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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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Endogenous LC3-I to LC3-II Conversion in BMMs Infected by ShigellaWild-type, caspase-1-deficient, Ipaf-deficient, or ASC-deficient BMMs were infected with Shigella WT, ΔvirG, or TTSS mutant S325. At 30 min after infection, the total lysates of BMMs were prepared and analyzed by western blotting using anti-LC3 antibody. The immunoblot for β-actin was indicated as an internal control.
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ppat-0030111-g006: Endogenous LC3-I to LC3-II Conversion in BMMs Infected by ShigellaWild-type, caspase-1-deficient, Ipaf-deficient, or ASC-deficient BMMs were infected with Shigella WT, ΔvirG, or TTSS mutant S325. At 30 min after infection, the total lysates of BMMs were prepared and analyzed by western blotting using anti-LC3 antibody. The immunoblot for β-actin was indicated as an internal control.

Mentions: Autophagy is induced by diverse death stimuli, including that associated with caspase-independent death, but the regulation of autophagy triggered by bacterial infection is poorly understood [32]. In pathogen-infected cells, autophagy appears to function as a host defense mechanism that can be subverted by certain virulent bacteria to enhance their intracellular replication [33–37]. To study the role of Ipaf and ASC in autophagy, we first examined whether autophagy is induced in Shigella-infected BMMs. To assess autophagy, wild-type and caspase-1-deficient BMMs were transfected with GFP-LC3 (Atg8, a marker protein of autophagy) using a retroviral vector, and the GFP-LC3 labeling pattern was visualized by fluorescence microscopy. Autophagy induced by amino acid starvation was not affected by caspase-1, because a similar number of GFP-LC3 aggregates that are typically associated with the formation of autophagosomal vesicles were observed in wild-type and caspase-1-, Ipaf-, and ASC-deficient BMMs (Figure 4A and 4B). As another approach, endogenous LC3-I to LC3-II conversion, which is an indicator of autophagosome maturation, was examined by western blotting after rapamycin treatment to induce autophagy [38–40]. Although LC3-II was more abundant than LC3-I in steady state in all BMMs, LC3 conversions (an increase of the amounts of LC3-II) were actually observed in wild-type and caspase-1-, Ipaf-, and ASC-deficient BMMs (Figure 4C), suggesting that rapamycin-induced autophagy is not affected by these genetic deficiencies. The cellular localization of GFP-LC3 was examined 30 min after infection with Shigella, since at this early time the membrane integrity of the majority of wild-type BMMs was retained (unpublished data). As shown in Figure 5A and 5B, nearly 20% of intracellular Shigella was associated with accumulated GFP-LC3 in wild-type BMMs, whereas the percentage increased to about 90% in caspase-1-deficient BMMs. No accumulation of GFP alone was observed in infected cells. These results indicate that the absence of caspase-1 promotes autophagosome maturation induced by Shigella infection. Interestingly, a large number of GFP-LC3-containing vesicles, which were not associated with bacteria, were observed in caspase-1-deficient BMMs infected with Shigella (Figure 5A), suggesting that endogenous autophagy was also activated during infection. The endogenous LC-I to LC3-II conversion was also detected by Shigella infection in caspase-1-deficient BMMs (Figure 6). In epithelial cells, internalized Shigella can escape from autophagy by secreting the IcsB effector, which interferes with the interaction of host Atg5 with bacterial surface protein VirG [41]. VirG is not only a bacterial factor essential for actin polymerization, but also a molecular target of host autophagy. Indeed, ΔvirG, a Shigella mutant lacking VirG, did not induce GFP-LC3 accumulation in epithelial cells [41]. Notably, we found that unlike that observed in epithelial cells, ΔvirG induced a similar level of GFP-LC3 aggregates as wild-type Shigella in BMMs (Figures 5A, 5B, and 6). These results indicate that factors other than VirG are involved in autophagy formation in caspase-1-deficient BMMs. In both wild-type and caspase-1-deficient BMMs, GFP-LC3 accumulation around the phagocytosed Shigella TTSS mutant (S325) and endogenous LC3 conversion were not induced (Figures 5A, 5B, and 6), suggesting that bacterial escape from the phagosome is required for autophagosome maturation in Shigella-infected BMMs.


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)

Endogenous LC3-I to LC3-II Conversion in BMMs Infected by ShigellaWild-type, caspase-1-deficient, Ipaf-deficient, or ASC-deficient BMMs were infected with Shigella WT, ΔvirG, or TTSS mutant S325. At 30 min after infection, the total lysates of BMMs were prepared and analyzed by western blotting using anti-LC3 antibody. The immunoblot for β-actin was indicated as an internal control.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0030111-g006: Endogenous LC3-I to LC3-II Conversion in BMMs Infected by ShigellaWild-type, caspase-1-deficient, Ipaf-deficient, or ASC-deficient BMMs were infected with Shigella WT, ΔvirG, or TTSS mutant S325. At 30 min after infection, the total lysates of BMMs were prepared and analyzed by western blotting using anti-LC3 antibody. The immunoblot for β-actin was indicated as an internal control.
Mentions: Autophagy is induced by diverse death stimuli, including that associated with caspase-independent death, but the regulation of autophagy triggered by bacterial infection is poorly understood [32]. In pathogen-infected cells, autophagy appears to function as a host defense mechanism that can be subverted by certain virulent bacteria to enhance their intracellular replication [33–37]. To study the role of Ipaf and ASC in autophagy, we first examined whether autophagy is induced in Shigella-infected BMMs. To assess autophagy, wild-type and caspase-1-deficient BMMs were transfected with GFP-LC3 (Atg8, a marker protein of autophagy) using a retroviral vector, and the GFP-LC3 labeling pattern was visualized by fluorescence microscopy. Autophagy induced by amino acid starvation was not affected by caspase-1, because a similar number of GFP-LC3 aggregates that are typically associated with the formation of autophagosomal vesicles were observed in wild-type and caspase-1-, Ipaf-, and ASC-deficient BMMs (Figure 4A and 4B). As another approach, endogenous LC3-I to LC3-II conversion, which is an indicator of autophagosome maturation, was examined by western blotting after rapamycin treatment to induce autophagy [38–40]. Although LC3-II was more abundant than LC3-I in steady state in all BMMs, LC3 conversions (an increase of the amounts of LC3-II) were actually observed in wild-type and caspase-1-, Ipaf-, and ASC-deficient BMMs (Figure 4C), suggesting that rapamycin-induced autophagy is not affected by these genetic deficiencies. The cellular localization of GFP-LC3 was examined 30 min after infection with Shigella, since at this early time the membrane integrity of the majority of wild-type BMMs was retained (unpublished data). As shown in Figure 5A and 5B, nearly 20% of intracellular Shigella was associated with accumulated GFP-LC3 in wild-type BMMs, whereas the percentage increased to about 90% in caspase-1-deficient BMMs. No accumulation of GFP alone was observed in infected cells. These results indicate that the absence of caspase-1 promotes autophagosome maturation induced by Shigella infection. Interestingly, a large number of GFP-LC3-containing vesicles, which were not associated with bacteria, were observed in caspase-1-deficient BMMs infected with Shigella (Figure 5A), suggesting that endogenous autophagy was also activated during infection. The endogenous LC-I to LC3-II conversion was also detected by Shigella infection in caspase-1-deficient BMMs (Figure 6). In epithelial cells, internalized Shigella can escape from autophagy by secreting the IcsB effector, which interferes with the interaction of host Atg5 with bacterial surface protein VirG [41]. VirG is not only a bacterial factor essential for actin polymerization, but also a molecular target of host autophagy. Indeed, ΔvirG, a Shigella mutant lacking VirG, did not induce GFP-LC3 accumulation in epithelial cells [41]. Notably, we found that unlike that observed in epithelial cells, ΔvirG induced a similar level of GFP-LC3 aggregates as wild-type Shigella in BMMs (Figures 5A, 5B, and 6). These results indicate that factors other than VirG are involved in autophagy formation in caspase-1-deficient BMMs. In both wild-type and caspase-1-deficient BMMs, GFP-LC3 accumulation around the phagocytosed Shigella TTSS mutant (S325) and endogenous LC3 conversion were not induced (Figures 5A, 5B, and 6), suggesting that bacterial escape from the phagosome is required for autophagosome maturation in Shigella-infected BMMs.

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