Developmental and Comparative Immunology 45 (2014) 214–218

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Intestinal autophagy activity is essential for host defense against Salmonella typhimurium infection in Caenorhabditis elegans Alexander Curt, Jiuli Zhang, Justin Minnerly, Kailiang Jia ⇑ Department of Biological Sciences, Florida Atlantic University, Jupiter, FL, USA

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Article history: Received 8 December 2013 Revised 17 March 2014 Accepted 17 March 2014 Available online 24 March 2014 Keywords: Autophagy bec-1 Salmonella C. elegans Pathogen-host interaction

a b s t r a c t Salmonella typhimurium infects both intestinal epithelial cells and macrophages. Autophagy is a lysosomal degradation pathway that is present in all eukaryotes. Autophagy has been reported to limit the Salmonella replication in Caenorhabditis elegans and in mammals. However, it is unknown whether intestinal autophagy activity plays a role in host defense against Salmonella infection in C. elegans. In this study, we inhibited the autophagy gene bec-1 in different C. elegans tissues and examined the survival of these animals following Salmonella infection. Here we show that inhibition of the bec-1 gene in the intestine but not in other tissues confers susceptibility to Salmonella infection, which is consistent with recent studies in mice showing that autophagy is involved in clearance of Salmonella in the intestinal epithelial cells. Therefore, the intestinal autophagy activity is essential for host defense against Salmonella infection from C. elegans to mice, perhaps also in humans. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Infectious diseases have been a major threat to humans throughout our history. Although great advances in medicine have been made and our understanding of these diseases have improved, infectious diseases are still the second leading cause of death worldwide (Fauci et al., 2005). In order to overcome these diseases, a better understanding of the mechanisms through which the pathogens act is needed. More than a decade ago the free-living soil nematode Caenorhabditis elegans was established as an invertebrate model organism for studying the host-pathogen interactions (Aballay and Ausubel, 2002; Kurz and Ewbank, 2003; Millet and Ewbank, 2004; Mylonakis and Aballay, 2005). The rapid life cycle and vast genetic resources available have made C. elegans a good model system for studying infections. In the laboratory, C. elegans is cultured at a standard temperature of 20 °C on agar plates with seeded bacterium Escherichia coli (strain OP50) as food (Brenner, 1974; Riddle et al., 1997). Under these conditions, the intestine is the primary site of the infection. C. elegans is susceptible to infections from a variety of pathogens (Couillault and Ewbank, 2002). Although initially believed to be only suitable for broad host range opportunistic pathogens, such as Pseudomonas aeruginosa, it has

⇑ Corresponding author. Address: Department of Biological Sciences, Florida Atlantic University, 5353 Parkside Drive, Jupiter, FL 33458, USA. Tel.: +1 561 799 8054; fax: +1 561 799 8061. E-mail address: [email protected] (K. Jia). http://dx.doi.org/10.1016/j.dci.2014.03.009 0145-305X/Ó 2014 Elsevier Ltd. All rights reserved.

been shown that highly specific pathogens like Salmonella typhimurium can infect and kill C. elegans as well (Aballay et al., 2003, 2000; Jia et al., 2009; Labrousse et al., 2000). S. typhimurium is a Gram-negative bacterium that causes foodborne illnesses in humans. It is able to establish an infection in the intestine of C. elegans that leads to early death for the infected worms (Aballay et al., 2003, 2000; Jia et al., 2009; Labrousse et al., 2000). C. elegans has conserved immune effectors such as antimicrobial peptides to defend against the Salmonella infection (Alegado and Tan, 2008; Millet and Ewbank, 2004). The C. elegans heat-shock transcription factor-1 (HSF-1) was reported to mediate the effects of the DAF-2 insulin-like signaling pathway in defense to Salmonella infection (Singh and Aballay, 2006). Overexpression of DAF-16 FOXO transcription factor, the major target of DAF-2 pathway, also confers resistance to Salmonella infection in C. elegans (Jia et al., 2009). By contrast, the intestinal GATA-type transcription factor ELT-2 is required independently of the DAF-2 signaling pathway for the expression of a variety of infection-response genes following Salmonella infection of C. elegans (Kerry et al., 2006). It was recently reported that autophagy is required for C. elegans immunity against Salmonella infection and functions downstream of the DAF-2 pathway (Jia et al., 2009). Autophagy is an evolutionarily conserved lysosomal degradation pathway that is present in all eukaryotes (Levine and Klionsky, 2004). It allows the cell to break down cytoplasmic proteins and organelles in order to maintain homeostasis (Levine and Klionsky, 2004) and plays a role in the innate immune response (Deretic,

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2005; Levine and Deretic, 2007). Autophagy has been shown to engulf and destroy invading Salmonella in several model systems including C. elegans, mammalian cells and mice (Benjamin et al., 2013; Birmingham et al., 2006; Conway et al., 2013; Jia et al., 2009). To date, most intestinal bacterial pathogens of C. elegans remain extracellular except S. typhimurium that have been shown to establish intracellular infection in the intestinal cells after autophagy is inhibited (Jia et al., 2009). However, it is unknown whether it is the intestinal autophagy activity or the cell non-autonomous function of autophagy that protects C. elegans against Salmonella infection. To answer this question, we examined the tissue-specific requirement of autophagy in host defense against Salmonella infection. We found that the intestinal-expressed autophagy gene bec-1 is essential for C. elegans to fight against Salmonella infection, which is consistent with a recent discovery in mice and that autophagy in the intestinal epithelial cells is required to protect from a Salmonella infection (Benjamin et al., 2013; Conway et al., 2013). Thus, these results indicate an evolutionarily conserved role of intestinal autophagy activity in protection against Salmonella infection from C. elegans to mice, which may be also true in humans. 2. Materials and methods

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night with shaking at 37 °C. Then 80 ll of Salmonella overnight culture was placed on each nematode growth medium (NGM) plate and incubated for 6 h at room temperature to dry the bacteria. The RNAi-treated L4 hermaphrodites and control animals were put on Salmonella bacteria to start the infection. Forty-eight hours later, the Salmonella-infected worms were transferred to fresh corresponding RNAi plates. The worms were transferred to fresh RNAi plates daily during the egg-lay period. After that, the worms were transferred every 3 days. The survival of worms was examined daily or every other day. When animals failed to respond to touch, they were scored as dead. Animals that died of internal-hatching or vulva rupture were excluded from the statistical analysis. All lifespan experiments were performed at least twice and similar results were obtained. 2.4. Statistical analyses of lifespan data The Kaplan–Meier method was used to estimate survival curves. The log-rank test was used to determine if there was a significant difference in survival between two groups. These analyses were performed with the GraphPad Prism 5 software program (GraphPad Software, Inc.). Every experiment was repeated at least once. The statistical information for two representative trials is shown in the Supplementary table (Table S1).

2.1. C. elegans strains The wild-type strain used in these studies was the C. elegans Bristol strain N2 (Brenner, 1974). The C. elegans mutant strains used were: WM27 rde-1(ne219)V (RNAi defective), NR222 rde1(ne219) V; kzIs9[pKK1260(lin-26p::nls::GFP) + pKK1253(lin-26p:: rde-1) + pRF6(rol-6(su1006)] (RNAi is only effective in the hypodermis), NR350 rde-1(ne219) V; kzIs20[pDM#715(hlh-1p::rde-1) + pTG95(sur-5p::nls::GFP)] (RNAi is only effective in body wall muscles), VP303 rde-1(ne219) V; kbIs7[nhx-2p::rde-1 + rol-6(su1006)] (RNAi is only effective in intestinal cells) (Durieux et al., 2011; Qadota et al., 2007) and HC75 ccIs4251I; sid-1(qt2) V (RNAi is only effective in intestinal cells) (Mouchiroud et al., 2011; Winston et al., 2007). Strains WM27 (herein referred to as rde-1), NR222 and NR350 were gifts from Dr. Hiroshi Qadota at Emory University. VP303 and HC75 (herein referred to as sid-1) were purchased from Caenorhabditis Genetics Center (CGC, University of Minnesota, Twin Cities). 2.2. C. elegans RNAi The bec-1 RNAi feeding plasmid used in this project has been previously shown to effectively knock down the expression of the bec-1 gene (Jia et al., 2009). The vector only RNAi plasmid was used as a control. The RNAi feeding was performed as described previously (Kamath et al., 2001). Briefly, larval stage four (L4) hermaphrodites were put on RNAi and control plates, and incubated at 20 °C for 36 h. Then the worms were transferred to fresh RNAi and control plates, respectively. After 24 h, the parental worms were removed and the progeny were allowed to develop. The L4 hermaphrodites of RNAi-treated progeny were collected for Salmonella infection experiments. 2.3. Salmonella infection and C. elegans lifespans at 20 °C The experiment was conducted as we reported previously (Jia et al., 2009). S. typhimurium ATCC14028s was used for the infection experiment. The Salmonella frozen stock was streaked on 5.5% XLD agar plates (xylose lysine desoxycholate, EMD Chemical Inc.) and the plate was incubated overnight at 37 °C. A single Salmonella colony was selected the next day to grow in 2 ml Luria Broth over-

3. Results and discussion To evaluate the tissue-requirement of autophagy in host defense against Salmonella infection in C. elegans, we used RNAi feeding to silence the C. elegans autophagy gene bec-1 which is the ortholog of yeast ATG6 and functions in autophagic vesicle nucleation (Levine and Klionsky, 2004). A bec-1::gfp reporter gene is expressed in multiple tissues including the hypodermis and intestine (Melendez et al., 2003). Autophagy genes have been found to be required for various longevity signals that regulate C. elegans lifespan (Jia and Levine, 2010). In the present study, we used 1 nM IPTG to induce RNAi to avoid the effect of autophagy deficiency on lifespan, which may mask the killing effect of Salmonella infection. This IPTG concentration has been shown to successfully induce bec-1 RNAi to inhibit autophagy and abrogate the pathogen resistance of daf-2 mutants (Jia et al., 2009). As shown in Fig. 1A, the bec-1 RNAi-treatment with 1 nM IPTG concentration has no influence on the lifespan of N2 wild-type animals. The median lifespans of bec-1 RNAi-treated N2 worms and control animals are 18 and 16 days, respectively (p = 0.4949, log-rank test) (Fig. 1A and Table S1). However, compared to the control animals the median lifespan of bec-1 RNAi-treated N2 worms is decreased by 27% after Salmonella infection (11 vs. 15 days) with the maximum lifespan shortened by 6 days (Fig. 1B and Table S1). Therefore, under this 1 nM IPTG experimental concentration, bec-1 RNAi treatment significantly increased the susceptibility of C. elegans to Salmonella infection consistent with previous studies (Jia et al., 2009). C. elegans rde-1 gene encodes a member of the PIWI/STING/Argonaute family of proteins that is an essential component of the RNAi machinery (Tabara et al., 1999). rde-1 mutant animals are resistant to RNAi (Tabara et al., 1999). As predicted bec-1 RNAi has no effect on the lifespan of rde-1 mutant animals non-infected or infected by Salmonella because they are defective for RNAi treatment (p = 0.5365 and p = 0.2245, log-rank tests, respectively) (Fig. 1C and D and Table S1). Both median and maximum lifespans of bec-1 RNAi-treated animals are very similar to that of control animals whether or not they are infected by Salmonella, which confirms the role of autophagy in fighting against Salmonella infection (Fig. 1C and D, Table S1).

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Fig. 1. Influence of bec-1 inhibition on the survival of C. elegans infected by Salmonella. Inhibition of the bec-1 gene confers susceptibility of C. elegans wild-type animals to Salmonella infection (A and B) but has no effect on the survival of RNAi-defective rde-1 mutants (C and D). rde-1 mutants with rde-1 rescued only in the hypodermis (for the strain NR222, RNAi is only effective in the hypodermis) are not more susceptible to Salmonella infection compared to vector only controls (E and F). The entire experiment was repeated at least once.

To ascertain the tissue requirement of bec-1 in host defense against Salmonella infection, we performed a tissue-specific RNAi feeding to knock down the expression of bec-1 gene in the hypodermis, body wall muscles and intestine, respectively (Durieux et al., 2011; Qadota et al., 2007). We used three different rde-1 mutants that had rde-1 rescued in specific tissues. First, we tested NR222 worms in which rde-1 is rescued in the hypodermis (leaving only the hypodermis sensitive to bec-1 RNAi treatment) (Qadota et al., 2007). bec-1 RNAi does not affect the lifespan of NR222 worms compared to the vector control (p = 0.7449, log-rank test). Both of these treatments resulted in a median lifespan of 13 days (Fig. 1E and Table S1). NR222 worms treated with bec-1 RNAi and then infected with Salmonella do not show a decrease in lifespan compared to the infected vector control worms (Fig. 1F and Table S1) (p = 0.3023, log-rank test). This indicates that autophagy in the hypodermis is not essential for protection against Salmonella infections. Next we examined the NR350 worms that are only sensitive to RNAi treatment in the body wall muscles (Qadota et al., 2007). Similar to the NR222 worms, the bec-1 RNAi-treated NR350 worms have no obvious change of lifespan compared to worms given the control vector RNAi (Fig. 2A and Table S1) (p = 0.0962, log-rank test). The bec-1 RNAi-treated and control worms have median lifespans of 16 and 15 days respectively, and maximum lifespans of 25 and 22 days respectively. We also found that there was no significant difference in lifespan between the bec-1 RNAi-treated and control-RNAi treated NR350 worms infected by Salmonella (Fig. 2B and Table S1) (p = 0.6510, log-rank test). From these results

we conclude that autophagy in the body wall muscle does not play an important role in providing resistance to Salmonella infections. To determine the role of intestinal autophagy in the innate immune response to Salmonella infection we used the VP303 strain, an rde-1 mutant with the rde-1 gene rescued only in the intestine (Durieux et al., 2011). As with the previous experiments, treatment with either bec-1 or vector RNAi resulted in no significant change in lifespan (Fig. 2C and Table S1) (p = 0.0665, log-rank test). Both bec-1 RNAi-treated worms and control animals have the same median lifespan of 12 days. Interestingly, when these RNAi-treated worms were infected with Salmonella there was a significant decrease in lifespan for the bec-1 RNAi-treated worms compared to the vector control (Fig. 2D and Table S1) (p = 0.0016, log-rank test). The bec-1 RNAi-treated worms infected by Salmonella had median and maximum lifespans of 9 and 14 days respectively, while the vector treated worms had a 10-day median lifespan and a 16-day maximum lifespan. This data indicates that autophagy in the intestine is needed for countering Salmonella infection. To confirm the results with the VP303 strain, we used sid-1 mutants to repeat this experiment. sid-1 mutants have been shown to allow gene inactivation by feeding intestinal-specific RNA because their dsRNA systemic spreading is inhibited (Mouchiroud et al., 2011; Winston et al., 2007). Similar to the VP303 strain, bec-1 RNAi treatment does not influence the lifespan of sid-1 mutants (p = 0.6702, log-rank test) (Fig. 2E and Table S1). However, bec-1 RNAi-treated sid-1 mutants die much faster than control worms after Salmonella infection. The median lifespan is decreased by 2 days, and the maximum lifespan is decreased by 8 days from

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F sid-1 + vector + Salmonella sid-1 + bec-1 RNAi + Salmonella

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Fig. 2. Intestinal bec-1 activity is required for host defense against Salmonella infection. Inhibition of bec-1 gene in the body wall muscles fails to influence the sensitivity of C. elegans to Salmonella infection (RNAi is only effective in body wall muscles in NR350 animals) (A and B). Intestinal bec-1 expression is required for defending against Salmonella infection. In both VP303 (C and D) and sid-1 (E and F) animals, RNAi is only effective in intestinal cells. The entire experiment was performed twice and similar results were observed.

36 to 28 days (Fig. 2F and Table S1). Taken together, these data indicate that autophagy acts cell autonomously in the intestine to defend against Salmonella infection in C. elegans, which is consistent with recent studies showing that autophagy proteins Atg16L1 and Atg5 are required for clearance of Salmonella bacteria in mouse intestinal epithelial cells (Benjamin et al., 2013; Conway et al., 2013). In C. elegans, almost all characterized intestinal bacterial pathogens remain extracellular except S. typhimurium that have been shown to establish intracellular infection in the intestinal cells (Jia et al., 2009). We used red fluorescent protein (RFP)-labeled Salmonella to examine whether intestinal inhibition of autophagy gene bec-1 would increase the intestinal replication of Salmonella and RFP-labeled Salmonella were not observed inside intestinal epithelial cells (data not shown). At present we cannot explain the different results obtained by using two different methods. Regardless, our genetic studies indicate an essential role of intestinal autophagy activity in host defense against Salmonella infection although the exact mechanism by which autophagy works warrants further studies. It has been published that mice defective in intestinal expression of the atg genes, Atg16L1 or Atg5, were found to have defects in the secretory function of the Paneth cell that is a key cell type involved in secretion of gut antimicrobial peptides (Deretic et al., 2008). Therefore, it is likely autophagy deficiency may decrease the secretion of antimicrobial peptides into the intestinal lumen, which in turn increased Salmonella accumulation in the intestinal lumen and caused the early death of infected worms. Recently, it was demonstrated that the C. elegans p38 MAP kinase (PMK-1) also acts in the intestinal epithelium to regulate

the immune response to a wide variety of bacterial pathogens (Shivers et al., 2009) and the pmk-1 loss-of-function mutant is hypersensitive to infections with a spectrum of pathogens including Salmonella (Aballay et al., 2003; Pukkila-Worley and Ausubel, 2012). It would be interesting to investigate whether PMK-1 regulates autophagy in C. elegans intestinal epithelial cells to remove Salmonella. We have previously shown that overexpression of the DAF-16 FOXO transcription factor induces autophagy and confers resistance to Salmonella infection dependent on the functional autophagy (Jia et al., 2009). Interestingly, intestinal expression of DAF-16 is required for resistance to P. aeruginosa (Evans et al., 2008). Although the role of autophagy in defending against P. aeruginosa in C. elegans has not been examined, it is possible that intestinal DAF-16 may also regulate autophagy in host response to P. aeruginosa infection. C. elegans has been used as a model organism to examine evolutionarily conserved components of innate immunity. In nature, C. elegans eats bacteria and fungi as food. Thus, the intestine is the primary site through which microorganisms infect C. elegans. It is anticipated that intestinal epithelial cells are important for C. elegans to defend against microorganism infections, as C. elegans does not have an adaptive immune system (Pukkila-Worley and Ausubel, 2012). Our data presented here show an evolutionarily conserved role of autophagy in the intestinal epithelial cells in defense against Salmonella infection in C. elegans. Autophagy is a cellular process that is pharmacologically regulated. C. elegans has been used for automated high-throughput screen of compounds and natural product extracts to look for new antimicrobials against E. faecalis (Moy et al., 2009). As C. elegans mainly takes up these

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compounds through the intestine, our data suggest that the C. elegans can be used in a compound screen to identify new chemicals regulating intestinal autophagy activities in defending against Salmonella infections, which have considerable therapeutic potential in the treatment of infections with Salmonella and other pathogens. Competing interests The authors have declared that no competing interests exist. Acknowledgements We thank Dr. Hiroshi Qadota (Emory University) for providing strains rde-1, NR222 and NR350, and Dr. Diane Baronas-Lowell for critical reading of the manuscript. An FAU Charles E. Schmidt College of Science seed grant and an Ellison Medical Foundation New Aging Scholarship to K. Jia supported this work. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.dci.2014.03.009. References Aballay, A., Ausubel, F.M., 2002. Caenorhabditis elegans as a host for the study of host-pathogen interactions. Curr. Opin. Microbiol. 5, 97–101. Aballay, A., Drenkard, E., Hilbun, L.R., Ausubel, F.M., 2003. Caenorhabditis elegans innate immune response triggered by Salmonella enterica requires intact LPS and is mediated by a MAPK signaling pathway. Curr. Biol. 13, 47–52. Aballay, A., Yorgey, P., Ausubel, F.M., 2000. Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Curr. Biol. 10, 1539–1542. Alegado, R.A., Tan, M.W., 2008. Resistance to antimicrobial peptides contributes to persistence of Salmonella typhimurium in the C. elegans intestine. Cell. Microbiol. 10, 1259–1273. Benjamin, J.L., Sumpter Jr., R., Levine, B., Hooper, L.V., 2013. Intestinal epithelial autophagy is essential for host defense against invasive bacteria. Cell Host Microbe 13, 723–734. Birmingham, C.L., Smith, A.C., Bakowski, M.A., Yoshimori, T., Brumell, J.H., 2006. Autophagy controls Salmonella infection in response to damage to the Salmonella-containing vacuole. J. Biol. Chem. 281, 11374–11383. Brenner, S., 1974. The Genetics of Caenorhabditis elegans. Genetics 77, 71–94. Conway, K.L., Kuballa, P., Song, J.H., Patel, K.K., Castoreno, A.B., Yilmaz, O.H., Jijon, H.B., Zhang, M., Aldrich, L.N., Villablanca, E.J., Peloquin, J.M., Goel, G., Lee, I.A., Mizoguchi, E., Shi, H.N., Bhan, A.K., Shaw, S.Y., Schreiber, S.L., Virgin, H.W., Shamji, A.F., Stappenbeck, T.S., Reinecker, H.C., Xavier, R.J., 2013. Atg16l1 is required for autophagy in intestinal epithelial cells and protection of mice from Salmonella infection. Gastroenterology 145, 1347–1357. Couillault, C., Ewbank, J.J., 2002. Diverse bacteria are pathogens of Caenorhabditis elegans. Infect. Immun. 70, 4705–4707. Deretic, V., 2005. Autophagy in innate and adaptive immunity. Trends Immunol. 26, 523–528. Deretic, V., Master, S., Singh, S., 2008. Autophagy gives a nod and a wink to the inflammasome and Paneth cells in Crohn’s disease. Dev. Cell 15, 641–642.

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