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Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages.

Suzuki T, Franchi L, Toma C, Ashida H, Ogawa M, Yoshikawa Y, Mimuro H, Inohara N, Sasakawa C, Nuñez G - PLoS Pathog. (2007)

Bottom Line: Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin.Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells.Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.

View Article: PubMed Central - PubMed

Affiliation: Division of Bacterial Pathogenesis, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan. t-suzuki@med.u-ryukyu.ac.jp

ABSTRACT
Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.

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Differential Regulation of Shigella-Induced Autophagy by Ipaf and ASCGFP-LC3-expressing Ipaf-deficient or ASC-deficient BMMs were infected with Shigella WT or TTSS mutant.(A) At 30 min after infection, the infected cells were immunostainted with Cy5-labeled anti-Shigella LPS antibody (colored red) and examined using a confocal microscope. The merged image with Cy5 bacteria and GFP fluorescence, and differential interference contrast (DIC) were also shown. Scale bars = 10 μm.(B) GFP-LC3-associated intracellular bacteria were quantified. Error bars represent mean ± SD.
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ppat-0030111-g007: Differential Regulation of Shigella-Induced Autophagy by Ipaf and ASCGFP-LC3-expressing Ipaf-deficient or ASC-deficient BMMs were infected with Shigella WT or TTSS mutant.(A) At 30 min after infection, the infected cells were immunostainted with Cy5-labeled anti-Shigella LPS antibody (colored red) and examined using a confocal microscope. The merged image with Cy5 bacteria and GFP fluorescence, and differential interference contrast (DIC) were also shown. Scale bars = 10 μm.(B) GFP-LC3-associated intracellular bacteria were quantified. Error bars represent mean ± SD.

Mentions: We next examined the role of Ipaf and ASC in autophagosome maturation in Shigella-infected BMMs. Similar to that observed in caspase-1-deficient BMMs, GFP-LC3 accumulation and endogenous LC3-I to LC3-II conversion were enhanced after Shigella infection in Ipaf-deficient BMMs when compared to wild-type cells (Figures 6, 7A, and 7B). Because caspase-1 activation induced by Shigella is deficient in caspase-1- and Ipaf-deficient BMMs, these results suggested that caspase-1 activation inhibits the induction of Shigella-induced autophagy. However, when ASC-deficient BMMs were infected with Shigella, the levels of autophagy associated with intracellular bacteria were similar to those observed in wild-type BMMs (Figures 6, 7A, and 6B), indicating that autophagosome maturation is not enhanced by ASC deficiency, even though caspase-1 activation is abrogated upon Shigella infection (Figure 2E). GFP-LC3-associated autophagic vesicles triggered by amino acid starvation and endogenous LC3-I to LC3-II conversion by rapamycin treatment were still induced in ASC-deficient BMMs, suggesting that the autophagic machinery is intact in the absence of ASC. Together with the results presented in Figure 3, these results indicate that Ipaf and ASC differentially regulate the induction of autophagy and suggest that autophagy in caspase-1- and Ipaf-deficient BMMs is associated with resistance to pyroptotic cell death.


Differential regulation of caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in Shigella-infected macrophages.

Suzuki T, Franchi L, Toma C, Ashida H, Ogawa M, Yoshikawa Y, Mimuro H, Inohara N, Sasakawa C, Nuñez G - PLoS Pathog. (2007)

Differential Regulation of Shigella-Induced Autophagy by Ipaf and ASCGFP-LC3-expressing Ipaf-deficient or ASC-deficient BMMs were infected with Shigella WT or TTSS mutant.(A) At 30 min after infection, the infected cells were immunostainted with Cy5-labeled anti-Shigella LPS antibody (colored red) and examined using a confocal microscope. The merged image with Cy5 bacteria and GFP fluorescence, and differential interference contrast (DIC) were also shown. Scale bars = 10 μm.(B) GFP-LC3-associated intracellular bacteria were quantified. Error bars represent mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1941748&req=5

ppat-0030111-g007: Differential Regulation of Shigella-Induced Autophagy by Ipaf and ASCGFP-LC3-expressing Ipaf-deficient or ASC-deficient BMMs were infected with Shigella WT or TTSS mutant.(A) At 30 min after infection, the infected cells were immunostainted with Cy5-labeled anti-Shigella LPS antibody (colored red) and examined using a confocal microscope. The merged image with Cy5 bacteria and GFP fluorescence, and differential interference contrast (DIC) were also shown. Scale bars = 10 μm.(B) GFP-LC3-associated intracellular bacteria were quantified. Error bars represent mean ± SD.
Mentions: We next examined the role of Ipaf and ASC in autophagosome maturation in Shigella-infected BMMs. Similar to that observed in caspase-1-deficient BMMs, GFP-LC3 accumulation and endogenous LC3-I to LC3-II conversion were enhanced after Shigella infection in Ipaf-deficient BMMs when compared to wild-type cells (Figures 6, 7A, and 7B). Because caspase-1 activation induced by Shigella is deficient in caspase-1- and Ipaf-deficient BMMs, these results suggested that caspase-1 activation inhibits the induction of Shigella-induced autophagy. However, when ASC-deficient BMMs were infected with Shigella, the levels of autophagy associated with intracellular bacteria were similar to those observed in wild-type BMMs (Figures 6, 7A, and 6B), indicating that autophagosome maturation is not enhanced by ASC deficiency, even though caspase-1 activation is abrogated upon Shigella infection (Figure 2E). GFP-LC3-associated autophagic vesicles triggered by amino acid starvation and endogenous LC3-I to LC3-II conversion by rapamycin treatment were still induced in ASC-deficient BMMs, suggesting that the autophagic machinery is intact in the absence of ASC. Together with the results presented in Figure 3, these results indicate that Ipaf and ASC differentially regulate the induction of autophagy and suggest that autophagy in caspase-1- and Ipaf-deficient BMMs is associated with resistance to pyroptotic cell death.

Bottom Line: Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin.Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells.Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.

View Article: PubMed Central - PubMed

Affiliation: Division of Bacterial Pathogenesis, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan. t-suzuki@med.u-ryukyu.ac.jp

ABSTRACT
Shigella infection, the cause of bacillary dysentery, induces caspase-1 activation and cell death in macrophages, but the precise mechanisms of this activation remain poorly understood. We demonstrate here that caspase-1 activation and IL-1beta processing induced by Shigella are mediated through Ipaf, a cytosolic pattern-recognition receptor of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, and the adaptor protein apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain (ASC). We also show that Ipaf was critical for pyroptosis, a specialized form of caspase-1-dependent cell death induced in macrophages by bacterial infection, whereas ASC was dispensable. Unlike that observed in Salmonella and Legionella, caspase-1 activation induced by Shigella infection was independent of flagellin. Notably, infection of macrophages with Shigella induced autophagy, which was dramatically increased by the absence of caspase-1 or Ipaf, but not ASC. Autophagy induced by Shigella required an intact bacterial type III secretion system but not VirG protein, a bacterial factor required for autophagy in epithelial-infected cells. Treatment of macrophages with 3-methyladenine, an inhibitor of autophagy, enhanced pyroptosis induced by Shigella infection, suggesting that autophagy protects infected macrophages from pyroptosis. Thus, Ipaf plays a critical role in caspase-1 activation induced by Shigella independently of flagellin. Furthermore, the absence of Ipaf or caspase-1, but not ASC, regulates pyroptosis and the induction of autophagy in Shigella-infected macrophages, providing a novel function for NLR proteins in bacterial-host interactions.

Show MeSH
Related in: MedlinePlus