Veterinary Microbiology 171 (2014) 160–164
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Enterotoxigenic Escherichia coli infection induces intestinal epithelial cell autophagy Yulong Tang a,*, Fengna Li a, Bie Tan a, Gang Liu a, Xiangfeng Kong a, Philip R. Hardwidge b, Yulong Yin a a Institute of Subtropical Agriculture, Chinese Academy of Sciences, Research Center of Healthy Breeding Livestock & Poultry, Hunan Engineering & Research Center of Animal & Poultry Science, Key Lab Agro-ecology Processing Subtropical Region, Scientific Observational and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Changsha, Hunan, People’s Republic of China b Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 December 2013 Received in revised form 17 March 2014 Accepted 22 March 2014
The morbidity and mortality in piglets caused by enterotoxigenic Escherichia coli (ETEC) results in large economic losses to the swine industry, but the precise pathogenesis of ETEC-associated diseases remains unknown. Intestinal epithelial cell autophagy serves as a host defense against pathogens. We found that ETEC induced autophagy, as measured by both the increased punctae distribution of GFP-LC3 and the enhanced conversion of LC3-I to LC3-II. Inhibiting autophagy resulted in decreased survival of IPEC-1 cells infected with ETEC. ETEC triggered autophagy in IPEC-1 cells through a pathway involving the mammalian target of rapamycin (mTOR), the extracellular signal-regulated kinases 1/2 (ERK1/2), and the AMP-activated protein kinase (AMPK). ß 2014 Elsevier B.V. All rights reserved.
Keywords: Autophagy Enterotoxigenic Escherichia coli IPEC-1
1. Introduction Enterotoxigenic Escherichia coli (ETEC) causes porcine post-weaning diarrhea and is also a source of human morbidity and mortality (Turner et al., 2006). ETEC virulence is associated with the production of colonization factors and with one or more enterotoxins (Devriendt et al., 2010). The intestinal epithelium deploys multiple innate defense mechanisms to fight microbial intruders, including rapid epithelial cell turnover, epithelial barrier integrity, expulsion of infected cells, and autophagy (Kim et al., 2010). Autophagy is a fundamental homeostatic process in which cytoplasmic targets are sequestered in double* Corresponding author. Tel.: +86 73184619767. E-mail addresses: [email protected] (Y. Tang), [email protected] (Y. Yin). http://dx.doi.org/10.1016/j.vetmic.2014.03.025 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
membraned autophagosomes and subsequently delivered to lysosomes for degradation. Autophagy is considered a vital part of host defenses against microbial infection by mediating the delivery of pathogens to lysosomes (Cemma and Brumell, 2012). Antibacterial autophagy (xenophagy) involves several important genes (ATGs) that are critical for autophagosome formation (Knodler and Celli, 2011). Microtubule-associated protein 1 light chain 3 (LC3) is the mammalian ortholog of yeast Atg8 and is required for autophagosome formation. The autophagy-related proteins Atg12 and Atg5, with Atg7 and Atg10, are involved in autophagosome conjugation (Nishida et al., 2009). Several signaling pathways regulate autophagy, including the mammalian target of rapamycin (mTOR), ERK1/2, class I phosphoinositide 3-kinase (PI3 K), Akt, and the AMPactivated protein kinase [AMPK (Saiki et al., 2011)]. Although the pathogenesis and virulence factors of ETEC have been well characterized, its ability to induce
Y. Tang et al. / Veterinary Microbiology 171 (2014) 160–164
autophagy in mammalian cells has not been documented. In this report, we present evidence that ETEC induces autophagy through the mTOR pathway.
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were selected (IPEC/EGFP-LC3 cells) and maintained in selection medium. 2.3. Bacterial infection
2. Materials and methods 2.1. Bacterial strains The ETEC strain SEC470 (F18, LT+, STa+ and STb+) was originally isolated from the anus of a piglet afflicted with diarrheal disease. It was cultured in Luria-Bertani broth or on agar at 3 8C (Chen et al., 2014). 2.2. Plasmid construction and stable transfection of IPEC cells The EGFP and porcine MAPILC3B genes were amplified from the mammalian expression vector pcDNA3.1-EGFP and from porcine intestinal epithelial cells, respectively. The EGFP-LC3 fusion fragment was obtained using overlap PCR and cloned into the multiple cloning site of pcDNA3.1 (Invitrogen). IPEC-1 porcine intestinal epithelial cells were grown in high-glucose Dulbecco’s modified Eagle medium (DMEM)-Hank F12 containing 5% fetal bovine serum, 50 mg/ml penicillin, 50 mg/ml streptomycin, and insulin (5 mg/ml), transferrin (5 mg/ml), selenium (5 ng/ml) (ITS, ScienCell), and 5 ng/ml of epidermal growth factor (EGF; Sigma). IPEC-1 cells were transfected with plasmids using Lipofectamine2000 (Life Technologies). Transfected cells were cultured overnight in complete medium, followed by culturing in selection medium containing 600 mg/ml of G418 (Sigma). Single clones stably expressing EGFP-LC3
IPEC-1 or IPEC/EGFP-LC3 cells were grown in 6-well tissue culture plates. ETEC SEC470 was cultured in LB for 6 h at 37 8C, harvested by centrifugation, washed twice in phosphate-buffered saline (PBS), and resuspended in fresh F12 medium. Confluent monolayers of IPEC-1 or IPEC/ EGFP-LC3 cells were inoculated with ETEC SEC470 at an MOI of 1:100 for 1–3 h. The chemicals used to treat IPEC-1 or IPEC/EGFP-LC3 cells included 0.5 mM rapamycin (12 h, Sigma), 4 mM 3-methyladenine (2 h, Sigma), 20 mM UO126 (2 h, Sigma) and 5 mM compound C (2 h, Merck). 2.4. Adhesion and invasion assays Adhesion and invasion assays were performed as described previously (Johnson et al., 2009). Confluent monolayers of IPEC-1 cells (105 cells/well) were infected with ETEC suspensions to obtain a multiplicity of infection (MOI) of 1:100. For adhesion assays, plates were centrifuged at 800 g for 10 min and incubated for 1 h at 37 8C and 5% CO2. Monolayers were then washed four times with PBS, trypsinized and disrupted by mild pipetting. Viable bacterial cells were determined by plating dilutions of the lysates onto agar. For invasion assays, infected monolayers were incubated for 1 h at 37 8C and 5% CO2 and then subjected to antibiotic killing of extracellular bacteria for 1 h at 37 8C. Viable intracellular bacteria were counted.
Fig. 1. ETEC induces autophagosome formation in IPEC-1 cells. (A) Stably transfected IPEC-1 cells expressing EGFP-LC3 were infected with ETEC or treated with rapamycin. LC3 is shown in green and DAPI is shown in blue. (B) Average number of punctae in each cell (n = 60–80). (C) Immunoblotting of LC3-I and LC3-II in IPEC-1 cells infected with ETEC or treated with 0.5 mM rapamycin. LC3-II expression was normalized to b-Actin expression.
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2.5. Confocal microscopy
2.7. Cell viability and cytotoxicity
IPEC/EGFP-LC3 cells were seeded on coverslips (Fisher) in 6-well tissue culture plates for overnight culture at 37 8C. For detection of autophagosomes, the cells were inoculated with ETEC or treated with 0.5 mM rapamycin for indicated times. Cell were washed with PBS, fixed and permeabilized with 80% cold acetone at 20 8C for 20 min, and washed again with PBS. Cells were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI). Fluorescence of EGFP-LC3 was observed under a laser scanning confocal microscope. The average number of EGFP-LC3 punctae per cell from at least 60–80 cells per sample was counted. Only cells with at least five dots were scored as positive.
Cell viability was determined using the MTT method according to the manufacturer’s instructions (Promega). IPEC-1 cells were seeded in 96-well plates at a density of 1 104 cells/well and were incubated at 37 8C in a CO2 incubator. After overnight incubation, the medium was replaced by fresh complete medium containing DMSO or 3-MA and incubated for up to 2 h. After ETEC infection, 10 ml of MTT was then added to each well. The plates were further incubated for 4 h at 37 8C. Absorbance at 490 nm was measured using a Thermomax microplate reader. Each treatment was performed in triplicate. Cytotoxicity was assayed by quantifying lactate dehydrogenase (LDH) release using the CytoTox96 kit (Roche). Absorbance was measured at 500 nm using a Thermomax microplate reader. The experiment was performed in triplicate.
2.6. Immunoblotting Immunoblotting assays were performed as described previously (Wang et al., 2012). Blots were developed using the ECL Plus detection system and Fujifilm LAS-3000 (Tokyo, Japan). Protein band density was normalized to b-actin signal and quantified using Quantity One software.
2.8. Statistics Data were expressed as means standard errors and analyzed using two-tailed Student’s t-tests. Experiments
Fig. 2. Inhibiting mTOR signaling induces autophagy through ERK1/2 and AMPK. (A) Immunoblotting of phosphorylated and total mTOR in IPEC-1 cells infected with ETEC or treated with rapamycin. (B) Immunoblotting of phosphorylated and total ERK1/2 LC3-II in IPEC-1 cells infected with ETEC. (C) IPEC-1 cells were treated with U0126 and then analyzed for LC3-I, LC3-II, ERK1/2, and p-ERK1/2 expression after ETEC infection. (D) Immunoblotting of phosphorylated and total AMPK after ETEC infection. (E) IPEC-1 cells were treated with compound C and then analyzed for LC3-I, LC3-II, AMPK and p-AMPK expression after ETEC infection.
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were performed in triplicate. P-values < 0.05 were considered statistically significant. 3. Results and discussion 3.1. ETEC infection increases autophagosome formation Porcine intestinal epithelial cells bind numerous ETEC strains to varying degrees. ETEC SEC470 adhered to IPEC-1 cells at a concentration of 105 bacterial/ml after 1 h infection. ETEC SEC470 is non-invasive, with intracellular bacteria observed in only 0.2% of infected cells. Transfecting EGFP-LC3 for fluorescence analysis is widely used to detect autophagosomes (Zhu et al., 2012). To investigate whether ETEC infection induces autophagosome formation, IPEC/EGFP-LC3 cells were infected with ETEC for 3 h. Confocal microscopy analysis showed that both ETEC and the autophagy inducer rapamycin induced a substantial increase in the number of cells containing EGFP-LC3 punctae, as compared with uninfected control cells (Fig. 1A). The number of punctae per cell was counted for quantitative assessment of autophagic activity in transfected IPEC-1 cells. Cells treated with ETEC or rapamycin showed a 4.9-fold or 5.7-fold increase, respectively, in the number of EGFP-LC3 punctae per cell as compared with control cells (Fig. 1B). During autophagy, cytosolic LC3-I is linked to phosphatidylethanolamine (PE) to form LC3-II and remain tightly bound to the autophagosomal membranes (Mizushima et al., 2010). To determine whether the observed autophagosomes-like vesicles are indeed related to autophagy, the LC3-II protein, another hallmark of autophagy, was examined by immunoblotting. ETECinfected cells had a significantly increased ratio of LC3II/b-actin as compared with uninfected cells at 2 and 3 h post-infection (Fig. 1C). Thus, it appears that ETEC infection induces autophagy in IPEC-1 cells.
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Towler and Hardie, 2007), and is a positive regulator of autophagy (Egan et al., 2011). ETEC infection significantly increased the amount of phosphorylated AMPK without affecting total AMPK abundance (Fig. 2D). Treating IPEC-1 cells with the AMPK inhibitor Compound C reduced LC3-II abundance (Fig. 2E). 3.3. Inhibiting autophagy increases ETEC-induced cell death To determine whether inhibiting autophagy would affect IPEC-1 cell viability during ETEC infection, we treated cells with 3-MA, a phostphatidylinositol-3-kinase (PI3K) inhibitor that is widely used for selective suppression of autophagy by blocking the formation of autophagomes. IPEC-1 cells were treated with 3-MA for 2 h to inhibit autophagy and then infected with ETEC for 1–3 h. Cells pre-treated with 3-MA displayed reduced viability during ETEC infection as determined using MTT assays (Fig. 3A). IPEC-1 cytotoxicity was also assayed by measuring lactate dehydrogenase (LDH) release. Cells treated with 3-MA released greater amounts of LDH during ETEC infection, as compared with infected cells not treated with 3-MA (Fig. 3B). ETEC are an important cause of diarrhea in man and animals. In neonatal and weaned piglets, ETEC-associated diarrhea is considered to be one of the most important diseases in swine husbandry. Here, we provide evidence that ETEC induces autophagosome formation in IPEC-1 cells as shown by the punctate GFP-LC3 distribution in the cytoplasm and the corresponding increase in the punctae counts. LC3-II protein abundance was also significantly
3.2. ETEC inhibits mTOR phosphorylation and ERK1/2 and AMPK contribute to autophagy during ETEC infection To understand further the underlying mechanism of ETEC-induced autophagy, we next investigated whether mTOR, a signaling protein that regulates autophagy was inhibited by ETEC. mTOR phosphorylation was significantly inhibited by both ETEC infection and by rapamycin treatment (Fig. 2A). There was no change in total mTOR abundance (Fig. 2A). The ERK1/2 pathway regulates host responses to ETEC infection (Wang et al., 2012) and plays a key role in regulating autophagy. We investigated whether ETEC induces autophagy by activating ERK1/2 signaling in IPEC-1 cells. ETEC infection increased ERK1/2 phosphorylation after 1 h (Fig. 2B), without affecting total ERK1/2 abundance. Inhibiting ERK1/2 phosphorylation with UO126 suppressed the conversion of LC3-I to LC3-II (Fig. 2C), suggesting ETEC induces autophagy in IPEC-1 cells through ERK1/2 signaling. AMPK plays a key role in the regulation of energy homeostasis, is activated by an elevated AMP/ATP ratio due to infection and heat shock (Arsenault et al., 2013;
Fig. 3. Inhibiting autophagy increases cell death in ETEC-infected IPEC-1 cells. (A) Cell viability was determined using MTT assay after ETEC infection in the presence or absence of 3-MA. (B) IPEC-1 cell cytotoxicity was analyzed by quantifying LDH release after ETEC infection in the presence or absence of 3-MA.
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higher in ETEC-infected cells as compared with uninfected cells. Autophagy may not only function as a defensive mechanism against invading pathogens but also may serve as a protective cellular response against toxins (Tan et al., 2009) and noninvasive pathogens. The cellular autophagic machinery appeared beneficial to IPEC-1 cells, as cells inhibited for autophagy showed enhanced cell death during ETEC infection. Overall, our data suggest that ETEC induces autophagy in IPEC-1 cells by inhibiting mTOR through the AMPK and ERK1/2 signaling pathways. Acknowledgements This research was jointly supported by National Basic Research Projects (2012CB124704 and 2009CB118800) and Comprehensive Strategic Cooperation projects from the Chinese Academy of Sciences and Guangdong Province (2012B091100210). References Arsenault, R.J., Napper, S., Kogut, M.H., 2013. Salmonella enterica Typhimurium infection causes metabolic changes in chicken muscle involving AMPK, fatty acid and insulin/mTOR signaling. Vet. Res. 44, 35. Cemma, M., Brumell, J.H., 2012. Interactions of pathogenic bacteria with autophagy systems. Curr. Biol. 22, R540–R545. Chen, X., Huan, H., Wan, T., Wang, L., Gao, S., Jiao, X., 2014. Antigenic determinants analysis and detection of virulence factors in F18 fimbriae Escherichia coli strains isolated from pigs. Acta Microbiol. Sin. 54, 236–242. Devriendt, B., Stuyven, E., Verdonck, F., Goddeeris, B.M., Cox, E., 2010. Enterotoxigenic Escherichia coli (K88) induce proinflammatory
responses in porcine intestinal epithelial cells. Dev. Comp. Immunol. 34, 1175–1182. Egan, D.F., Shackelford, D.B., Mihaylova, M.M., Gelino, S., Kohnz, R.A., Mair, W., Vasquez, D.S., Joshi, A., Gwinn, D.M., Taylor, R., Asara, J.M., Fitzpatrick, J., Dillin, A., Viollet, B., Kundu, M., Hansen, M., Shaw, R.J., 2011. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331, 456– 461. Johnson, A.M., Kaushik, R.S., Francis, D.H., Fleckenstein, J.M., Hardwidge, P.R., 2009. Heat-labile enterotoxin promotes Escherichia coli adherence to intestinal epithelial cells. J. Bacteriol. 191, 178–186. Kim, M., Ashida, H., Ogawa, M., Yoshikawa, Y., Mimuro, H., Sasakawa, C., 2010. Bacterial interactions with the host epithelium. Cell Host Microbe 8, 20–35. Knodler, L.A., Celli, J., 2011. Eating the strangers within: host control of intracellular bacteria via xenophagy. Cell. Microbiol. 13, 1319–1327. Mizushima, N., Yoshimori, T., Levine, B., 2010. Methods in mammalian autophagy research. Cell 140, 313–326. Nishida, K., Kyoi, S., Yamaguchi, O., Sadoshima, J., Otsu, K., 2009. The role of autophagy in the heart. Cell Death Differ. 16, 31–38. Saiki, S., Sasazawa, Y., Imamichi, Y., Kawajiri, S., Fujimaki, T., Tanida, I., Kobayashi, H., Sato, F., Sato, S., Ishikawa, K., Imoto, M., Hattori, N., 2011. Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition. Autophagy 7, 176–187. Tan, Y.K., Kusuma, C.M., St John, L.J., Vu, H.A., Alibek, K., Wu, A., 2009. Induction of autophagy by anthrax lethal toxin. Biochem. Biophys. Res. Commun. 379, 293–297. Towler, M.C., Hardie, D.G., 2007. AMP-activated protein kinase in metabolic control and insulin signaling. Circ. Res. 100, 328–341. Turner, S.M., Scott-Tucker, A., Cooper, L.M., Henderson, I.R., 2006. Weapons of mass destruction: virulence factors of the global killer enterotoxigenic Escherichia coli. FEMS Microbiol. Lett. 263, 10–20. Wang, X., Gao, X., Hardwidge, P.R., 2012. Heat-labile enterotoxin-induced activation of NF-kappaB and MAPK pathways in intestinal epithelial cells impacts enterotoxigenic Escherichia coli (ETEC) adherence. Cell. Microbiol. 14, 1231–1241. Zhu, B., Xu, F., Li, J., Shuai, J., Li, X., Fang, W., 2012. Porcine circovirus type 2 explores the autophagic machinery for replication in PK-15 cells. Virus Res. 163, 476–485.
Contents lists available at ScienceDirect
Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic
Enterotoxigenic Escherichia coli infection induces intestinal epithelial cell autophagy Yulong Tang a,*, Fengna Li a, Bie Tan a, Gang Liu a, Xiangfeng Kong a, Philip R. Hardwidge b, Yulong Yin a a Institute of Subtropical Agriculture, Chinese Academy of Sciences, Research Center of Healthy Breeding Livestock & Poultry, Hunan Engineering & Research Center of Animal & Poultry Science, Key Lab Agro-ecology Processing Subtropical Region, Scientific Observational and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Changsha, Hunan, People’s Republic of China b Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, KS, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 December 2013 Received in revised form 17 March 2014 Accepted 22 March 2014
The morbidity and mortality in piglets caused by enterotoxigenic Escherichia coli (ETEC) results in large economic losses to the swine industry, but the precise pathogenesis of ETEC-associated diseases remains unknown. Intestinal epithelial cell autophagy serves as a host defense against pathogens. We found that ETEC induced autophagy, as measured by both the increased punctae distribution of GFP-LC3 and the enhanced conversion of LC3-I to LC3-II. Inhibiting autophagy resulted in decreased survival of IPEC-1 cells infected with ETEC. ETEC triggered autophagy in IPEC-1 cells through a pathway involving the mammalian target of rapamycin (mTOR), the extracellular signal-regulated kinases 1/2 (ERK1/2), and the AMP-activated protein kinase (AMPK). ß 2014 Elsevier B.V. All rights reserved.
Keywords: Autophagy Enterotoxigenic Escherichia coli IPEC-1
1. Introduction Enterotoxigenic Escherichia coli (ETEC) causes porcine post-weaning diarrhea and is also a source of human morbidity and mortality (Turner et al., 2006). ETEC virulence is associated with the production of colonization factors and with one or more enterotoxins (Devriendt et al., 2010). The intestinal epithelium deploys multiple innate defense mechanisms to fight microbial intruders, including rapid epithelial cell turnover, epithelial barrier integrity, expulsion of infected cells, and autophagy (Kim et al., 2010). Autophagy is a fundamental homeostatic process in which cytoplasmic targets are sequestered in double* Corresponding author. Tel.: +86 73184619767. E-mail addresses: [email protected] (Y. Tang), [email protected] (Y. Yin). http://dx.doi.org/10.1016/j.vetmic.2014.03.025 0378-1135/ß 2014 Elsevier B.V. All rights reserved.
membraned autophagosomes and subsequently delivered to lysosomes for degradation. Autophagy is considered a vital part of host defenses against microbial infection by mediating the delivery of pathogens to lysosomes (Cemma and Brumell, 2012). Antibacterial autophagy (xenophagy) involves several important genes (ATGs) that are critical for autophagosome formation (Knodler and Celli, 2011). Microtubule-associated protein 1 light chain 3 (LC3) is the mammalian ortholog of yeast Atg8 and is required for autophagosome formation. The autophagy-related proteins Atg12 and Atg5, with Atg7 and Atg10, are involved in autophagosome conjugation (Nishida et al., 2009). Several signaling pathways regulate autophagy, including the mammalian target of rapamycin (mTOR), ERK1/2, class I phosphoinositide 3-kinase (PI3 K), Akt, and the AMPactivated protein kinase [AMPK (Saiki et al., 2011)]. Although the pathogenesis and virulence factors of ETEC have been well characterized, its ability to induce
Y. Tang et al. / Veterinary Microbiology 171 (2014) 160–164
autophagy in mammalian cells has not been documented. In this report, we present evidence that ETEC induces autophagy through the mTOR pathway.
161
were selected (IPEC/EGFP-LC3 cells) and maintained in selection medium. 2.3. Bacterial infection
2. Materials and methods 2.1. Bacterial strains The ETEC strain SEC470 (F18, LT+, STa+ and STb+) was originally isolated from the anus of a piglet afflicted with diarrheal disease. It was cultured in Luria-Bertani broth or on agar at 3 8C (Chen et al., 2014). 2.2. Plasmid construction and stable transfection of IPEC cells The EGFP and porcine MAPILC3B genes were amplified from the mammalian expression vector pcDNA3.1-EGFP and from porcine intestinal epithelial cells, respectively. The EGFP-LC3 fusion fragment was obtained using overlap PCR and cloned into the multiple cloning site of pcDNA3.1 (Invitrogen). IPEC-1 porcine intestinal epithelial cells were grown in high-glucose Dulbecco’s modified Eagle medium (DMEM)-Hank F12 containing 5% fetal bovine serum, 50 mg/ml penicillin, 50 mg/ml streptomycin, and insulin (5 mg/ml), transferrin (5 mg/ml), selenium (5 ng/ml) (ITS, ScienCell), and 5 ng/ml of epidermal growth factor (EGF; Sigma). IPEC-1 cells were transfected with plasmids using Lipofectamine2000 (Life Technologies). Transfected cells were cultured overnight in complete medium, followed by culturing in selection medium containing 600 mg/ml of G418 (Sigma). Single clones stably expressing EGFP-LC3
IPEC-1 or IPEC/EGFP-LC3 cells were grown in 6-well tissue culture plates. ETEC SEC470 was cultured in LB for 6 h at 37 8C, harvested by centrifugation, washed twice in phosphate-buffered saline (PBS), and resuspended in fresh F12 medium. Confluent monolayers of IPEC-1 or IPEC/ EGFP-LC3 cells were inoculated with ETEC SEC470 at an MOI of 1:100 for 1–3 h. The chemicals used to treat IPEC-1 or IPEC/EGFP-LC3 cells included 0.5 mM rapamycin (12 h, Sigma), 4 mM 3-methyladenine (2 h, Sigma), 20 mM UO126 (2 h, Sigma) and 5 mM compound C (2 h, Merck). 2.4. Adhesion and invasion assays Adhesion and invasion assays were performed as described previously (Johnson et al., 2009). Confluent monolayers of IPEC-1 cells (105 cells/well) were infected with ETEC suspensions to obtain a multiplicity of infection (MOI) of 1:100. For adhesion assays, plates were centrifuged at 800 g for 10 min and incubated for 1 h at 37 8C and 5% CO2. Monolayers were then washed four times with PBS, trypsinized and disrupted by mild pipetting. Viable bacterial cells were determined by plating dilutions of the lysates onto agar. For invasion assays, infected monolayers were incubated for 1 h at 37 8C and 5% CO2 and then subjected to antibiotic killing of extracellular bacteria for 1 h at 37 8C. Viable intracellular bacteria were counted.
Fig. 1. ETEC induces autophagosome formation in IPEC-1 cells. (A) Stably transfected IPEC-1 cells expressing EGFP-LC3 were infected with ETEC or treated with rapamycin. LC3 is shown in green and DAPI is shown in blue. (B) Average number of punctae in each cell (n = 60–80). (C) Immunoblotting of LC3-I and LC3-II in IPEC-1 cells infected with ETEC or treated with 0.5 mM rapamycin. LC3-II expression was normalized to b-Actin expression.
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2.5. Confocal microscopy
2.7. Cell viability and cytotoxicity
IPEC/EGFP-LC3 cells were seeded on coverslips (Fisher) in 6-well tissue culture plates for overnight culture at 37 8C. For detection of autophagosomes, the cells were inoculated with ETEC or treated with 0.5 mM rapamycin for indicated times. Cell were washed with PBS, fixed and permeabilized with 80% cold acetone at 20 8C for 20 min, and washed again with PBS. Cells were counterstained with 40 ,6-diamidino-2-phenylindole (DAPI). Fluorescence of EGFP-LC3 was observed under a laser scanning confocal microscope. The average number of EGFP-LC3 punctae per cell from at least 60–80 cells per sample was counted. Only cells with at least five dots were scored as positive.
Cell viability was determined using the MTT method according to the manufacturer’s instructions (Promega). IPEC-1 cells were seeded in 96-well plates at a density of 1 104 cells/well and were incubated at 37 8C in a CO2 incubator. After overnight incubation, the medium was replaced by fresh complete medium containing DMSO or 3-MA and incubated for up to 2 h. After ETEC infection, 10 ml of MTT was then added to each well. The plates were further incubated for 4 h at 37 8C. Absorbance at 490 nm was measured using a Thermomax microplate reader. Each treatment was performed in triplicate. Cytotoxicity was assayed by quantifying lactate dehydrogenase (LDH) release using the CytoTox96 kit (Roche). Absorbance was measured at 500 nm using a Thermomax microplate reader. The experiment was performed in triplicate.
2.6. Immunoblotting Immunoblotting assays were performed as described previously (Wang et al., 2012). Blots were developed using the ECL Plus detection system and Fujifilm LAS-3000 (Tokyo, Japan). Protein band density was normalized to b-actin signal and quantified using Quantity One software.
2.8. Statistics Data were expressed as means standard errors and analyzed using two-tailed Student’s t-tests. Experiments
Fig. 2. Inhibiting mTOR signaling induces autophagy through ERK1/2 and AMPK. (A) Immunoblotting of phosphorylated and total mTOR in IPEC-1 cells infected with ETEC or treated with rapamycin. (B) Immunoblotting of phosphorylated and total ERK1/2 LC3-II in IPEC-1 cells infected with ETEC. (C) IPEC-1 cells were treated with U0126 and then analyzed for LC3-I, LC3-II, ERK1/2, and p-ERK1/2 expression after ETEC infection. (D) Immunoblotting of phosphorylated and total AMPK after ETEC infection. (E) IPEC-1 cells were treated with compound C and then analyzed for LC3-I, LC3-II, AMPK and p-AMPK expression after ETEC infection.
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were performed in triplicate. P-values < 0.05 were considered statistically significant. 3. Results and discussion 3.1. ETEC infection increases autophagosome formation Porcine intestinal epithelial cells bind numerous ETEC strains to varying degrees. ETEC SEC470 adhered to IPEC-1 cells at a concentration of 105 bacterial/ml after 1 h infection. ETEC SEC470 is non-invasive, with intracellular bacteria observed in only 0.2% of infected cells. Transfecting EGFP-LC3 for fluorescence analysis is widely used to detect autophagosomes (Zhu et al., 2012). To investigate whether ETEC infection induces autophagosome formation, IPEC/EGFP-LC3 cells were infected with ETEC for 3 h. Confocal microscopy analysis showed that both ETEC and the autophagy inducer rapamycin induced a substantial increase in the number of cells containing EGFP-LC3 punctae, as compared with uninfected control cells (Fig. 1A). The number of punctae per cell was counted for quantitative assessment of autophagic activity in transfected IPEC-1 cells. Cells treated with ETEC or rapamycin showed a 4.9-fold or 5.7-fold increase, respectively, in the number of EGFP-LC3 punctae per cell as compared with control cells (Fig. 1B). During autophagy, cytosolic LC3-I is linked to phosphatidylethanolamine (PE) to form LC3-II and remain tightly bound to the autophagosomal membranes (Mizushima et al., 2010). To determine whether the observed autophagosomes-like vesicles are indeed related to autophagy, the LC3-II protein, another hallmark of autophagy, was examined by immunoblotting. ETECinfected cells had a significantly increased ratio of LC3II/b-actin as compared with uninfected cells at 2 and 3 h post-infection (Fig. 1C). Thus, it appears that ETEC infection induces autophagy in IPEC-1 cells.
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Towler and Hardie, 2007), and is a positive regulator of autophagy (Egan et al., 2011). ETEC infection significantly increased the amount of phosphorylated AMPK without affecting total AMPK abundance (Fig. 2D). Treating IPEC-1 cells with the AMPK inhibitor Compound C reduced LC3-II abundance (Fig. 2E). 3.3. Inhibiting autophagy increases ETEC-induced cell death To determine whether inhibiting autophagy would affect IPEC-1 cell viability during ETEC infection, we treated cells with 3-MA, a phostphatidylinositol-3-kinase (PI3K) inhibitor that is widely used for selective suppression of autophagy by blocking the formation of autophagomes. IPEC-1 cells were treated with 3-MA for 2 h to inhibit autophagy and then infected with ETEC for 1–3 h. Cells pre-treated with 3-MA displayed reduced viability during ETEC infection as determined using MTT assays (Fig. 3A). IPEC-1 cytotoxicity was also assayed by measuring lactate dehydrogenase (LDH) release. Cells treated with 3-MA released greater amounts of LDH during ETEC infection, as compared with infected cells not treated with 3-MA (Fig. 3B). ETEC are an important cause of diarrhea in man and animals. In neonatal and weaned piglets, ETEC-associated diarrhea is considered to be one of the most important diseases in swine husbandry. Here, we provide evidence that ETEC induces autophagosome formation in IPEC-1 cells as shown by the punctate GFP-LC3 distribution in the cytoplasm and the corresponding increase in the punctae counts. LC3-II protein abundance was also significantly
3.2. ETEC inhibits mTOR phosphorylation and ERK1/2 and AMPK contribute to autophagy during ETEC infection To understand further the underlying mechanism of ETEC-induced autophagy, we next investigated whether mTOR, a signaling protein that regulates autophagy was inhibited by ETEC. mTOR phosphorylation was significantly inhibited by both ETEC infection and by rapamycin treatment (Fig. 2A). There was no change in total mTOR abundance (Fig. 2A). The ERK1/2 pathway regulates host responses to ETEC infection (Wang et al., 2012) and plays a key role in regulating autophagy. We investigated whether ETEC induces autophagy by activating ERK1/2 signaling in IPEC-1 cells. ETEC infection increased ERK1/2 phosphorylation after 1 h (Fig. 2B), without affecting total ERK1/2 abundance. Inhibiting ERK1/2 phosphorylation with UO126 suppressed the conversion of LC3-I to LC3-II (Fig. 2C), suggesting ETEC induces autophagy in IPEC-1 cells through ERK1/2 signaling. AMPK plays a key role in the regulation of energy homeostasis, is activated by an elevated AMP/ATP ratio due to infection and heat shock (Arsenault et al., 2013;
Fig. 3. Inhibiting autophagy increases cell death in ETEC-infected IPEC-1 cells. (A) Cell viability was determined using MTT assay after ETEC infection in the presence or absence of 3-MA. (B) IPEC-1 cell cytotoxicity was analyzed by quantifying LDH release after ETEC infection in the presence or absence of 3-MA.
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higher in ETEC-infected cells as compared with uninfected cells. Autophagy may not only function as a defensive mechanism against invading pathogens but also may serve as a protective cellular response against toxins (Tan et al., 2009) and noninvasive pathogens. The cellular autophagic machinery appeared beneficial to IPEC-1 cells, as cells inhibited for autophagy showed enhanced cell death during ETEC infection. Overall, our data suggest that ETEC induces autophagy in IPEC-1 cells by inhibiting mTOR through the AMPK and ERK1/2 signaling pathways. Acknowledgements This research was jointly supported by National Basic Research Projects (2012CB124704 and 2009CB118800) and Comprehensive Strategic Cooperation projects from the Chinese Academy of Sciences and Guangdong Province (2012B091100210). References Arsenault, R.J., Napper, S., Kogut, M.H., 2013. Salmonella enterica Typhimurium infection causes metabolic changes in chicken muscle involving AMPK, fatty acid and insulin/mTOR signaling. Vet. Res. 44, 35. Cemma, M., Brumell, J.H., 2012. Interactions of pathogenic bacteria with autophagy systems. Curr. Biol. 22, R540–R545. Chen, X., Huan, H., Wan, T., Wang, L., Gao, S., Jiao, X., 2014. Antigenic determinants analysis and detection of virulence factors in F18 fimbriae Escherichia coli strains isolated from pigs. Acta Microbiol. Sin. 54, 236–242. Devriendt, B., Stuyven, E., Verdonck, F., Goddeeris, B.M., Cox, E., 2010. Enterotoxigenic Escherichia coli (K88) induce proinflammatory
responses in porcine intestinal epithelial cells. Dev. Comp. Immunol. 34, 1175–1182. Egan, D.F., Shackelford, D.B., Mihaylova, M.M., Gelino, S., Kohnz, R.A., Mair, W., Vasquez, D.S., Joshi, A., Gwinn, D.M., Taylor, R., Asara, J.M., Fitzpatrick, J., Dillin, A., Viollet, B., Kundu, M., Hansen, M., Shaw, R.J., 2011. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy. Science 331, 456– 461. Johnson, A.M., Kaushik, R.S., Francis, D.H., Fleckenstein, J.M., Hardwidge, P.R., 2009. Heat-labile enterotoxin promotes Escherichia coli adherence to intestinal epithelial cells. J. Bacteriol. 191, 178–186. Kim, M., Ashida, H., Ogawa, M., Yoshikawa, Y., Mimuro, H., Sasakawa, C., 2010. Bacterial interactions with the host epithelium. Cell Host Microbe 8, 20–35. Knodler, L.A., Celli, J., 2011. Eating the strangers within: host control of intracellular bacteria via xenophagy. Cell. Microbiol. 13, 1319–1327. Mizushima, N., Yoshimori, T., Levine, B., 2010. Methods in mammalian autophagy research. Cell 140, 313–326. Nishida, K., Kyoi, S., Yamaguchi, O., Sadoshima, J., Otsu, K., 2009. The role of autophagy in the heart. Cell Death Differ. 16, 31–38. Saiki, S., Sasazawa, Y., Imamichi, Y., Kawajiri, S., Fujimaki, T., Tanida, I., Kobayashi, H., Sato, F., Sato, S., Ishikawa, K., Imoto, M., Hattori, N., 2011. Caffeine induces apoptosis by enhancement of autophagy via PI3K/Akt/mTOR/p70S6K inhibition. Autophagy 7, 176–187. Tan, Y.K., Kusuma, C.M., St John, L.J., Vu, H.A., Alibek, K., Wu, A., 2009. Induction of autophagy by anthrax lethal toxin. Biochem. Biophys. Res. Commun. 379, 293–297. Towler, M.C., Hardie, D.G., 2007. AMP-activated protein kinase in metabolic control and insulin signaling. Circ. Res. 100, 328–341. Turner, S.M., Scott-Tucker, A., Cooper, L.M., Henderson, I.R., 2006. Weapons of mass destruction: virulence factors of the global killer enterotoxigenic Escherichia coli. FEMS Microbiol. Lett. 263, 10–20. Wang, X., Gao, X., Hardwidge, P.R., 2012. Heat-labile enterotoxin-induced activation of NF-kappaB and MAPK pathways in intestinal epithelial cells impacts enterotoxigenic Escherichia coli (ETEC) adherence. Cell. Microbiol. 14, 1231–1241. Zhu, B., Xu, F., Li, J., Shuai, J., Li, X., Fang, W., 2012. Porcine circovirus type 2 explores the autophagic machinery for replication in PK-15 cells. Virus Res. 163, 476–485.
