (B); two times for (C) and (D); and one of the representative experiments with similar results is shown.
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ppat.1005121.g001: Identification of NleA and NleE as inhibitors of host IL-1β secretion.Differentiated THP-1 cells were infected for 1 hr followed by addition of gentamicin to terminate extracellular bacteria. Cells were further incubated for 6 hrs, and culture supernatant was collected for ELISA analysis. (A) IL-1β secretion from EPEC-infected cells. Cells were uninfected or infected with WT, ΔescF (T3SS-deficient strain) and TOE-A6 (non-LEE effector-deleted strain), and the amount of IL-1β in the culture supernatant was measured. (B) TOE-A4 and TOE-A5 isogenic mutants lost their inhibitory property on IL-1β secretion. Cells were infected with WT and TOE-A strains (A1-A6), followed by measurement of the amount of IL-1β secreted in culture supernatants. (C and D) Complementation test for TOE-A4 or TOE-A5 identified NleE and NleA as inhibitors. TOE-A4 possessing nleE, or nleB, or espL were used for infection of THP-1 cells along with the parental strain (C). Alternatively, TOE-A5 possessing nleA, or nleF, or nleH2 were used for infection of THP-1 cells along with the parental strain (D). * p < 0.05 by Student’s t-test. Experiments were done in triplicate and repeated independently three times for (A) and (B); two times for (C) and (D); and one of the representative experiments with similar results is shown.

Mentions: The inflammasome plays a major role in the secretion of IL-1β, one of the essential inflammatory cytokines for host defenses against enteropathogens. To determine whether pathogenic Escherichia coli could modulate inflammasome activity with effectors, we first induced THP-1 to differentiate into a macrophage-like cells and primed cells with LPS to promote synthesis of pro-IL-1β. LPS-primed dTHP-1 (differentiated THP-1) were uninfected (UI) or infected with wild type EPEC E2348/69 (WT), a ΔescF isogenic mutant (T3SS-defective strain), and TOE-A6 (an E2348/69 strain that lacks all non-LEE-effector genes); we then measured the amount of IL-1β using an ELISA assay. Compared with WT, we found that although the ΔescF mutant reduced the amount of secreted cytokine, TOE-A6 caused an increase in IL-1β secretion (Fig 1A), despite of comparatively lesser degree of bacterial exposure of TOE-A6 (S1 Fig). The reduction of IL-1β secretion from the ΔescF mutant-infected cells is consistent with previous reports in which the T3SS of EPEC/EHEC could elicit an inflammasome response [20]. The significant increase in IL-1β release from the cells infected by TOE-A6 suggested that deleted non-LEE effector(s) might participate in the suppression. To elucidate potential inhibitory effector(s), we performed a screening experiment by infecting dTHP-1 with the EPEC derivatives TOE-A1 to TOE-A6, which are strains with serial deletions of clusters of effector genes [21]. After 1 hr of infection followed by 6 hrs of further incubation, we used an ELISA method to measure the IL-1β secretion from cells infected with TOE-A1 to TOE-A6 strains. We found that there was a significant increase in the TOE-A4 and TOE-A5 strains compared with TOE-A3 and TOE-A4, respectively (Fig 1B). TOE-A4 was derived from TOE-A3 by the deletion of the IE6 region, which contains a cluster of effector genes (nleE, nleB1, and espL), resulting in a lack of a total of nine effector genes (nleB2, nleH1, espJ, nleG, nleC, nleD, nleE, nleB1 and espL). TOE-A5 was generated upon the removal of PP6 from TOE-A4, leading to additional loss in the effector genes nleH, nleA, and nleF. The results of the screening suggested that some of nleE, nleB1, espL, nleH, nleA, and nleF might be involved in the interference of IL-1β secretion. To identify the inhibitory effectors, we performed the rescue test by using plasmids expressing one of these effectors. Plasmids harboring each individual effector gene with a FLAGx3 epitope at the C-terminus were introduced into TOE-A4 or TOE-A5, yielding TOE-A4/pFLAG3-NleE (TOE-A4/nleE), TOE-A4/ pFLAG3-nleB1 (TOE-A4/nleB1), TOE-A4/ pFLAG3-espL (TOE-A4/espL), TOE-A5/ pFLAG3-nleH (TOE-A5/nleH), TOE-A5/ pFLAG3-nleA (TOE-A5/nleA), and TOE-A5/ pFLAG3-nleF (TOE-A5/nleF). We then infected dTHP-1 with these established strains and compared the level of IL-1β secretion to their respective parent strains. As shown in Fig 1C and 1D, TOE-A4/nleE and TOE-A5/nleA exhibited significant suppression of IL-1β release from the host. The expression of other effectors in TOE-A4 or TOE-A5 failed to reduce the secretion levels. These results indicated that at least two non-LEE-effectors, NleA and NleE, are capable of inhibiting host IL-1β secretion.


Enteropathogenic Escherichia coli Uses NleA to Inhibit NLRP3 Inflammasome Activation.

Yen H, Sugimoto N, Tobe T - PLoS Pathog. (2015)

Identification of NleA and NleE as inhibitors of host IL-1β secretion.Differentiated THP-1 cells were infected for 1 hr followed by addition of gentamicin to terminate extracellular bacteria. Cells were further incubated for 6 hrs, and culture supernatant was collected for ELISA analysis. (A) IL-1β secretion from EPEC-infected cells. Cells were uninfected or infected with WT, ΔescF (T3SS-deficient strain) and TOE-A6 (non-LEE effector-deleted strain), and the amount of IL-1β in the culture supernatant was measured. (B) TOE-A4 and TOE-A5 isogenic mutants lost their inhibitory property on IL-1β secretion. Cells were infected with WT and TOE-A strains (A1-A6), followed by measurement of the amount of IL-1β secreted in culture supernatants. (C and D) Complementation test for TOE-A4 or TOE-A5 identified NleE and NleA as inhibitors. TOE-A4 possessing nleE, or nleB, or espL were used for infection of THP-1 cells along with the parental strain (C). Alternatively, TOE-A5 possessing nleA, or nleF, or nleH2 were used for infection of THP-1 cells along with the parental strain (D). * p < 0.05 by Student’s t-test. Experiments were done in triplicate and repeated independently three times for (A) and (B); two times for (C) and (D); and one of the representative experiments with similar results is shown.
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ppat.1005121.g001: Identification of NleA and NleE as inhibitors of host IL-1β secretion.Differentiated THP-1 cells were infected for 1 hr followed by addition of gentamicin to terminate extracellular bacteria. Cells were further incubated for 6 hrs, and culture supernatant was collected for ELISA analysis. (A) IL-1β secretion from EPEC-infected cells. Cells were uninfected or infected with WT, ΔescF (T3SS-deficient strain) and TOE-A6 (non-LEE effector-deleted strain), and the amount of IL-1β in the culture supernatant was measured. (B) TOE-A4 and TOE-A5 isogenic mutants lost their inhibitory property on IL-1β secretion. Cells were infected with WT and TOE-A strains (A1-A6), followed by measurement of the amount of IL-1β secreted in culture supernatants. (C and D) Complementation test for TOE-A4 or TOE-A5 identified NleE and NleA as inhibitors. TOE-A4 possessing nleE, or nleB, or espL were used for infection of THP-1 cells along with the parental strain (C). Alternatively, TOE-A5 possessing nleA, or nleF, or nleH2 were used for infection of THP-1 cells along with the parental strain (D). * p < 0.05 by Student’s t-test. Experiments were done in triplicate and repeated independently three times for (A) and (B); two times for (C) and (D); and one of the representative experiments with similar results is shown.
Mentions: The inflammasome plays a major role in the secretion of IL-1β, one of the essential inflammatory cytokines for host defenses against enteropathogens. To determine whether pathogenic Escherichia coli could modulate inflammasome activity with effectors, we first induced THP-1 to differentiate into a macrophage-like cells and primed cells with LPS to promote synthesis of pro-IL-1β. LPS-primed dTHP-1 (differentiated THP-1) were uninfected (UI) or infected with wild type EPEC E2348/69 (WT), a ΔescF isogenic mutant (T3SS-defective strain), and TOE-A6 (an E2348/69 strain that lacks all non-LEE-effector genes); we then measured the amount of IL-1β using an ELISA assay. Compared with WT, we found that although the ΔescF mutant reduced the amount of secreted cytokine, TOE-A6 caused an increase in IL-1β secretion (Fig 1A), despite of comparatively lesser degree of bacterial exposure of TOE-A6 (S1 Fig). The reduction of IL-1β secretion from the ΔescF mutant-infected cells is consistent with previous reports in which the T3SS of EPEC/EHEC could elicit an inflammasome response [20]. The significant increase in IL-1β release from the cells infected by TOE-A6 suggested that deleted non-LEE effector(s) might participate in the suppression. To elucidate potential inhibitory effector(s), we performed a screening experiment by infecting dTHP-1 with the EPEC derivatives TOE-A1 to TOE-A6, which are strains with serial deletions of clusters of effector genes [21]. After 1 hr of infection followed by 6 hrs of further incubation, we used an ELISA method to measure the IL-1β secretion from cells infected with TOE-A1 to TOE-A6 strains. We found that there was a significant increase in the TOE-A4 and TOE-A5 strains compared with TOE-A3 and TOE-A4, respectively (Fig 1B). TOE-A4 was derived from TOE-A3 by the deletion of the IE6 region, which contains a cluster of effector genes (nleE, nleB1, and espL), resulting in a lack of a total of nine effector genes (nleB2, nleH1, espJ, nleG, nleC, nleD, nleE, nleB1 and espL). TOE-A5 was generated upon the removal of PP6 from TOE-A4, leading to additional loss in the effector genes nleH, nleA, and nleF. The results of the screening suggested that some of nleE, nleB1, espL, nleH, nleA, and nleF might be involved in the interference of IL-1β secretion. To identify the inhibitory effectors, we performed the rescue test by using plasmids expressing one of these effectors. Plasmids harboring each individual effector gene with a FLAGx3 epitope at the C-terminus were introduced into TOE-A4 or TOE-A5, yielding TOE-A4/pFLAG3-NleE (TOE-A4/nleE), TOE-A4/ pFLAG3-nleB1 (TOE-A4/nleB1), TOE-A4/ pFLAG3-espL (TOE-A4/espL), TOE-A5/ pFLAG3-nleH (TOE-A5/nleH), TOE-A5/ pFLAG3-nleA (TOE-A5/nleA), and TOE-A5/ pFLAG3-nleF (TOE-A5/nleF). We then infected dTHP-1 with these established strains and compared the level of IL-1β secretion to their respective parent strains. As shown in Fig 1C and 1D, TOE-A4/nleE and TOE-A5/nleA exhibited significant suppression of IL-1β release from the host. The expression of other effectors in TOE-A4 or TOE-A5 failed to reduce the secretion levels. These results indicated that at least two non-LEE-effectors, NleA and NleE, are capable of inhibiting host IL-1β secretion.

Bottom Line: It was found that the reduction is not because of blocked NF-κB activity; instead, the reduction results from inhibited caspase-1 activation by NleA.We further showed that the effector interacts with regions of NLRP3 containing the PYD and LRR domains.Additionally, NleA was found to associate with non-ubiquitinated and ubiquitinated NLRP3 and to interrupt de-ubiquitination of NLRP3, which is a required process for inflammasome activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Informatics, Graduate School of Medicine, Osaka University, Osaka, Japan.

ABSTRACT
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are related strains capable of inducing severe gastrointestinal disease. For optimal infection, these pathogens actively modulate cellular functions through the deployment of effector proteins in a type three secretion system (T3SS)-dependent manner. In response to enteric pathogen invasion, the Nod-like receptor pyrin domain containing (NLRP) inflammasome has been increasingly recognized as an important cytoplasmic sensor against microbial infection by activating caspase-1 and releasing IL-1β. EPEC and EHEC are known to elicit inflammasome activation in macrophages and epithelial cells; however, whether the pathogens actively counteract such innate immune responses is unknown. Using a series of compound effector-gene deletion strains of EPEC, we screened and identified NleA, which could subdue host IL-1β secretion. It was found that the reduction is not because of blocked NF-κB activity; instead, the reduction results from inhibited caspase-1 activation by NleA. Immunostaining of human macrophage-like cells following infection revealed limited formation of inflammasome foci with constituents of total caspase-1, ASC and NLRP3 in the presence of NleA. Pulldown of PMA-induced differentiated THP-1 lysate with purified MBP-NleA reveals that NLRP3 is a target of NleA. The interaction was verified by an immunoprecipitation assay and direct interaction assay in which purified MBP-NleA and GST-NLRP3 were used. We further showed that the effector interacts with regions of NLRP3 containing the PYD and LRR domains. Additionally, NleA was found to associate with non-ubiquitinated and ubiquitinated NLRP3 and to interrupt de-ubiquitination of NLRP3, which is a required process for inflammasome activation. Cumulatively, our findings provide the first example of EPEC-mediated suppression of inflammasome activity in which NieA plays a novel role in controlling the host immune response through targeting of NLRP3.

No MeSH data available.


Related in: MedlinePlus