Title: An essential complementary role of NF-κB pathway to microbicidal oxidants in Drosophila gut immunity
Abstract: Article20 July 2006free access An essential complementary role of NF-κB pathway to microbicidal oxidants in Drosophila gut immunity Ji-Hwan Ryu Ji-Hwan Ryu Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Eun-Mi Ha Eun-Mi Ha Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Chun-Taek Oh Chun-Taek Oh Laboratory of Innate Immunology, Institut Pasteur Korea, Seoul, South Korea Search for more papers by this author Jae-Hong Seol Jae-Hong Seol School of Biological Science, Seoul National University, Seoul, South Korea Search for more papers by this author Paul T Brey Paul T Brey Unité de Biochimie et Biologie Moléculaire, Institut Pasteur, Paris, France Search for more papers by this author Ingnyol Jin Ingnyol Jin Department of Microbiology, Kyungpook National University, Daegu, South Korea Search for more papers by this author Dong Gun Lee Dong Gun Lee Department of Microbiology, Kyungpook National University, Daegu, South Korea Search for more papers by this author Jaesang Kim Jaesang Kim Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Daekee Lee Daekee Lee Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Won-Jae Lee Corresponding Author Won-Jae Lee Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Ji-Hwan Ryu Ji-Hwan Ryu Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Eun-Mi Ha Eun-Mi Ha Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Chun-Taek Oh Chun-Taek Oh Laboratory of Innate Immunology, Institut Pasteur Korea, Seoul, South Korea Search for more papers by this author Jae-Hong Seol Jae-Hong Seol School of Biological Science, Seoul National University, Seoul, South Korea Search for more papers by this author Paul T Brey Paul T Brey Unité de Biochimie et Biologie Moléculaire, Institut Pasteur, Paris, France Search for more papers by this author Ingnyol Jin Ingnyol Jin Department of Microbiology, Kyungpook National University, Daegu, South Korea Search for more papers by this author Dong Gun Lee Dong Gun Lee Department of Microbiology, Kyungpook National University, Daegu, South Korea Search for more papers by this author Jaesang Kim Jaesang Kim Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Daekee Lee Daekee Lee Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Won-Jae Lee Corresponding Author Won-Jae Lee Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea Search for more papers by this author Author Information Ji-Hwan Ryu1, Eun-Mi Ha1, Chun-Taek Oh2, Jae-Hong Seol3, Paul T Brey4, Ingnyol Jin5, Dong Gun Lee5, Jaesang Kim1, Daekee Lee1 and Won-Jae Lee 1 1Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seoul, South Korea 2Laboratory of Innate Immunology, Institut Pasteur Korea, Seoul, South Korea 3School of Biological Science, Seoul National University, Seoul, South Korea 4Unité de Biochimie et Biologie Moléculaire, Institut Pasteur, Paris, France 5Department of Microbiology, Kyungpook National University, Daegu, South Korea *Corresponding author. Division of Molecular Life Science and National Creative Research Initiative Center for Symbiosystem, Ewha Woman's University, Seodaemun-Gu Daehyun-Dong 11-1, Seoul 120-750, South Korea. Tel.: +82 2 3277 3349; Fax: +82 2 3277 3760; E-mail: [email protected] The EMBO Journal (2006)25:3693-3701https://doi.org/10.1038/sj.emboj.7601233 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In the Drosophila gut, reactive oxygen species (ROS)-dependent immunity is critical to host survival. This is in contrast to the NF-κB pathway whose physiological function in the microbe-laden epithelia has yet to be convincingly demonstrated despite playing a critical role during systemic infections. We used a novel in vivo approach to reveal the physiological role of gut NF-κB/antimicrobial peptide (AMP) system, which has been 'masked' in the presence of the dominant intestinal ROS-dependent immunity. When fed with ROS-resistant microbes, NF-κB pathway mutant flies, but not wild-type flies, become highly susceptible to gut infection. This high lethality can be significantly reduced by either re-introducing Relish expression to Relish mutants or by constitutively expressing a single AMP to the NF-κB pathway mutants in the intestine. These results imply that the local 'NF-κB/AMP' system acts as an essential 'fail-safe' system, complementary to the ROS-dependent gut immunity, during gut infection with ROS-resistant pathogens. This system provides the Drosophila gut immunity the versatility necessary to manage sporadic invasion of virulent pathogens that somehow counteract or evade the ROS-dependent immunity. Introduction Gastrointestinal epithelia face an exceptional challenge among various organ tissues in that they are in constant contact with a countless number of microbes (Macpherson and Harris, 2004; Sansonetti, 2004; Macdonald and Monteleone, 2005). Therefore, this microbial-laden mucosal tissue must be armed with an efficient innate microbial control system. (Ganz, 2003; Bevins, 2004; Lehrer, 2004). In Drosophila gut, intestinal redox homeostasis, via the infection-induced de novo generation of oxygen-dependent innate immune effectors such as reactive oxygen species (ROS) by dual oxidase (Duox) and their elimination by immune-regulated catalase, is finely regulated to mediate pathogen–host interaction (Ha et al, 2005a, 2005b). The function of this immune system is critical in the host survival during natural gut infections resulting for example from the ingestion of microbe-contaminated foods (Ha et al, 2005a, 2005b). Natural gut infections can also trigger the immune deficiency (IMD)/NF-κB pathway in the intestine, which results in the de novo synthesis of innate immune effectors including antimicrobial peptides (AMPs) via the activation of p105-like NF-κB, Relish (Ferrandon et al, 1998; Tzou et al, 2000; Onfelt Tingvall et al, 2001). Despite the central role of the NF-κB/AMP pathway in host survival during the systemic immune response, which follows microbial infection in the hemocoel (Silverman and Maniatis, 2001; Boutros et al, 2002; Hoffmann and Reichhart, 2002; Hultmark, 2003; Brennan and Anderson, 2004; Lemaitre, 2004), its exact physiological function in intestinal innate immunity has not yet been convincingly demonstrated at the organism level. This is probably attributed to the fact that other effective defense systems such as ROS-dependent innate immunity are also operating in the gut and effectively controlling the majority of infections. Thus, at least under infectious conditions with a fairly wide spectrum of microbes, the epithelial NF-κB/AMP pathway appears to be less essential for host survival, as all known NF-κB mutant flies are totally resistant to natural gut infection (Ha et al, 2005a, 2005b). Nevertheless, given that AMPs have been demonstrated in vitro to be capable of killing a wide variety of microbes (Hertu et al, 1998), we hypothesized that epithelial AMPs operating via the NF-κB pathway may constitute an essential antimicrobial defense within the gastrointestinal tract. We further hypothesized that this defense system may possibly exhibit a complementary and/or synergistic action in combination with the other efficient immune effectors, ROS, in Drosophila gut immunity. Clear in vivo data supporting or undermining this hypothesis is lacking at present, perhaps mainly owing to the absence of a suitable experimental model. In the present study, we show that intestinal NF-κB/AMP-dependent innate immunity becomes crucial to host survival when the host encounters pathogenic microbes that somehow escape ROS-dependent innate immunity. These results imply that the epithelia of Drosophila developed two evolutionally distinct innate immune effectors, ROS and AMPs. Such 'dual-effector' system in the Drosophila gastrointestinal epithelium makes it difficult for pathogens to completely resist or circumvent the host immunity thus insuring host survival. Results IMD/NF-κB pathway is required for host protection against gut infection by ROS-resistant microbes, but not by normal ROS-sensitive microbes Recently, we have demonstrated that the ROS-dependent immune system, rather than the NF-κB-dependent innate immune system, is crucial to the survival of the host during the majority of host–microbe interactions in the gastrointestinal tract of Drosophila (Ha et al, 2005a, 2005b). These observations also imply that, during continuous gut–microbe interactions, one of the principal tactics of microbes may involve the evasion of or resistance to the host's ROS system, thereby securing a foothold for proliferation within the host. To investigate whether ROS resistance is the major virulent mechanism of microbes, it would be ideal to establish the natural infection conditions with a microbe that exhibits a marked resistance to ROS. However, at present, no orally transmitted and ROS-resistant natural pathogens for Drosophila are known. Therefore, we used the KNU5377 yeast strain, isolated from a natural environment and highly resistant to various types of exogenous stresses (Kwak et al, 2003). In the ROS resistance test using various concentrations of hydrogen peroxide, the KNU5377 showed a much higher survival rate when compared to a standard yeast strain (W303) (Figure 1A). We then performed gut infection using a standard yeast strain (W303) and a ROS-resistant strain (KNU5377). Contrary to our expectation, the flies were totally resistant to ROS-resistant KNU5377 infection, and no significant difference in host mortality was observed between ROS-sensitive W303 infection and ROS-resistant KNU5377 infection (Figure 1B). This result suggests that another form of gut immune system may be also operating as a complementary system to ROS-dependent immunity for the efficient control of ROS-resistant microbes. Figure 1.IMD/NF-κB-dependent innate immunity is indispensable for host protection from ROS-resistant pathogen. (A) KNU5377 yeast is a ROS-resistant yeast strain. The standard yeast strain (W303) and the KNU5377 strain were exposed to 10 mM H2O2 for different times (0, 30, 60, 90 and 120 min). The aliquots were spotted on YPD agar plates, and were incubated at 30°C in order to determine their survival rates. (B) Wild-type flies are equally resistant to both normal and ROS-resistant yeasts. The adult male flies were subjected to natural infection with W303 or KNU5377. (C) IMD/NF-κB pathway mutant flies are susceptible to KNU5377 but not to W303. The IMD/NF-κB pathway mutant flies (DreddB118, key1 and RelishE20) and the Toll/NF-κB pathway mutant flies (spzrm7 and Dif1) were subjected to natural infection with W303 or KNU5377. (D) Flies exhibiting impaired regulation of the Toll pathway showed wild-type level resistance against KNU5377. Loss-of-function flies for Toll pathway (J4 and Pelle-RNAi/+; Da-GAL4/+) or gain-of-function flies for Toll pathway (cactA2) were subjected to natural infection with W303 or KNU5377. The Pelle-RNAi/+; Da-GAL4/+ flies used in this study showed severely reduced level of infection-induced Drosomycin gene expression following systemic infection (data not shown). The flies exhibiting impaired potential for both Toll and IMD pathways (Dredd; Pelle-RNAi/+; Da-GAL4/+) were also used in this experiment. These flies showed similar immune susceptibility to that of flies carrying IMD pathway mutation alone. In all cases, survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05). Download figure Download PowerPoint As natural infection is also known to activate local intestinal NF-κB pathway, we hypothesized that Drosophila relies on the intestinal NF-κB-dependent innate immunity as the second line of defense for the efficient host protection against ROS-resistant microbes. If this were the case, ROS-resistant microbial strains should prove to be more pathogenic to NF-κB pathway mutant flies than to normal flies as host survival would be largely dependent on the intestinal NF-κB pathway-dependent innate immunity. In an attempt to assess this hypothesis, we fed various IMD/NF-κB pathway mutant flies (p105-like NF-κB mutant (RelishE20), caspase mutant (DreddB118) and Drosophila IκB kinase γ mutant (key1)) on either the KNU5377 strain or the W303 strain. Consistent with our hypothesis, high mortality levels were observed in these NF-κB pathway mutant flies only when they fed on KNU5377 strain (Figure 1C). No significant mortality was observed in the NF-κB pathway mutant flies fed on the W303 strain (Figure 1C). Interestingly, enhanced levels of KNU5377-induced mortality were observed only in the IMD/NF-κB pathway mutant flies (DreddB118, key1 and RelishE20) but not in the Toll/NF-κB pathway mutant flies (spzrm7 and Dif1) (Figure 1C). To rule out partially redundant function of three NF-κB molecules (Dif and Dorsal for Toll pathway and Relish for IMD pathway) in the gut immunity, we also checked the KNU5377-induced mortality using the flies carrying Dif and Dorsal double mutation (J4), mutant flies exhibiting constitutive activation of Dif and Dorsal (cactA2) and the knockdown flies for Toll pathway generated by introducing Pelle-RNAi using ubiquitously expressing Daughterless (Da)-GAL4 driver (Pelle-RNAi/+; Da-GAL4/+). In all cases, the impaired regulation of the Toll pathway (either gain-of-function or loss-of-function) showed wild-type resistance (Figure 1D). Furthermore, the flies exhibiting reduced potential for both Dif/Dorsal-mediated Toll and Relish-mediated IMD pathway (DreddB118; Pelle-RNAi/+; Da-GAL4/+) showed similar immune susceptibility to that of flies carrying IMD pathway mutation alone (DreddB118, key1 or RelishE20) (Figure 1C and D). This result is consistent with that NF-κB activity in the epithelia is controlled primarily via the IMD/NF-κB pathway but not via the Toll/NF-κB pathway (Ferrandon et al, 1998; Tzou et al, 2000; Onfelt Tingvall et al, 2001; Ha et al, 2005b). To exclude possible crosstalk between NF-κB activation and ROS production in the gut, we tested whether the ROS production or ROS-generating Duox enzyme expression is affected in the gain-of-function or loss-of-function mutant flies of NF-κB pathways. The result showed that infection-induced ROS production and Duox induction were not significantly affected in any of the tested NF-κB pathway mutant flies (Supplementary Figure 1). Conversely, Duox-RNAi flies exhibiting reduced infection-induced ROS production showed normal NF-κB target gene activation (Supplementary Figure 2). These results strongly suggest that NF-κB-dependent immunity and ROS-dependent immunity function independently as two separate defense systems but they play complementary roles in gut immunity. Furthermore, our results demonstrate that IMD/NF-κB pathway is essential for host protection against gut infection with ROS-resistant microbes, but not with normal ROS-sensitive microbes. The gut IMD/NF-κB pathway, but not the systemic IMD/NF-κB pathway, is required for host protection from the gut infection with ROS-resistant microbes In order to corroborate that the observed increase in KNU5377-induced mortality in the IMD/NF-κB pathway mutant flies was due to a lack of intestinal NF-κB pathway potential, we examined the effect of tissue-specific re-establishment of Relish expression on the survival of RelishE20 using two different tissue-specific GAL4 drivers. We used the caudal (cad)-GAL4 driver for the re-introduction of Relish expression in the intestine because cad expression is effectively restricted to the posterior midgut and proventriculus (Mlodzik and Gehring, 1987). Cad is also expressed in the salivary glands and ejaculatory duct (Ryu et al, 2004), but not in the fat body as demonstrated by green fluorescence protein (GFP) expression pattern in cad-GAL4/UAS-EGFP flies (data not shown). To introduce Relish expression in the fat body/hemocytes (the main immune tissue of systemic immunity), we used the c564-GAL4 driver. The c564-GAL4 strain did not express GAL4 in the intestine as determined by GFP expression patterns in the flies carrying c564-GAL4/UAS-EGFP (data not shown). Importantly, the re-introduction of Relish expression primarily in the intestines of RelishE20 mutant flies (flies carrying UAS-Relish/cad-GAL4; RelishE20), but not in the fat body/hemocytes of RelishE20 mutant flies (flies carrying UAS-Relish/c564-GAL4; RelishE20), resulted in a dramatic upswing in the survival rates after the ingestion of ROS-resistant KNU5377 strain (Figure 2A). In a control experiment, the re-introduction of Relish in the RelishE20 by c564-GAL4, but not by cad-GAL4, efficiently protected host in the case of systemic infections (Figure 2B). This result showed that the IMD/NF-κB pathway is required in a tissue-specific manner depending on the route of infection and that the survival of the flies during KNU5377 invasion is dependent specifically on the intestinal IMD/NF-κB pathway. Figure 2.The susceptibility of RelishE20 flies to natural KNU5377 infection can be ameliorated via the re-introduction of Relish in the intestine but not in the fat body. For the rescue experiment, the RelishE20 flies were crossed with flies carrying UAS-Relish. The cad-GAL4 and c564-GAL4 drivers were used for intestine-specific and fat body/hemocyte-specific Relish expression, respectively. The genotypes of the flies used in this study were as follows: control (cad-GAL4/+); RelishE20 (cad-GAL4/+; RelishE20); RelishE20+Relish (intestine) (cad-GAL4/UAS-Relish; RelishE20); RelishE20+Relish (fat body/hemocytes) (c564-GAL4/UAS-Relish; RelishE20). Natural gut infection (A) and septic infection (B) were performed with KNU5377 and Erwinia carotovora carotovora 15 (Ecc15), respectively. In all cases, survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05). Download figure Download PowerPoint ROS-removing activity can act as a virulence factor to the host lacking IMD/NF-κB pathway potential The fact that KNU5377 is not a modified food-type yeast but instead an environmental isolate resistant to various stresses raises doubts as to whether the pathogenicity of this microbe is solely or mainly attributable to its ROS resistance. To further confirm that microbe's capacity for ROS resistance such as ROS-removing activity can be a major virulence factor to the host lacking NF-κB pathway potential, we engineered normal bacteria to overexpress a single ROS-removing enzyme, which would confer a higher potential pathogenicity due to increased ROS resistance. We used Salmonella enterica serotype Typhimurium (SL1344) and SL1344 overexpressing antioxidant KatN gene (SL1344-KatN) for natural gut infection. The KatN gene is one of the candidate genes responsible for Salmonella virulence, encoding a non-haem catalase responsible for ROS resistance (Robbe-Saule et al, 2001). To test whether KatN is involved in the removal of host's intestinal ROS, we measured the in vivo intestinal ROS level following SL1344-KatN infection. The result showed that infection-induced intestinal ROS level was significantly lower following SL1344-KatN infection, compared to that following SL1344 infection (Figure 3A). This result clearly showed that the bacterial virulent genes such as antioxidant enzyme KatN can efficiently antagonize the microbicidal ROS at the organism level. When we fed NF-κB pathway mutant flies on either the SL1344 strain or the SL1344-KatN, we observed high mortality levels in the flies fed on SL1344-KatN strain (Figure 3B). No significant mortality was observed in the NF-κB pathway mutant flies fed on either the SL1344 strain or the SL1344 overexpressing mutant form of KatN (SL1344-KatN-mut) (Figure 3B). We also observed that overexpression of KatN gene is sufficient to render non-pathogenic Escherichia coli strain (DH5α) highly virulent to NF-κB pathway mutant flies (Figure 3B). Furthermore, the remarkable levels of Salmonella KatN-induced mortality seen in the RelishE20 flies were completely abolished as a result of the re-introduction of the Relish gene expression in the intestine (Figure 3C). Taken together, these results demonstrate that intestinal NF-κB-dependent innate immunity plays an essential role in protecting the host against attacks by pathogens resistant to the ROS-dependent innate immunity. Figure 3.ROS-removing activity can act as a virulence factor to the host lacking IMD/NF-κB pathway potential. (A) KatN-overexpressing Salmonella can significantly decrease infection-induced host's ROS level. The total in vivo intestinal ROS levels were quantified (Ha et al, 2005a) with flies both before and after natural infection with control Salmonella (SL1344) or KatN-overexpressing Salmonella (SL1344-KatN). The ROS level in the uninfected control intestine was taken arbitrarily to be 100, and the results are presented as relative levels. Results are expressed as the mean and the standard deviations of three different experiments. (B) IMD/NF-κB pathway mutant flies are susceptible to bacteria overexpressing the Salmonella KatN catalase. Natural infection was performed with Salmonella enterica serotype Typhimurium (SL1344), SL1344 overexpressing Salmonella catalase, KatN (SL1344-KatN), SL1344 overexpressing mutant form of KatN (SL1344-KatN-mut), E. coli DH5α strain (E. coli) and DH5α strain overexpressing KatN (E. coli-KatN). (C) The susceptibility of RelishE20 flies to natural SL1344-KatN infection can be greatly ameliorated via the re-introduction of Relish in the intestine. The genotypes of the flies used in this study are described in Figure 2. In all cases, survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05). Download figure Download PowerPoint Gut AMP is required for host protection against gut infection by ROS-resistant microbes We next investigated the molecular mechanism by which the intestinal IMD/NF-κB pathway protects the host from ROS-resistant pathogens. In Drosophila epithelia, the IMD/NF-κB pathway is believed to be essential for the full expression of immune effector genes, including AMPs (Ferrandon et al, 1998; Tzou et al, 2000; Onfelt Tingvall et al, 2001). In the case of systemic infections, the importance of AMP has been supported by the observation that constitutive expression of a single AMP can restore resistance to systemic infection to the wild-type level in Toll and IMD pathway mutants (Tzou et al, 2002). Although the epithelial AMPs are believed to constitute an important host defense system that inhibits the onset of local microbial proliferation in Drosophila (Brey et al, 1993; Ferrandon et al, 1998; Tzou et al, 2000; Onfelt Tingvall et al, 2001; Ryu et al, 2004), the exact in vivo role of epithelial AMPs has not yet been demonstrated at an organism level owing to the lack of suitable experimental models. We questioned if the high level of pathogen-induced mortality in the IMD/NF-κB pathway mutant flies was due to the absence of NF-κB-dependent local AMP expression, which would ostensibly result in microbial over-proliferation and host death in the end. We attempted to ameliorate the survival rates of DreddB118 flies by inducing the tissue-specific expression of the AMP Cecropin (Cec) A1 gene in the intestine. The Drosophila Cec gene was selected for this experiment because Cec exhibits broad microbicidal activity against both bacteria and yeast (Gazit et al, 1994; Ekengren and Hultmark, 1999) and because the Cec gene is also rapidly induced in the intestine as the result of natural gut infection with yeasts via the IMD/NF-κB pathway (Figure 4A). Our in vitro antimicrobial activity assay revealed that both KNU5377 and W303 strains were equally susceptible to low concentrations of synthetic Cec A1 (Figure 4B) although the KNU5377 strain exhibited a much higher resistance to ROS than the W303 strain (Figure 1A). These results demonstrate that KNU5377 has different in vitro sensitivities to two distinct immune effectors, ROS and AMP. Consistently, both SL1344 and ROS-resistant SL1344-KatN strains were also equally susceptible to synthetic Cec A1 (Figure 4C). Our in vivo rescue experiment revealed that intestine-specific Cec expression in the DreddB118 flies (DreddB118;UAS-Cec/cad-GAL4) was sufficient to confer protection against natural KNU5377 or SL1344-KatN infection in the host lacking a functional IMD/NF-κB pathway (Figure 4D and E). Figure 4.Intestinal AMP expression is indispensable for host protection from attack of ROS-resistant pathogens. (A) Natural yeast infection induces Cecropin (Cec) expression in the midgut via IMD/NF-κB pathway. Wild-type flies (WT) or IMD/NF-κB pathway mutant flies (DreddB118) were subjected to natural infection (0 and 12 h) with either the W303 or KNU5377 strains. Quantitative real-time PCR analysis of Cec gene transcription was performed using dissected midguts. Cec expression in the tissues of uninfected WT flies was taken arbitrarily as 1, and the results are shown as relative expressions. Results are expressed as the means±s.d. (P<0.05) of three different experiments. (B) Both KNU5377 and W303 strains are found to be equally susceptible to low concentrations of synthetic Cec A1 peptide. Yeast cells were incubated with serially diluted Cec A1 peptide at 28°C for 18 h. Antifungal activity was performed as described in Materials and methods. Results are expressed as the means±s.d. (P<0.05) of three different experiments. (C) Both SL1344 and SL1344-KatN strains are equally susceptible to synthetic Cec A1. To measure the antibacterial activity, inhibition zone assay were performed with serially diluted Cec A1, as described in Materials and methods. (D) The susceptibility of the DreddB118 flies to natural KNU5377 infection can be dramatically ameliorated by the ectopic expression of Cec A1 in the intestine. For the rescue experiment, DreddB118 flies were crossed with flies carrying UAS-Cec A1. The cad-GAL4 driver was used for intestine-specific Cec expression. The genotypes of the flies used in this study were as follows: control (cad-GAL4/+); DreddB118 (DreddB118; cad-GAL4/+); DreddB118+Cec (DreddB118; cad-GAL4/UAS-Cec). Natural infection was performed with KNU5377. Survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05). (E) The susceptibility of IMD/NF-κB pathway mutant flies to ROS-resistant Salmonella infection can be dramatically ameliorated by ectopic Cec A1 expression in the intestine. The genotypes of the flies used in this study are shown in panel (D). Natural infection was performed with SL1344-KatN. Survival in three or more independent cohorts of about 25 flies each was monitored over time. Results are expressed as the means±s.d. (P<0.05). Download figure Download PowerPoint Gut IMD/AMP system is required for the efficient clearance of ROS-resistant microbes in the intestine In order to further verify that the natural infection-induced mortality of DreddB118 flies was due to uncontrolled microbial proliferation in the absence of AMPs and that the host protection seen in DreddB118;UAS-Cec/cad-GAL4 flies was due to Cec-mediated antimicrobial activity, we attempted to assess the persistence of the ROS-resistant microbes in the intestines of the control, DreddB118 and DreddB118; UAS-Cec/cad-GAL4 flies. First, it was shown that the KNU5377 counts in the intestines of DreddB118 flies were ∼100 times higher than those measured in the control flies (Figure 5A and Supplementary Figure 3). Next, the levels of KNU5377 found in the intestines of DreddB118 flies were reduced to control levels via the introduction of intestinal Cec expression into the DreddB118 flies (Figure 5A and Supplementary Figure 3). The results are consistent with that the marked KNU5377 proliferation was due to the absence of AMPs in the intestines of the DreddB118 flies. In a separate experiment, we used the GFP-tagged E. coli DH5α (E. coli-GFP) or E. coli-GFP overexpressing KatN (E. coli-KatN-GFP), which allowed us to follow in real time in vivo microbial persistence in the intestines of the hosts. In the control flies, we observed no significant microbial persistence following the ingestion of eit