Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut

Article

Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut Graphical Abstract

Authors Eun-Kyoung Kim, Kyung-Ah Lee, Do Young Hyeon, ..., Daehee Hwang, Youngjoo Kwon, Won-Jae Lee

Correspondence [email protected]

In Brief Although the gut microbiome is generally symbiotic or commensal, some microbiome members become pathogenic under certain circumstances. Kim et al. show that bacterial nucleoside catabolism of gut luminal uridine to uracil and ribose is required for the commensalto-pathogen transition by boosting interbacterial communications and virulence gene expression.

Highlights d

Bacterial nucleoside catabolism converts gut luminal uridine to uracil and ribose

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Uridine-derived uracil is required for DUOX-dependent ROS generation

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Uridine-derived ribose induces bacterial quorum sensing and virulence gene expression

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Bacterial nucleoside catabolism is required for commensalto-pathogen transition

Kim et al., 2020, Cell Host & Microbe 27, 1–13 March 11, 2020 ª 2020 Elsevier Inc. https://doi.org/10.1016/j.chom.2020.01.025

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

Cell Host & Microbe

Article Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut Eun-Kyoung Kim,1,2 Kyung-Ah Lee,1,2,3 Do Young Hyeon,1 Minsoo Kyung,1 Kyu-Yeon Jun,4 Seung Hee Seo,4 Daehee Hwang,1 Youngjoo Kwon,4 and Won-Jae Lee1,2,3,5,* 1School

of Biological Sciences, Seoul National University, Seoul 08826, South Korea Creative Research Initiative Center for Hologenomics, Seoul National University, Seoul 08826, South Korea 3Institute of Molecular Biology and Genetics, Seoul National University, Seoul 151-742, South Korea 4College of Pharmacy, Graduate School of Pharmaceutical Sciences, Ewha Woman’s University, Seoul 120-750, South Korea 5Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2020.01.025 2National

SUMMARY

Although the gut microbiome is generally symbiotic or commensal, some microbiome members become pathogenic under certain circumstances. However, the factors driving this pathogenic switch are largely unknown. Pathogenic bacteria can generate uracil that triggers host dual oxidase (DUOX) to produce antimicrobial reactive oxygen species (ROS). We show that pathogens generate uracil and ribose upon nucleoside catabolism of gut luminal uridine, which triggers not only host defenses but also inter-bacterial communication and pathogenesis in Drosophila. Uridine-derived uracil triggers DUOXdependent ROS generation, whereas ribose induces bacterial quorum sensing (QS) and virulence gene expression. Genes implicated in nucleotide metabolism are found in pathogens but not commensal bacteria, and their genetic ablation blocks QS and the commensal-to-pathogen transition in vivo. Furthermore, commensal bacteria lack functional nucleoside catabolism, which is required to achieve gut-microbe symbiosis, but can become pathogenic by enabling nucleotide catabolism. These findings reveal molecular mechanisms governing the commensal-to-pathogen transition in different contexts of host-microbe interactions. INTRODUCTION Gut-microbe interactions are complex and evolutionarily conserved phenomena found in all metazoans (Buchon et al., 2013; Clemente et al., 2012; Lee and Hase, 2014; McFall-Ngai et al., 2013). Although it is clear that the gut microbial community is deeply involved in diverse ranges of host physiology (Rooks and Garrett, 2016; Schretter et al., 2018; Sekirov et al., 2010; Shin et al., 2011; Storelli et al., 2018), the detailed molecular mechanisms of the host-microbe interactions determining sym-

biosis or dysbiosis are largely unknown. This is in part due to the lack of genetic model systems on both the host and microbial sides. In this regard, Drosophila has provided a powerful genetic model of gut-microbe interactions in understanding symbiotic, dysbiotic, or pathogenic interactions between the gut and microbes (Charroux et al., 2018; Douglas, 2018; Guo et al., 2014; Hang et al., 2014; Houtz et al., 2019; Lee and Brey, 2013; Mistry et al., 2016). Drosophila has a relatively simple commensal community in which the families of Lactobacillaceae and Acetobacteraceae are predominant (Iatsenko et al., 2018; Ryu et al., 2008; Shin et al., 2011; Storelli et al., 2011). These commensal bacteria affect different aspects of host physiology such as immunity, development, metabolism, and behavior (Kamareddine et al., 2018; Ryu et al., 2008; Schretter et al., 2018; Shin et al., 2011; Iatsenko et al., 2018; Masuzzo et al., 2019). In addition to these symbiotic gut-microbe interactions, gut epithelia could have pathogenic interactions with naturally occurring opportunistic pathogens such as Erwinia carotovora subspecies carotovora 15 (Ecc15) and Pseudomonas entomophila (Basset et al., 2000; Houtz et al., 2019; Vodovar et al., 2005). During gut-microbe interactions, gut innate immunity is readily activated to control microbes (Lee and Brey, 2013; Lemaitre and Hoffmann, 2007). Genetic analyses of Drosophila gut immunity demonstrated that dual oxidase (DUOX, a member of the nicotinamide adenine dinucleotide phosphate oxidase family) plays a pivotal role in antagonizing invading pathogens by producing microbicidal reactive oxygen species (ROS) (Ha et al., 2005; Lee et al., 2013). In addition to this ROS-based immunity, the immune deficiency (IMD) pathway is also involved in the gut antimicrobial response by producing antimicrobial peptides (AMPs) (Lemaitre et al., 1995; Lemaitre et al., 1996). Prior research revealed that ROS-based immunity synergistically works with AMP-based immunity during gut-microbe interactions (Ryu et al., 2006). Both immune systems are specifically activated in response to microbial challenges. Microbe-associated molecular patterns (MAMPs) such as peptidoglycan (PGN) are known to activate the IMD pathway (Lemaitre and Hoffmann, 2007; Royet and Dziarski, 2007). For example, diaminopimelic acid (DAP)-type PGN, a common structure found in the membranes of Gramnegative bacteria, is recognized by PGN recognition proteins that subsequently initiate IMD pathway activation for AMP Cell Host & Microbe 27, 1–13, March 11, 2020 ª 2020 Elsevier Inc. 1

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

production (Royet and Dziarski, 2007). However, DAP-type PGN is not a pathogen-specific molecular pattern because it is commonly found in both symbionts such as Acetobacter and pathogens such as Ecc15. As both bacteria can activate the IMD pathway (Lee et al., 2013), this pathway does not sufficiently explain the mechanism by which gut epithelia distinguish symbionts from pathogens. Interestingly, prior research found that DUOX is activated in response to Ecc15 but remains quiescent in response to Acetobacter (Lee et al., 2013), indicating that DUOX-dependent ROS generation is a pathogen-specific event. Further analysis revealed that bacterial-derived uracil acts as a ligand for DUOX and that pathogenic bacteria, but not commensal symbiotic bacteria, release uracil (Lee et al., 2013). These data indicate that bacterial-derived uracil is a pathogen-specific signature used by the host to distinguish pathogens from symbionts. Uracil can act as a ligand to initiate complex signaling pathways in enterocytes for DUOX activation (Ha et al., 2009; Lee et al., 2015, 2018). These DUOX-activating signaling pathways include the PLCb-Ca2+ pathway, hedgehog-cadherin 99C pathway, and target of rapamycin-autophagy 1-mediated lipolytic pathway (Ha et al., 2009; Lee et al., 2015, 2018). In addition to their antimicrobial role, DUOX-dependent ROS also act as ligands for the reactive chemical receptor TrpA1, which in turn facilitates pathogen-clearing defecation (Du et al., 2016). Despite the central importance of bacterial-derived uracil as a main ligand for DUOX-dependent immunity, it is presently unknown how and why pathogenic bacteria, but not commensal bacteria, release uracil. The present study aimed to identify pathogen-specific factors involved in uracil production and release, and understand the role of these factors in different contexts of gut-microbe interactions. We revealed that uracil and ribose are produced by the catabolic activity of bacterial nucleoside hydrolase (NH) using extracellular uridine in the gut lumen. We demonstrated that unlike pathogens, symbionts do not produce uracil because of the lack of functional NH activity, thereby avoiding DUOX activation. Furthermore, we revealed a role of NH-generated ribose as a signal for bacterial cell-to-cell communication and pathogen virulence, which are required for the commensal-to-pathogen transition in vivo. Our results highlight the importance of bacterial nucleoside catabolism in different contexts of gut-microbe interactions. RESULTS Pathogens Drive ‘‘Uridine-in and Uracil-out’’ Metabolic Flux Previously, regarding Drosophila gut immunity, it was found that enteric pathogens actively release pro-inflammatory uracil and that the host in turn recognizes uracil as a pathogen-sensing mechanism for DUOX-dependent microbicidal ROS generation (Lee et al., 2013). However, it is unclear which bacterial metabolism is involved in uracil excretion and why this bacterial metabolism operates inside the gut epithelia (i.e., during the enteric infection process). To gain insight into the intestinal niche-specific modulation of bacterial metabolism, we performed capillary electrophoresis (CE) coupled with time-of-flight mass spectrometry (TOF-MS) using the Drosophila midgut luminal fluid following gut infection with Ecc15, a well-known Drosophila opportunistic 2 Cell Host & Microbe 27, 1–13, March 11, 2020

pathogen. In the gut luminal metabolomes following Ecc15 infection, the most significant changes were observed in metabolites belonging to the nucleotide metabolic pathway (Figures 1A and 1B; Table S1). Interestingly, following Ecc15 infection, we observed a strong increase in the levels of nucleobases (e.g., uracil) accompanying a strong decrease in those of nucleosides (e.g., uridine), a molecule of uracil attached to a ribose ring (Figure 1C). This observation suggests that uridine in the gut lumen is rapidly absorbed by bacterial cells (i.e., uridine-in) for the possible catabolic conversion of uridine into ribose and uracil, and that uracil is then secreted by these cells (i.e., uracil-out). To further investigate the possible ‘‘uridine-in and uracil-out’’ metabolic flux, we incubated Ecc15 in minimal medium containing uridine. The uracil and uridine levels in the culture supernatants were measured by using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The result showed that uracil levels in the culture supernatant gradually increase after the incubation of Ecc15, whereas uridine completely disappeared from the culture supernatant (Figure 1D). We next examined whether excreted uracil is directly derived from absorbed extracellular uridine. For this purpose, we incubated Ecc15 in medium containing radioactive uridine carrying isotope in the nucleobase moiety (15N-uridine) or ribose moiety (13C-uridine). Following incubation, we measured 15N-uridine, 13 C-uridine, 15N-uracil, and 13C-ribose levels in the culture supernatant. We found that both 15N-uridine and 13C-uridine levels gradually decreased in the culture supernatant during 4 h of incubation (Figure 1E). During this period, 15N-uracil content in the culture supernatant progressively increased, indicating that secreted uracil from Ecc15 cells is indeed derived from absorbed 15 N-uridine. However, in contrast to the nucleobase moiety, 13 C-ribose was undetectable in the culture supernatant (Figure 1E), suggesting that the ribose moiety, following the cleavage of uridine inside the bacterial cells, is not secreted by Ecc15 cells. Taken together, we could conclude that pathogenic Ecc15 is capable of absorbing and catabolizing extracellular uridine into ribose and uracil. In addition, uracil, but not ribose, is secreted from the bacterial cells. Pathobionts, but Not Symbionts, Drive ‘‘Uridine-in and Uracil-out’’ Metabolic Flux for DUOX Activation In Drosophila, different bacteria in the Acetobacteraceae family are major components of its gut-associated microbiota in natural environments as well as laboratory conditions (Chandler et al., 2011; Ryu et al., 2008). Among gut microbes, most bacteria are beneficial (referred to as symbionts), whereas others are conditionally pathogenic, especially when they become dominant (referred to as pathobionts). Previously, we found that pathobiont members of the Acetobacteraceae family such as Gluconobacter morbifer secrete uracil, unlike symbiont members of Acetobacteraceae such as Acetobacter pomorum, suggesting that uracil secretion might be a characteristic of pathobionts (Lee et al., 2013). In the NCBI database for microbial genomes, the genomic information of approximately 100 species in the Acetobacteraceae family is currently available. Among these genome-sequenced Acetobacteraceae members, we selected ten bacteria and obtained them from original sources. Using these bacteria, we examined the relationship between the bacterial ‘‘uridine-in and uracil-out’’ flux and intestinal DUOX

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

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activation. We found that seven bacteria exhibit ‘‘uridine-in and uracil-out’’ metabolic flux, whereas three bacteria do not (Figure 2A). Importantly, all seven bacteria displaying ‘‘uridine-in and uracil-out’’ flux were capable of inducing intestinal DUOXdependent ROS generation (Figure 2B). These data indicate that the ‘‘uridine-in and uracil-out’’ flux of the bacteria correlates perfectly with their ability to induce DUOX activation in the gut. Based on this finding, we could divide these Acetobacteraceae members into two distinct groups based on their effect on the host intestine, namely a pathobiont group capable of driving ‘‘uridine-in and uracil-out’’ flux resulting in DUOX activation, and a symbiont group devoid of such metabolic flux resulting in the absence of DUOX activation. Comparative Genomics between the Pathobiont and Symbiont Members of Acetobacteraceae To understand the molecular mechanism of the pathobiont-specific ‘‘uridine-in and uracil-out’’ metabolic flux, we

Figure 1. Pathogens Drive ‘‘Uridine-in and Uracil-out’’ Metabolic Flux (A) Heatmap representation of the metabolomics profile following enteric Ecc15 infection. Flies were orally infected with sucrose solution in the absence (None) or presence of Ecc15 (Ecc15) for 2 h. Gut luminal fluids were subjected to metabolomic analysis using capillary electrophoresis coupled with time-of-flight mass spectrometry. In total, 152 metabolites were detected. Each metabolite was categorized in eight different metabolic processes. The number of metabolites in each metabolic process is indicated. The color bar shows the gradient of log2 fold changes of metabolite levels after enteric infection relative to control at each time point. The gray bar denotes metabolites that were not detected in the analysis. (B) Nucleotide metabolic processes are enriched by the up- and downregulated metabolites after enteric infection. The bars represent log10(p value) where the p value determined using DAVID software indicates the significance of the processes being enriched by up- and downregulated metabolites. (C) Heatmap showing the differential levels of upand downregulated metabolites involved in nucleotide metabolism. The color bar shows the gradient of log2 fold changes of metabolite levels after enteric infection relative to the control levels at each time point. Uridine and uracil are indicated by arrows. (D and E) Uracil is derived from extracellular uridine by Ecc15. Ecc15 was incubated in M9 minimal medium containing uridine (D) or isotope-labeled uridines (E). Positions of radioactive N and C are indicated by red. Uridine, uracil, and ribose molecules in the culture supernatants were analyzed at different time points using LCMS/MS analysis. Data are presented as the mean ± SD of at least three experiments. ND, not detected.

performed comparative genomics focusing on nucleoside metabolism. Comparative genomic analyses revealed that nucleoside metabolic pathways, especially pyrimidine utilization and purine and pyrimidine conversion, are distinct between the symbiont and pathobiont groups (Figure 2C). Importantly, four genes, namely NH (converting nucleoside into nucleobase and ribose), nucleoside permease (NupC, transporting nucleosides inside the cells), adenine deaminase (AdeC, converting adenine to hypoxanthine), and cytidine deaminase (Cdd, converting cytidine to uridine), are present only in pathobiont members (Figure 2C). Based on this, we could conclude that the pathobiont-specific NupC and NH genes provide a possible mechanism by which pathobionts drive ‘‘uridine-in and uracilout’’ metabolic flux for DUOX activation (Figure 2D). Our comparative genomics could also provide a possible explanation for why symbionts, devoid of NupC and NH genes, were unable to import extracellular uridine and why they could not generate uracil from uridine. Cell Host & Microbe 27, 1–13, March 11, 2020 3

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

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NH Activity of Pathobiont Is Required for Uracil Excretion and Subsequent DUOX-Dependent ROS Generation To demonstrate the direct involvement of NH activity in uracil release from pathobionts, we generated a mutant of the pathobiont G. morbifer lacking NH activity (GmoDNH) by site-directed mutagenesis. When we examined uracil release from bacterial cells, we found that the uracil release observed in the parental wild-type strain (GmoWT) was completely abolished in GmoDNH (Figure 3A). The impaired uracil-releasing ability of GmoDNH was restored by re-introducing the NH gene (GmoDNH_NH) (Figure 3A). Consistent with the absence of uracil release, GmoDNH cannot induce intestinal ROS generation, unlike GmoWT or GmoDNH_NH (Figure 3B). These results demonstrate that bacterial NH activity is required for uracil excretion and subsequent DUOX-dependent ROS generation. NH Activity of Pathobiont Acts as a Colitogenic Factor Responsible for Gut Pathology and Early Host Death To further examine whether the NH activity of pathobionts is the direct cause of gut pathology, we compared the rates of gut cell damage between germ-free (GF) flies mono-associated with GmoWT or GmoDNH. The results indicated that the high gut cell apoptosis rates observed in GF flies mono-associated with 4 Cell Host & Microbe 27, 1–13, March 11, 2020

Figure 2. Pathobionts, but Not Symbionts, Drive ‘‘Uridine-in and Uracil-out’’ Metabolic Flux (A and B) Pathobiont members of Acetobacteraceae, but not symbiont members, secrete uracil for DUOX activation. Bacteria were incubated in mannitol medium containing 15N-labeled uridine for 8 h, and 15N-labeled uracil in the culture supernatant was quantified using LC-MS/MS analysis (A). Intestinal DUOX-dependent ROS generation was measured using R19S dye following the oral ingestion of the indicated bacteria (approximately 1 3 1010 CFU) for 1.5 h (B). Data are presented as the mean ± SD. ND, not detected; NS, not significant; ***p < 0.001 (analyzed using ANOVA). (C) Heatmap analysis of genes involved in nucleotide metabolic pathways using whole-genome information. Genes of different bacteria were compared with corresponding genes of G. morbifer, and the percentage of amino acid sequence identity was shown as blue color gradient bar. Red color denotes genes that are absent (i.e., less than 40% identity) in the given bacterial genome. Note that only four genes (NH, NupC, AdeC, and Cdd) indicated by arrows were present in all pathobiont members but absent in all symbiont members. (D) Diagram of the nucleoside import and catabolic pathway for uracil production and release in pathobiont members.

GmoWT were almost completely abolished in the case of GmoDNH mono-association (Figure 3C). Consistent with the gut cell pathology, GmoWT-associated flies exhibited a shorter lifespan than GmoDNH-associated flies (Figure 3D). Therefore, the elimination of NH activity is sufficient to induce a bacterial phenotypic shift from pathobiont to commensal bacteria. However, reintroduction of NH activity into mutant pathobionts (GmoDNH_NH) is sufficient to restore all host pathologies such as gut cell damage and high mortality, similar to the levels observed in GmoWT-associated flies (Figures 3C and 3D). Taken together, we conclude that the NH activity of pathobiont acts as a colitogenic factor that is responsible for chronic DUOX activation and high host mortality. Absence of Nucleoside Catabolism of Acetobacteraceae Is Required for Gut-Microbe Symbiosis Based on the aforementioned results, we hypothesized that the absence of core genes involved in uridine catabolism (e.g., NH and NupC), as observed in symbionts, makes it possible to peacefully interact with the host gut by avoiding chronic DUOX activation. To test this hypothesis, we engineered an Acetobacter pasteurianus symbiont by introducing the NH and NupC genes to generate a strain (ApaNH_NupC). In contrast to its parental wild-type strain (i.e., ApaWT), ApaNH_NupC can drive ‘‘uridine-in and uracil-out’’ metabolic flux and subsequent DUOXdependent ROS generation (Figures 3E and 3F). In the case of GF flies mono-associated with ApaNH_NupC, we found that gut cell apoptosis was greatly increased, resulting in higher mortality

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

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Figure 3. Bacterial NH Activity Is Responsible for Gut Dysbiosis

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compared with the findings in GF flies mono-associated with ApaWT (Figures 3G and 3H). These results revealed that ectopic expression of NH and NupC in symbionts is sufficient to establish uracil-producing ability, which is responsible for the bacterial phenotypic shift from symbiont to pathobiont. Taken together, we could conclude that the absence of NH-dependent nucleoside catabolism is required for gut-microbe symbiosis by avoiding chronic DUOX activation. Enteric Pathogens Catabolize Gut Luminal Uridine for Uracil Excretion in a NH-Dependent Manner In addition to gut microbiota, the Drosophila gut naturally interacts with opportunistic pathogens such as Ecc15. As Ecc15 can induce ‘‘uridine-in and uracil-out’’ metabolic flux in vitro (Figures 1D and 1E), we next investigated the role of uridine catabolism in pathogenic bacteria. We first examined genes involved in nucleoside catabolism. As the genomic information of Ecc15 is not yet available, we established draft genome sequence information via a whole-genome shotgun strategy (DDBJ/EMBL/ GenBank under the accession WNLC00000000). Analysis of the Ecc15 genome revealed that the bacterium carries two NH

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(A–D) NH activity of pathobionts acts as a colitogenic factor responsible for gut pathology and early host death. NH gene of the pathobiont G. morbiferWT was deleted to generate G. morbiferDNH. Complementation of the G. morbiferDNH mutant was achieved by introducing the NH gene to generate G. morbiferDNH_NH. In vitro uracil-secreting ability using 15N-labeled uridine (A), ROS-producing ability (B), gut cell apoptosis (C), and host survival (D) were determined. (E–H) Absence of nucleoside catabolism in Acetobacteraceae is required for gut-microbe symbiosis. To induce uracil-secreting ability, NH and NupC genes from G. morbifer were introduced to A. pasteurianusWT to generate A. pasteurianusNupC_NH. In vitro uracil-secreting ability using 15N-labeled uridine (E), ROS-producing ability (F), gut cell apoptosis (G), and host survival (H) were determined. The data (C, D, G, and H) were obtained using germ-free flies mono-associated with indicated bacterial strains. Data were analyzed using ANOVA (A, B, and F) or Welch’s t test (E). Values are presented as the mean ± SD (***p < 0.001) of at least three independent experiments. In (C and G), each dot represents an individual fly, and horizontal lines denote mean value. ***p < 0.001 (ANOVA). In (D and H), log-rank analysis (Kaplan– Meier method) for lifespan revealed a significant difference in survival between G. morbiferWT- and G. morbiferDNH-associated GF flies (***p < 0.001) (D) and between A. pasteurianusWT- and A. pasteurianusNH_NupC-associated GF flies (*p < 0.05) (H). ND, not detected; NS, not significant.

genes. In addition to these genes, Ecc15 also carries two nucleoside phosphorylase (NP) genes capable of converting nucleoside into nucleobase and ribose 1-phosphate. When we generated quadruple mutant Ecc15 (Ecc15D4) lacking all four of these genes, we found that the strain could not drive ‘‘uridine-in and uracil-out’’ metabolic flux in vitro (Figure 4A), demonstrating that NH and NP activity is required for the catabolism of extracellular uridine. We next investigated whether ‘‘uridine-in and uracil-out’’ metabolic flux also operates in vivo; i.e., when pathogens are introduced into the gut lumen. For this purpose, we generated enteric infection via oral feeding of Ecc15. Uracil and uridine levels in luminal fluids were measured before and after enteric infection. In the absence of enteric infection, uridine levels were high, whereas uracil was almost undetectable, resulting in an extremely low uracil/uridine ratio in the gut lumen (Figure 4B). Importantly, we found a strong increase in uracil level accompanying a strong decrease in uridine level, resulting in a high uracil/uridine ratio following enteric infection (Figure 4B). These data demonstrate that Ecc15 releases uracil in the gut lumen in vivo. However, enteric infection did not further increase Cell Host & Microbe 27, 1–13, March 11, 2020 5

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

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Figure 4. Nucleoside Catabolic Activity of Ecc15 Is Required for Uracil Secretion and Bacterial Metabolic Regulation

(A) Nucleoside catabolic activity is required for in vitro uracil secretion from Ecc15. Two NH genes and two NP genes of Ecc15WT were deleted to generate Ecc15D4. Complementation of Ecc15D4 was achieved by introducing the NH gene to generate Ecc15D4_NH. Bacteria were incubated in M9 minimal medium containing uridine for the indicated time, and uridine and uracil levels in the culture supernatant were quantified using LC-MS/ MS analysis. (B) Nucleoside catabolic activity is required for D E F in vivo uracil secretion from Ecc15. Flies were orally infected with the indicated bacteria for 2 h. Gut luminal fluids were used to quantify uracil and uridine levels via LC-MS/MS analysis. (C) Nucleoside catabolic activity is required for in vivo ROS generation following enteric infection. Intestinal DUOX-dependent ROS generation was measured using R19S dye following oral ingestion of the indicated bacteria (approximately 1 3 1010 CFU) for 1.5 h. (D) Uracil secretion in vivo is observed following enteric infection with different pathogens. Flies were orally infected with the different pathogens (approximately 2 3 109 for P. aeruginosa and approximately 1 3 1010 CFU for other bacteria) for G H 2 h. Gut luminal fluids were analyzed to quantify uracil and uridine levels via LC-MS/MS analysis. (E and F) Relative proportions of 320 differentially expressed genes between Ecc15 and Ecc15D4 according to their associated gene ontology biological processes (GOBPs). The GOBP term at levels 1 (E) and 2–4 (F) were used for general cellular processes and metabolic processes, respectively. (G) Biological processes enriched by the upregulated or downregulated genes in Ecc15D4. The bars represent log10(p value) in which the p value from DAVID software denotes the significance of the processes being enriched by the up- and downregulated genes. (H) Heatmap showing the differential expression of the up- and downregulated genes involved in different metabolic processes. The color bar shows the gradient of log2 fold changes of mRNA expression levels of the Ecc15D4 strain relative to those of the Ecc15 strain. In (B–D), data were analyzed using ANOVA. Values are presented as the mean ± SD (*p < 0.05, **p < 0.01, ***p < 0.001) of at least three independent experiments. NS, not significant; ND, not detected.

the uracil/uridine ratio when Ecc15D4 was used for enteric infection (Figure 4B). When we introduced the NH gene into the Ecc15D4 mutant (i.e., Ecc15D4_NH), low uracil/uridine ratio observed in the case of Ecc15D4 infection was greatly restored, resulting in a high uracil/uridine ratio (Figure 4B). These results demonstrate that the NH activity of Ecc15 pathogen is responsible for in vivo nucleoside catabolism by using luminal uridine to generate and secrete uracil into the gut lumen. We further found that Ecc15D4 is unable to induce DUOX-dependent ROS generation (Figure 4C), confirming that nucleoside catabolic ability perfectly correlates with DUOX-activating ability. Interestingly, infection-induced increases of the uracil/uridine ratio were also observed when different pathogens (i.e., Salmonella typhimurium, Serratia marcescens, Shigella flexneri, Escherichia coli O157, and Pseudomonas aeruginosa) were introduced into the gut lumen (Figure 4D). Taken together, ‘‘nucleosides-in and 6 Cell Host & Microbe 27, 1–13, March 11, 2020

nucleobases-out’’ metabolic flux is likely a common characteristic of different pathogens that occurs via bacterial nucleoside catabolism but is absent in Drosophila commensal bacteria. Nucleoside Catabolic Activity Is Required for Downregulating Primary Metabolic Pathways and Upregulating Quorum Sensing Given that nucleoside catabolism is a pathogen-specific metabolic event that is absent in symbionts, we next investigated the role of nucleoside catabolism in the physiology of pathogenic bacteria. We performed high-resolution RNA-seq analysis using Ecc15 and Ecc15D4. We found that several genes were dramatically up- and downregulated in the absence of nucleoside catabolic activity. Functional enrichment analysis revealed that the absence of nucleoside catabolic activity most significantly enhanced ‘‘metabolic process’’ (65.2%) among different

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

biological processes (Figure 4E). Notably, cellular amino acid metabolic processes (28.2%), nucleobase-containing small molecule metabolic processes (22.0%), and carbohydrate metabolic processes (19.2%) were most frequently over-activated in Ecc15D4, when compared with Ecc15 (Figures 4F–4H). However, the quorum sensing (QS) process was downregulated in Ecc15D4 (Figure 4H). These results suggest that nucleoside catabolism is required for the downregulation of primary metabolic activities and upregulation of the QS process. Nucleoside Catabolic Activity Is Required for the Full Production of Acyl Homoserine Lactone-type QuorumSensing Molecules Previously, it was demonstrated that primary metabolic activities are higher in the bacterial QS mutant strain than in the control strain (An et al., 2014). Consistent with this, we also found that genes involved in primary metabolisms are largely upregulated in Ecc15D4 having downregulated QS (Figure 4H), indicating that QS activation is required to maintain proper level of core metabolism. Therefore, we hypothesized that nucleoside catabolic activity may be required for QS, and that impaired QS activity caused by the absence of nucleoside catabolism may be responsible for the over-activated primary metabolic processes observed in Ecc15D4. To test this hypothesis, we directly measured the levels of QS molecules in Ecc15D4 and Ecc15. Ecc15 is known to produce two types of QS molecules: acyl homoserine lactone (AHL) produced by ExpI, and autoinducer-2 (AI-2) produced by LuxS enzymes (Barnard and Salmond, 2007). To measure these molecules, we used bioluminescentbased QS biosensors, specifically the E. coli pSB401 (Winson et al., 1998) and Vibrio harveyi MM32 strains (Miller et al., 2004), which emit bioluminescence upon AHL and AI-2 recognition, respectively. The result indicated that ExpI-dependent AHL levels were much higher in the Ecc15 culture supernatant than in the Ecc15D4 culture supernatant (Figure 5A). However, no differences were observed in terms of LuxS-dependent AI-2 levels between Ecc15 and Ecc15D4 culture supernatants (Figure S2). These results indicate that Ecc15D4 has impaired production of AHL-type QS molecules but not AI-2-type QS molecules. Consistent with these findings, quantitative analysis using LCMS/MS further revealed that the production of N-(3-oxo-hexanoyl)-L-homoserine lactone (3-oxo-C6-HSL), a major AHL-type in Ecc15 (Barnard and Salmond, 2007), is greatly impaired in Ecc15D4 (Figure 5B), indicating that nucleoside catabolic activity is required for the full production of AHL-type QS molecules. Ribose Generated via Nucleoside Catabolism Activates Quorum Sensing in an RbsR-Dependent Manner As Ecc15D4 exhibits reduced AHL production, we examined the mechanism by which nucleoside catabolism affects AHL production and AHL-dependent QS signaling. We found that the expression of two global regulators of QS, namely ExpR (a QSdependent transcriptional regulator that acts downstream of ExpI) and VqsM (an AraC-type global regulator of QS signaling and virulence) (Liang et al., 2014; Po˜llumaa et al., 2012), are greatly impaired in Ecc15D4 (Figures 4H and 5C). Given that the ExpR-AHL complex induces QS pathway activation including AHL production, the reduced AHL production observed in Ecc15D4 is likely attributable to the reduced expres-

sion of ExpR. These results indicate that nucleoside catabolic activity is required for the full activation of global QS regulators. We next investigated the mechanism by which nucleoside catabolism affects the expression of key QS regulators. As nucleoside catabolic activity can generate intracellular ribose from extracellular uridine, we hypothesized that the ribosemodulated transcription regulator, RbsR (Mauzy and Hermodson, 1992), is somehow involved in the transcriptional regulation of QS regulators. To test this hypothesis, we examined ExpR and VqsM expression in the absence of RbsR by generating Ecc15DRbsR. We found that ExpR and VqsM expression was greatly abolished in Ecc15DRbsR, as observed in Ecc15D4 (Figure 5C), indicating that RbsR is required for the full expression of these QS regulators. Consistent with these data, we found that AHL-dependent bioluminescence activity was greatly diminished in Ecc15DRbsR, as observed in Ecc15D4 (Figure 5D). Taken together, we could conclude that ribose generated by nucleoside catabolic activity upregulates QS regulators and induces QS pathway activation in an RbsR-dependent manner. Nucleoside Catabolic Activity Is Required for the Full Expression of Pathogen Virulence Genes Ecc15 is an entomopathogen that can persist in the Drosophila gut. It is known that Erwinia virulence factor (evf) acts as a major virulence gene, and it is responsible for the bacterial pathogenesis (Basset et al., 2003). At present, it is unknown whether evf is under the control of the QS system. To investigate whether the evf gene is under the control of a specific QS pathway, we generated bacteria carrying a mutation in an ExpI (Ecc15DExpIR) or LuxS pathway (Ecc15DLuxS), rendering them incapable of producing AHL-type QS and AI-2-type QS molecules, respectively. When we introduced the reporter plasmid carrying GFP under control of the evf promoter region (Pevf::GFP) into these QS mutants, we found that Pevf::GFP reporter activity is severely downregulated in Ecc15DExpIR but is normally expressed in Ecc15DLuxS (Figure 5F). These results demonstrate that a major virulence gene expression is primarily under the control of the AHL-type QS system but not the AI-2 type QS system. Given that nucleoside catabolism acts upstream of the AHL-dependent QS pathway, we next examined whether nucleoside catabolism is required for full activation of AHL-dependent evf genes in vitro. The result indicated that Pevf::GFP reporter activity and evf gene expression were severely impaired in Ecc15D4 and Ecc15DRbsR compared with that in Ecc15 (Figures 5E and 5F). Taken together, we could conclude that nucleoside catabolic activity and subsequent RbsR activation are required for QSdependent virulence gene expression. Nucleoside Catabolic Activity Is Required for QuorumSensing-Dependent Virulence In Vivo The aforementioned data indicate that nucleoside catabolism is required for bacterial QS activation and QS-dependent virulence gene expression in vitro. To investigate whether nucleoside catabolism-controlled QS activation indeed occurs in vivo, i.e., within a gut lumen, we first examined AHL production following enteric infection. To visualize AHL production in the gut, we performed the oral Ecc15 infection together with the QS biosensor E. coli pSB401 strain. Live in vivo imaging detected high levels of AHL-dependent bioluminescence in the gut lumen following Cell Host & Microbe 27, 1–13, March 11, 2020 7

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

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Figure 5. Nucleoside Catabolic Activity Is Required for AHL-Dependent QS Activation and Virulence Gene Expression in an RbsRDependent Manner

(A) Nucleoside catabolism is required for the production of AHL-type QS molecules. Culture supernatants of Ecc15 or Ecc15D4 at different growth time points were incubated with the E. coli pSB401 strain (bioluminescent-based AHL-type QS biosensor). Bioluminescence was measured using a luminometer. For the bioluminescence imaging, the pSB401 strain was incubated with the bacterial culture supernatant (obtained after 10.5 h of culture) for 2 h. The Ecc15 mutant lacking the AHLsynthesizing ExpI enzyme (Ecc15DExpIR) was used as a negative control. Data are presented as the mean ± SD of at least three experiments. (B) Measurement of bacterial-produced 3-oxoC D C6-HSL. Culture supernatants of Ecc15 or Ecc15D4 at different growth time points were used to quantify 3-oxo-C6-HSL levels via LC-MS/MS. Data are presented as the mean ± SD of at least three experiments. (C) Nucleoside catabolism and ribose-modulated transcription regulators are required for the expression of global QS regulators. The expression levels of the ExpR and VqsM genes were analyzed by qPCR analysis using Ecc15WT, Ecc15D4, and Ecc15DRbsR. Target gene expression in Ecc15WT was taken arbitrarily as 1, and the results are shown as relative levels of expression. E The bars, means ± SD (***p < 0.001) of at least three independent experiments. (D) RbsR is required for the production of AHLtype QS molecules. Culture supernatants of Ecc15 F or Ecc15DRbsR at different growth time points were incubated with the E. coli pSB401 strain. Bioluminescence was measured using a luminometer. For the bioluminescence imaging, the pSB401 strain was incubated with the bacterial culture supernatant (obtained after 10.5 h of culture) for 2 h. The Ecc15 mutant lacking nucleoside catabolic activity (Ecc15D4) was used as a control. Data are presented as the mean ± SD of at least three experiments. (E and F) Evf expression is under the control of the nucleoside catabolism, RbsR, and ExpIR-dependent QS system. The expression level of evf was analyzed using qPCR (E) and Pevf::GFP reporter activity (F). Target gene expression in Ecc15WT was taken arbitrarily as 1, and the results were shown as relative levels of expression. The bars, means ± SD (***p < 0.001) of at least three independent experiments.

Ecc15 ingestion (Figure 6A). Conversely, AHL-dependent bioluminescence was barely detectable following Ecc15D4 ingestion (Figure 6A), indicating that in vivo QS activation within a gut lumen is dependent on nucleoside catabolic activity. We further examined QS-dependent evf expression in the bacteria when they entered the gut lumen. For this purpose, we prepared two Pevf::GFP reporter bacteria (Ecc15-Pevf::GFP and Ecc15D4-Pevf::GFP) at the mid-exponential growth phase. At this growth point, Pevf::GFP expression levels were basal and similar between the two reporter bacteria (data not shown). Importantly, when these bacteria were introduced into the gut lumen, we found striking differences in terms of Pevf::GFP expression between Ecc15-Pevf::GFP and Ecc15D4-Pevf::GFP (Figure 6B). We observed intense GFP expression in Ecc15Pevf::GFP bacteria, mostly in the anterior midgut, whereas only weak GFP expression was observed for Ecc15D4-Pevf::GFP 8 Cell Host & Microbe 27, 1–13, March 11, 2020

(Figure 6B). Taken together, these data demonstrate that the nucleoside catabolic activity of Ecc15 is required for QS and virulence gene expression during in vivo interactions between the gut and microbes. Elimination of Bacterial Nucleoside Catabolic Activity Is Sufficient to Induce Pathogen-to-Symbiont Transition Previously, we found that symbiotic gut bacteria such as Acetobacter affect diverse features of host physiology such as development and metabolism (Shin et al., 2011). Under conditions such as protein malnutrition, GF flies are fully viable, but they exhibit delayed developmental speed and growth stunting (Figures 6C–6E and S3). These developmental defects are completely rescued in the presence of normal microbiota (i.e., conventionally reared flies) or A. pomorum (i.e., A. pomorumassociated GF flies) (Figures 6C–6E and S3). However, when

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

Figure 6. Nucleoside Catabolic Activity Is Required for Bacterial QS and Commensal-to-Pathogen Transition in the Gut

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(A) Nucleoside catabolic activity is required for in vivo QS activation in the gut lumen. Flies were orally infected with Ecc15WT or Ecc15D4 together with the bioluminescence reporter E. coli pSB401 strain. Bioluminescence imaging in live flies was obtained at 1.5 h after infection. Each dot represents an individual fly, and horizontal lines denote mean values. ***p < 0.001 (ANOVA). Representative images are shown. (B) Nucleoside catabolic activity is required for in vivo virulence gene activation in the gut lumen. Ecc15WT or Ecc15D4 carrying Pevf::GFP reporter was used for oral infection for 2 h using larvae or adults. Fluorescence intensity of the individual gut was measured via confocal microscopy. Each dot represents an individual animal, and horizontal lines denote mean values. ***p < 0.001(ANOVA). A representative confocal image is shown. (C) Impact of bacterial nucleoside catabolism on the developmental rate. Bacteria of the indicated genotypes were added to germ-free embryos, and the percentage of pupa formation was monitored every 12 h. Note that Ecc15D4 enhances the host developmental rate similar to the growth-promoting symbiont A. pomorum. (D and E) Impact of bacterial nucleoside catabolism on animal size. Bacteria of the indicated genotypes were added to germ-free embryos, and larval size was measured at 108 h after egg laying. Each dot represents an individual animal, and horizontal lines denote mean values. ***p < 0.001 (ANOVA). NS, not significant. Representative images are shown (E).

DISCUSSION

Ecc15 was added to GF flies, Ecc15-associated GF flies displayed more drastic pathological changes than GF flies, including growth arrest and severe larval lethality (>50% lethality) (Figure 6C). To assess whether nucleoside catabolism-induced QS activation is involved in this pathogenesis, we generated GF embryos mono-associated with Ecc15D4 or Ecc15DRbsR. Strikingly, these Ecc15D4- or Ecc15DRbsR-associated GF flies exhibited completely normal larval development (Figures 6C–6E and S3) comparable to that observed in symbiont (A. pomorum)-associated GF flies. These results indicate that eliminating bacterial nucleoside catabolic ability in pathogens is sufficient to drive the pathogen-to-symbiont transition in the Drosophila gut. Furthermore, when we re-introduced the NH gene into Ecc15D4, we observe the reappearance of host pathology (Figures 6C–6E), demonstrating that nucleoside catabolic activity is required for pathogen virulence. Taken together, we could conclude that Ecc15 uses host-derived uridine nucleosides to boost inter-bacterial communication, which is required for the commensal-to-pathogen transition in the Drosophila gut.

Microbe-harboring gut epithelia face an immunological dilemma in which they should control the pathogen while preserving peaceful cohabitation with symbionts. The innate immune system, consisting of pathogen recognition by MAMPs and subsequent activation of antimicrobial responses to antagonize pathogens, is well-illustrated in mucosal immunity of different metazoans including Drosophila (Janeway and Medzhitov, 2002; Kawai and Akira, 2010; Lemaitre and Hoffmann, 2007). Given that all microbes possess MAMPs without exception, current knowledge of the innate immune system based on MAMP recognition fails to explain how the gut distinguishes pathogens from symbionts and specifically activates antimicrobial responses to pathogens. We previously demonstrated that pathogens and pathobionts release uracil metabolite that acts as a ligand for DUOX activation, whereas symbionts do not release uracil, thereby avoiding DUOX activation (Lee et al., 2013). These findings raise an interesting possibility that microbe-contacting mucosal gut epithelia recognize pathogens by sensing pathogen-specific metabolic activity, capable of producing pathogen-specific metabolites such as uracil, to induce microbicidal ROS generation. However, the existence of pathogen-specific Cell Host & Microbe 27, 1–13, March 11, 2020 9

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

Figure 7. A Model for the Role of Bacterial Nucleoside Catabolism on Different Gut-Microbe Interactions Pathogens could drive ‘‘uridine-in and uracil-out’’ metabolic flux by using NupC and NH. On the pathogen side, uridine-derived ribose activates RbsR for bacterial cell-to-cell communication (via QS activation) and QS-dependent pathogenesis. On the host side, host enterocyte recognizes the presence of pathogens by sensing uridine-derived uracil. Uracil in turn acts as a ligand for DUOX activation and subsequent microbicidal ROS generation. Note the arms races between host defense (uracil-dependent microbicidal ROS) and pathogen virulence (ribose-dependent evf toxin). Pathobiont members could drive ‘‘uridine-in and uracil-out’’ metabolic flux by using NupC and NH, which results in chronic DUOX activation, gut cell damage, and early host death. However, symbiont members devoid of nucleoside catabolic activity could interact with enterocytes without DUOX activation. Note that all members of Acetobacteraceae (pathobionts or symbionts) lack QS system.

metabolic activity has never been described, making it difficult to explain why pathogens specifically release uracil. In this study, we uncovered that pathogens exhibit unique metabolic activities capable of driving ‘‘uridine-in and uracilout’’ metabolic flux (Figure 7). Our metabolomics and comparative genomics analyses revealed that enteric pathogens and pathobionts could absorb and catabolize gut luminal fluid uridine. This uridine catabolic ability is attributed to NH enzymes involved in nucleoside catabolism. It is presently unclear why uridine levels are high in the gut lumen and from where gut luminal uridine originates. High uridine levels were also detected in GF flies (Figure S4), indicating that gut luminal uridine does not originate from gut bacteria. Furthermore, we found similar levels of uridine in the gut lumen of GF flies fed a synthetic diet lacking uridine for 10 days (Figure S4). Although it is unknown why uridine exists at such high levels in the gut lumen, our results indicate that gut luminal uridine is not derived from gut microbes but is likely host-origin derived from enterocytes. Gut-associated bacteria are generally considered symbiotic or commensal. However, pathobiont members of commensal communities can be pathogenic under certain circumstances, a phenomenon best known as dysbiosis (Ryu et al., 2008). Dys10 Cell Host & Microbe 27, 1–13, March 11, 2020

biosis is a conserved phenomenon observed from Drosophila to mammals (Guo et al., 2014; Henao-Mejia et al., 2012; Ryu et al., 2008; Wu et al., 2017; Zmora et al., 2017). In Drosophila, although Acetobacteraceae members are considered natural gut symbionts, it has been observed that certain Acetobacteraceae members, especially G. morbifer, can act as pathobionts when they become dominant (Lee et al., 2013; Ryu et al., 2008). One of the key questions that needs to be answered is why some members of Acetobacteraceae are symbionts whereas others are pathobionts. Our comparative genomics and metabolic pathway analyses of symbiont and pathobiont members revealed that the utilization capacity of exogenous uridine, i.e., NupC-dependent uridine import and NH-dependent uridine hydrolysis, is a key feature of pathobionts that is absent in symbionts (Figure 2). Elimination of NH activity in pathobionts is sufficient to abolish DUOX activation, gut cell apoptosis, and early host death, thereby inducing a bacterial phenotypic shift from pathobiont to symbiont (Figure 3). Therefore, NH-dependent catabolism of extracellular uridine acts as a primary bacterial colitogenic factor by inducing chronic oxidative stress via DUOX overactivation. Inversely, a phenotypic shift from symbiont to pathobiont was also observed when we ectopically expressed NupC and NH in symbionts (Figure 3). Our data describe a fascinating example of how specific bacterial metabolic activity within members of the same bacterial family can act as a determining factor for gut-microbe symbiosis or dysbiosis (Figure 7). Transcriptome analysis revealed that the absence of uridine catabolism positively or negatively regulates a broad range of bacterial metabolic processes (Figure 4). Specifically, the upregulation of primary metabolic activities and downregulation of the QS process are observed in Ecc15D4, indicating the existence of an inverse correlation between primary metabolic activities and QS. In this regard, it is interesting to note that the primary metabolic activities were higher in the bacterial QS mutant strain than in the control strain (An et al., 2014; Goo et al., 2015), suggesting that QS functions as a metabolic brake. Consistent with this notion, we found similar metabolic patterns (i.e., upregulation of primary metabolic genes) between Ecc15D4 and QS mutant bacteria (Figure S1), further supporting a link between uridine catabolism and QS. How is uridine catabolism involved in QS? Unlike uracil, ribose is not secreted from bacterial cells following uridine catabolism (Figure 1). Although the exact mechanism of nucleoside catabolism-dependent QS regulation remains to be elucidated, RbsR is likely activated by uridine-derived ribose, which is required for the full expression of critical genes involved in QS activation such as ExpR (Figure 5). Downregulation of the ExpR and ExpI system in Ecc15D4 may explain the lower production of AHL (Figure 5). Bacterial cell-to-cell communication via QS plays a pivotal role in bacterial pathogenesis. Although the in vitro regulation of QS is relatively well-established, its in vivo regulation within host tissue is poorly understood. In this study, we uncovered an in vivo QS regulatory system in which nucleoside catabolic activity is required for in vivo QS activation and the subsequent virulence gene expression within the gut lumen (Figure 6). The importance of nucleoside catabolism was further demonstrated by the finding that elimination of uridine catabolic ability, as observed in Ecc15D4, is sufficient

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

to induce a phenotypic shift from a life-threatening pathogen to a growth-promoting symbiont. Regarding the bacterial side, bacteria must recognize whether they are in the right location (i.e., gut epithelia in the case of enteric pathogens) to ensure their successful infection. We found that uridine in the gut lumen is readily utilized by bacterial nucleoside catabolism, resulting in ribose production and subsequent QS activation. Therefore, gut-associated uridine may act as a location indicator for bacteria, which initiates QS activation and pathogenesis (Figure 7). In this context, it is interesting to note that significant amounts of nucleosides have been detected in the biological fluids such as blood, urine, saliva, and cerebrospinal fluids (Eckle et al., 2013; Eells and Spector, 1983; Hsu et al.,
ska et al., 2010), suggesting that other pathogens 2009; Szyman may also use NH-dependent QS modulation to induce their virulence. Indeed, we also found that eliminating the NH activity of the airway pathogen P. aeruginosa PAO1 strain was sufficient to decrease AHL-type QS molecule production and QS-dependent virulence factor activity (Figure S5). Although host-pathogen interactions are highly variable among different pathogens and hosts, it would be interesting to investigate whether NHdependent pathogenesis also operates in diverse ranges of host-pathogen interactions beyond Drosophila-Ecc15. Given that many pathogens possess NH-dependent nucleoside catabolism and that QS activation is required for bacterial virulence, the discovery of QS modulation by host-derived nucleosides and bacterial NH activity will greatly help to better understand the molecular mechanism of the commensal-to-pathogen shift in diverse host-microbe interactions.

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Measurement of Pyocyanin Production Measurement of Elastase Activity QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY B

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SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. chom.2020.01.025. ACKNOWLEDGMENTS This work was supported by grants from the National Creative Research Initiative programs of the National Research Foundation, South Korea (2015R1A3A2033475). E.-K.K. and K.-A.L. were supported by NRF2017R1D1A1B03034197 and NRF-2019R1I1A1A01059606, respectively. AUTHOR CONTRIBUTIONS E.-K.K. and W.-J.L. conceived and designed the experiments. E.-K.K., K.-A.L., M.K., K.-Y.J., and S.H.S. performed the experiments. E.-K.K., D.Y.H., D.H., Y.K., and W.-J.L. analyzed the data. E.-K.K and W.-J.L. wrote the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Received: November 30, 2019 Revised: January 3, 2020 Accepted: January 29, 2020 Published: February 19, 2020 SUPPORTING CITATIONS

STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d

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KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Bacterial Strains B Fly Rearing B Bacterial Culture Conditions METHOD DETAILS B Metabolomic Analyses B Quantitative Analysis of Metabolites Using LC-MS/MS B In Vivo ROS Measurement B Comparative Genomics B Construction of Bacterial Strains B Generation of Gnotobiotic Animals B Lifespan Analysis B Apoptosis Assay B mRNA Sequencing and Data Analysis B Identification of Differentially Expressed Genes (DEGs) B Real-Time qPCR Analysis B Bioluminescence Assays B LC-MS/MS Analysis of 3-oxo-C6-HSL B Measurement of Pevf::gfp Expression In Vitro and In Vivo B Bioluminescence Imaging of Live Drosophila B Measurement of Host Developmental Rate

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Piper, M.D., Blanc, E., Leita˜o-Gonc¸alves, R., Yang, M., He, X., Linford, N.J., Hoddinott, M.P., Hopfen, C., Soultoukis, G.A., Niemeyer, C., et al. (2014). A holidic medium for Drosophila melanogaster. Nat. Methods 11, 100–105. €e, T., and Ma €e, A. (2012). Quorum sensing and expression Po˜llumaa, L., Alama of virulence in pectobacteria. Sensors (Basel) 12, 3327–3349. Rooks, M.G., and Garrett, W.S. (2016). Gut microbiota, metabolites and host immunity. Nat. Rev. Immunol. 16, 341–352. Royet, J., and Dziarski, R. (2007). Peptidoglycan recognition proteins: pleiotropic sensors and effectors of antimicrobial defences. Nat. Rev. Microbiol. 5, 264–277. Ryu, J.H., Ha, E.M., Oh, C.T., Seol, J.H.T., Brey, P.T., Jin, I., Lee, D.G., Kim, J., Lee, D., and Lee, W.J. (2006). An essential complementary role of NF-kappaB pathway to microbicidal oxidants in Drosophila gut immunity. EMBO J. 25, 3693–3701. Ryu, J.H., Kim, S.H., Lee, H.Y., Bai, J.Y., Nam, Y.D., Bae, J.W., Lee, D.G., Shin, S.C., Ha, E.M., and Lee, W.J. (2008). Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science 319, 777–782. Schretter, C.E., Vielmetter, J., Bartos, I., Marka, Z., Marka, S., Argade, S., and Mazmanian, S.K. (2018). A gut microbial factor modulates locomotor behaviour in Drosophila. Nature 563, 402–406. Sekirov, I., Russell, S.L., Antunes, L.C., and Finlay, B.B. (2010). Gut microbiota in health and disease. Physiol. Rev. 90, 859–904. Shin, S.C., Kim, S.H., You, H., Kim, B., Kim, A.C., Lee, K.A., Yoon, J.H., Ryu, J.H., and Lee, W.J. (2011). Drosophila microbiome modulates host developmental and metabolic homeostasis via insulin signaling. Science 334, 670–674. Smith, K.M., Bu, Y., and Suga, H. (2003). Induction and inhibition of Pseudomonas aeruginosa quorum sensing by synthetic autoinducer analogs. Chem. Biol. 10, 81–89.

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Cell Host & Microbe 27, 1–13, March 11, 2020 13

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Bacterial and Virus Strains Erwinia carotovora subsp. carotovora15 (Ecc15WT)

Lemaitre B, EPFL

N/A

Ecc15DNH1,NH2,deoD,udp (Ecc15D4)

This study

N/A

Ecc15DrbsR (Ecc15DrbsR)

This study

N/A

Ecc15DexpIR (Ecc15DexpIR)

This study

N/A

Ecc15DluxS (Ecc15DluxS)

This study

N/A

Ecc15Drih1Drih2DdeoDDudp harboring pTac3Plac::NH (Ecc15D4_NH)

This study

N/A N/A

Commensalibacter intestini A911

(Ryu et al., 2008)

Gluconobacter morbifer G707 (GmoWT)

(Ryu et al., 2008)

N/A

GmoDNH (GmoDNH)

This study

N/A

GmoDNH harboring pCM62-PntpII::NH (GmoDNH_NH)

This study

N/A

Acetobacter pomorum

(Ryu et al., 2008)

N/A

Acetobacter pasteurianus SKU1108 (ApaWT)

Matsushita K, Yamaguchi University

NBRC101655

Apa harboring pCM62-PntpII::NH-NupC (ApaNH_NupC)

This study

N/A

Acetobacter tropicalis SKU1100

Matsushita K, Yamaguchi University

N/A

Gluconobacter frateurii

NITE Biological Resource Center

NBRC103465

Gluconobacter thailandicus

NITE Biological Resource Center

NBRC3255

Komagataeibacter medellinensis

NITE Biological Resource Center

NBRC3283

Gluconobacter oxidans 621H

Daffonchio D, King Abdullah University

N/A

Asaia SF2.1

Daffonchio D, King Abdullah University

N/A

Salmonella typhimurium

Ryu JH, Yonsei University

N/A

Serratia marcescens Db11

Ferrandon D, Institut de Biologie Mole´culaire et Cellulaire du CNRS

N/A

Shigella flexneri 2a

Kweon MN, Asan Medical Institute

N/A

Escherichia coli O157:H7

Park S, Sungkyunkwan University

N/A

Pseudomonas aeruginosa PAO1

Yoon SS, Yonsei University

N/A

PAO1DpqsL (PAO1)

This study

N/A

PAO1DpqsLDNH (PAO1 DNH)

This study

N/A

PAO1DpqsLDrih harboring pJN-Ptac::NH (PAO1 DNH_NH)

This study

N/A

E.coli JM109 harboring pSB401

(Winson et al., 1998)

N/A

E.coli JM109 harboring pSB536

(Winson et al., 1998)

N/A

E.coli JM109 harboring pSB1142

(Winson et al., 1998)

N/A

Vibrio harveyi MM32

Song JJ, Korea University College of Medicine

N/A

HOCl-specific rhodamine-based R19S dye

(Lee et al., 2013)

N/A

Uridine

Sigma-Aldrich

Cat#U3750

Uracil

Sigma-Aldrich

Cat#U0750

Chemicals, Peptides, and Recombinant Proteins

D-ribose

Sigma-Aldrich

Cat#R7500

13

Cambridge Isotope

Cat#CLM-3630

13

Cambridge Isotope

Cat#CLM-768

15

Cambridge Isotope

Cat#NLM-812

C-Uridine C-ribose N-Uridne

(Continued on next page)

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Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

15

Cambridge Isotope

Cat# NLM-637

Trizol

Thermo Fisher Scientific

Cat#15596026

N-(b-Ketocaproyl)-L-homoserine lactone

Sigma-Aldrich

Cat#K3007

Elastin-Congo red

Sigma-Aldrich

Cat#E0502

N-Uracil

Critical Commercial Assays SensiFAST SYBR Hi-ROX kit

Bioline

Cat#BIO-92020

TURBO DNA-free kit

Ambion

Cat#AM1907

Click-iTTM Plus TUNEL Assay Kit

Invitrogen

Cat#C10618

RNeasy MiniElute kit

QIAGEN

Cat#74204

Erwinia carotovora subsp. carotovora15 (Ecc15) genome

This study

DDBJ/ENA/GenBank: WNLC00000000

Raw and analyzed data

This study

GEO: GSE140194

This laboratory

N/A

This study

N/A

pTac3-Plac::NH

This study

N/A

pCM62-PntpII::NH

This study

N/A

pCM62-PntpII::NH-NupC

This study

N/A

pTac3-Pevf::gfp

This study

N/A

pJN-Ptac::NH

This study

N/A

Cutadapt v.2.6

(Martin, 2011)

http://cutadapt.readthedocs.org RRID:SCR_011841

TopHat v.2.1.1

(Trapnell et al., 2009)

https://ccb.jhu.edu/software/tophat/ index.shtml RRID:SCR_013035

HTSeq v.0.6.1

(Anders et al., 2015)

https://htseq.readthedocs.io/en/ release_0.11.1 RRID:SCR_005514

ZEN2.5 lite

Carlzeiss

https://www.zeiss.com/microscopy/ int/downloads.html RRID:SCR_013672

GraphPad Prism 8.2.0

GraphPad Software

https://www.graphpad.com/scientificsoftware/prism/ RRID:SCR_002798

DAVID v.6.8

(Huang da et al., 2009)

https://david.ncifcrf.gov/home.jsp RRID:SCR_001881

Deposited Data

Experimental Models: Organisms/Strains Drosophila melanogaster (w1118) Oligonucleotides Primers for real-time qPCR: see Table S2 Recombinant DNA

Software and Algorithms

LEAD CONTACT AND MATERIALS AVAILABILITY All unique reagents generated in this study are available from the Lead Contact without restriction. However, we may require a completed Materials Transfer Agreement if there is potential for commercial application. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Won-Jae Lee ([email protected]). EXPERIMENTAL MODEL AND SUBJECT DETAILS Bacterial Strains Bacterial strains used in this study are summarized in the Key Resources Table. Fly Rearing The Drosophila melanogaster (w1118) was used as control animals. Conventional flies were reared at 25 C on Bloomington Drosophila stock center’s standard cornmeal medium. Germ-free animals or germ-free animals mono-associated with a specific bacterial strain Cell Host & Microbe 27, 1–13.e1–e6, March 11, 2020 e2

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were reared on autoclaved standard cornmeal medium or holidic medium (Piper et al., 2014). For the experiments of larval development, protein malnutrition diet containing low yeast (1.5% yeast) was used. In this medium, bokinin and propionic acid were omitted. Adult female flies (5- to 6-day-old) were used for our in vivo ROS measurement, metabolomics analyses and confocal image analyses. Bacterial Culture Conditions E. coli strain 10B or SM10l strains, cultured in Luria-Bertani (LB) medium at 37 C, were used for all cloning procedures. Ecc15 strains were cultured in LB or minimal M9 medium at 30 C. Different antibiotics were used at appropriate concentrations; ampicillin (100 mg/mL), kanamycin (30 mg/mL), apramycin (25 mg/mL), tetracycline (20 mg/mL), rifampicin (50 mg/mL) and chloramphenicol (35 mg/mL). Aceatobacteraceae strains were cultured in mannitol medium at 30 C. For the conjugation of A. pasteurianus, A. pasteurianus was cultured in YPGD medium (0.5% of glycerol, 0.5% of polypepton, 0.5% glucose and 0.5% yeast extract). Following conjugation, bacteria were selected in YPGD medium supplemented with tetracycline (20 mg/mL) and acetic acid (0.1%). V. harveyi MM32 was cultured in AB broth (Bassler et al., 1994) at 30 C. All other bacteria used in this study were cultured in LB medium at 37 C. METHOD DETAILS Metabolomic Analyses Gut luminal fluids were obtained from 150 dissected female midguts (5- to 6-days old) before and after oral infection (1010 CFU Ecc15 for 2 h). Dissected midguts were chopped into 4-5 fragments that were further incubated in ice-cold PBS solution for 10 min to obtain diffused luminal fluids. Diffused luminal fluids were centrifuged (1 min at 4,000 rpm) and filtered through 0.2 mm cellulose acetate filter (ADVANTEC) for the metabolomics analysis (Human Metabolome Technologies Inc.). The metabolites were measured from two biological replicates per sample [gut luminal fluids from non-infected gut (NI fluids) and those from infected gut (I fluids)] in the Cation and Anion modes by Agilent CE-TOFMS system (Agilent Technologies Inc.) Peaks detected in CE-TOFMS analysis were extracted using automatic integration software (MasterHands ver. 2.16.0.15 developed at Keio University) in order to obtain peak information including m/z, migration time (MT), and peak area. The abundances of 158 metabolites detected in NI and I fluids were calculated as their peak areas. We then identified differentially expressed (DE) metabolites between NI and I fluids as the ones with absolute log2-fold-changes > 1 (2-fold) or the ones detected only in either NI or I fluids. To identify metabolite groups (e.g., nucleotide and amino acid) enriched by the DE metabolites, we performed the enrichment analysis using Fisher’s exact test and selected the groups with p-value < 0.05. Quantitative Analysis of Metabolites Using LC-MS/MS Uridine, uracil and D-ribose from bacterial culture supernatants or gut luminal fluids (obtained from 30 midgus) were analyzed by using Agilent 6460 triple quadrupole mass spectrometry (Agilent Technologies, USA) with an electrospray interface. The separation was achieved on the column of XSelect HSS T3 2.5 mm (2.1 3 100 mm; 2.5 mm particle size; Island Waters). For the analysis of uridine or uracil, the samples were mixed with 1 mL of the internal standard (5-bromouracil, 500 mg /mL in water) and extracted with 1 mL of 9:1 ethyl acetate:isopropyl alcohol (v:v) by vortex-mixing for 5 min. Each sample was centrifuged for 10 min at 4 C with 13,000 rpm. An aliquot of the upper organic layer (800 mL) was evaporated to dryness under nitrogen gas at 36 C. The residue was dissolved in 50 mL of water containing 0.2% acetic acid and vortexed for 5 min. The supernatants of each sample and standard solutions were injected into the LC-MS/MS system. The column temperature was maintained at 30 C and the injection volume was set to 1 mL. The mobile phase (A) contained 0.2% acetic acid in water. The mobile phase (B) contained 0.2% acetic acid in acetonitrile. The flow rate was 0.2 mL/min in gradient mode: 0-5 min, 0% (B); 10 min, 15% (B); 10.1-15 min, 0% (B). For analysis of ribose, the samples were mixed with 50 mL of the internal standard (6-13C-fucose, 500 ng/mL in water), followed by the sequential addition of 100 mL of 5 mM 3-amino-9-ethylcarbazole (AEC) in methanol, 50 mL of 10 mM NaCNBH3 solution, and 10 mL of acetic acid (used as a catalyst). The reaction mixture was incubated at 60 C for 60 min. Each tube was then cooled on ice for 1 min. Then, 300 mL of water and 300 mL of dichloromethane-hexane (2:1, v/v) solvent were added. Each sample was vortexed for 5 min and centrifuged at 10,000 rpm for 5 min. A 100 mL aliquot of the upper aqueous phase was transferred to the LC-MS/MS system. The column temperature was maintained at 40 C, and the injection volumes were set at 1 mL. The mobile phase (A) was 0.1% formic acid in water and mobile phase (B) was 0.1% formic acid in acetonitrile. The flow rate was 0.2 mL/min in gradient mode: 0 min, 25% (B); 8 min, 100% (B); and 8.1-15 min, 25% (B). The total run time was 15 min. In Vivo ROS Measurement To measure DUOX-dependent ROS generation in vivo, The R2 region of the intestine was stained with HOCl-specific rhodaminebased R19S dye as described previously (Lee et al., 2013). DUOX activity was shown as percentage of R19S-positive gut.

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Comparative Genomics Amino acid sequences of G. morbifer for 52 proteins involved in nucleotide metabolic pathways were collected from the NCBI protein sequence database. To evaluate the conservation of these proteins in different bacteria, the sequences in G. morbifer were mapped into the protein sequences of the 9 different Acetobacteraceae members using BLASTP program (Altschul et al., 1990) and the proteins with the lowest E-values in the 9 bacteria were selected as potential orthologs. For each ortholog, the percent identity was calculated as the number of the matched amino acids divided by the total number of amino acids. Construction of Bacterial Strains In-frame deletion mutants of Ecc15 (Ecc15D4, Ecc15DRbsR, Ecc15DluxS and Ecc15DExpIR) and P. aeruginosa PAO1 were generated using the pDM4 suicide vector as described (Milton et al., 1996). For the complementation of the Ecc15D4 mutant, the full-length NH gene was cloned into the pTac3 vector under the control of a lac promoter to generate pTac3-Plac::NH. Ecc15D4_NH strain was generated by introducing pTac3-Plac::NH into Ecc15D4 strain. In-frame deletion mutant of G. morbifer was generated using homologous recombination. The disruption plasmids were demethylated by using dam-/dcm- E.coli (NEB, C2925i) and introduced into G. morbifer by electroporation. The deletion of the target gene was confirmed by PCR and sequencing analysis. For the complementation of G. morbiferDNH, the P. aeruginosa NH gene was placed under control of the constitutive PntpII promoter in pCM62 vector to generate pCM62-PntpII::NH. The plasmid pCM62-PntpII::NH was demethylated and introduced into G. morbiferDNH to generate G. morbiferDNH_NH. For the generation of A. pasteurianusNH_NupC strain, NupC and NH genes from G. morbifer were placed under control of the constitutive PntpII promoter in pCM62 to generate pCM62-PntpII::NH_NupC. A. pasteurianus was transformed with pCM62-PntpII:: NH_NupC by a triparental mating method using E. coli HB101/pKR2013 (Matsutani et al., 2013) to generate A. pasteurianusNH_NupC. Generation of Gnotobiotic Animals Germ-free animals were generated exactly as described previously (Ryu et al., 2008). To generate gnotobiotic Drosophila, bacterial cells (approximately 2x107 cells) were washed two times with sterile PBS and added to axenic food vials containing germ-free embryos. Lifespan Analysis To measure the lifespan, germ-free flies were mono-associated with G. morbiferDNH, G. morbiferDNH_NH, or A. pasteurianusNH_NupC. G. morbifer and A. pasteurianus carrying mock vector (pCM62 carrying tetracycline resistant marker) were used as controls. Gnotobiotic flies were transferred to fresh axenic medium supplemented with 20 mg/mL tetracycline every 4-5 days. Survival in three or more independent cohorts comprised of approximately 25 flies each was monitored over time. Apoptosis Assay Midguts from 45-day-old female gnotobiotic flies were used for the apoptosis assay. The midguts were dissected in PBS and the apoptosis assay was performed using Click-iTTM Plus TUNEL Assay Kit (Invitrogen Inc. C10618). The apoptosis index was determined the percentage of apoptotic cell numbers by total cell numbers. mRNA Sequencing and Data Analysis Ecc15 and Ecc15D4 were grown in the M9 medium supplemented with 1 mM uridine to late-exponential phase (OD600 = 1.5) at 30 C. RNA was extracted using TRIzol (Invitrogen) and treated with 2 U of TURBO DNase (Ambion) for 30 min at 37 C. Finally, RNA was purified and concentrated with the RNeasy MiniElute kit (QIAGEN). cDNA libraries were generated with Ribo-Zero Bacteria TruseqTM Stranded Total RNA H/M/R Prep Kit (Illumina) according to the manufacturer’s protocol. The cDNA libraries were sequenced using an Illumina Hi-Seq 2500. The Illumina CASAVA pipeline (version 1.8.2) was used for base calling, and cutadapt (version 2.6) was used for discarding reads containing the Illumina adaptor sequences. The resulting reads were mapped onto Ecc15 contig sequences using TopHat aligner (version 2.1.1) with the default options. After the alignment, we counted the mapped reads for gene features using HTSeq (version 0.6.1) and estimated fragments per kilobase of transcript per million fragments mapped (FPKM) by normalizing the counts using the library size and gene length. The raw data of mRNA sequencing were deposited at the Gene Expression Omnibus database (GSE140194). Identification of Differentially Expressed Genes (DEGs) We first identified ‘expressed’ genes as the ones with FPKM > 1 in either Ecc15 or Ecc15D4. For these expressed genes, the FPKM values were converted to log2-FPKM after adding one to the FPKM values. The log2-FPKM values were then normalized using the quantile normalization method (Bolstad et al., 2003). We then identified DEGs between Ecc15 and Ecc15D4 as the ones with absolute log2-fold-changes > 0.58 (1.5-fold). To identify cellular processes represented by the DEGs, we performed the enrichment analysis of gene ontology biological processes (GOBPs) for the genes using Fisher’s exact test and selected the GOBPs with p-value < 0.05.

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Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

Real-Time qPCR Analysis Bacteria were cultured in the M9 medium supplemented with 1 mM uridine. Fluorescence real-time PCR was performed to quantify gene expression using SYBR Green (Bioline). The rpoB gene was used as an internal control. Bioluminescence Assays The lux-based biosensors, E. coli pSB401 (Winson et al., 1998) and Vibrio harveyi MM32 (Freeman and Bassler, 1999), were used to determine AHL and AI-2 levels, respectively. Bioluminescence was measured using MicroLumat Plus LB 96V microplate luminometer (Berthold Technologies, UK). Data were expressed as relative light units (RLU) of luminescence values from each sample. E. coli pSB401 was incubated with the bacterial culture supernatant for 2 h, and bioluminescence imaging was taken using NightOWL II LB 983 imaging system (Berthold Technologies, UK). LC-MS/MS Analysis of 3-oxo-C6-HSL Ecc15 and Ecc15D4 were grown in the M9 medium supplemented with 1 mM uridine. Bacterial cells were centrifuged for 5 min at 6,000 rpm, and then culture supernatant (500 ml) was used for the quantitative analysis of 3-oxo-C6-HSL. Liquid-liquid extraction was performed to extract 3-oxo-C6-HSL from the bacterial culture supernatants. 500 mL of each of standard solution and filtrated bacteria culture supernatants were taken and mixed with 5 mL of C7-HSL (100 pg/mL in water) followed by extraction with 1 mL of ethyl acetate. A total of 800 mL of the upper organic layer was only taken and vaporized to dryness under nitrogen gas at 40 C. The residue was dissolved in 250 mL of water containing 0.1% formic acid. After sonication for 2 min and vortex-mixing for 5 min, the supernatants were analyzed using Agilent 6460 triple quadrupole mass spectrometry (Agilent Technologies, USA) with an electrospray interface. The separation was carried out in the column of XSelect HSS T3 2.5 mm (2.1 3 100 mm; 2.5 mm particle size; Island Waters) with maintaining the column temperature at 30 C. The mobile phase (A) was 0.1% formic acid in water and mobile phase (B) was 0.1% formic acid in acetonitrile. The optimized chromatographic method held the initial mobile phase composition 10% (B) for 1 min, then linearly increased to 90% (B) in 6 min, finally returned to 10% (B) in 7.5 min and equilibrated for 7.5 min. The total run time was 15 min. The retention times of 3-oxo-C6-HSL and C7-HSL (used as an internal standard) were 5.87 and 7.62, respectively. The qualitative and quantitative analysis was performed by Agilent Mass Hunter software (USA). Measurement of Pevf::gfp Expression In Vitro and In Vivo The evf promoter (608 bp) fused to GFP was cloned into pTac vector to generate pTac-Pevf::gfp. To analyze the evf gene expression in vitro and in vivo, pTac-Pevf::gfp was introduced into Ecc15WT, Ecc15D4, Ecc15DRbsR, Ecc15DExpIR and Ecc15DLuxS. Bacteria were cultured in M9 medium supplemented with 1mM uridine and GFP signals were analyzed by using SPECTRAL Ami X molecular imager (Spectral Instruments Imaging). To analyze bacterial evf gene expression in the Drosophila gut, larvae or adult flies were orally infected with 1010 CFUs of bacteria (Ecc15WT or Ecc15D4) carrying Pevf::GFP reporter for 2 h. The whole guts were dissected in PBS and formalin fixed. Confocal fluorescence images of the individual gut were analyzed by using a Zeiss LSM 700 confocal microscope. GFP levels in selected region of interest (midgut R2 region) were quantified using ZEN software. Bioluminescence Imaging of Live Drosophila Ecc15WT, Ecc15D4 and E. coli pSB401 at the mid-exponential phase (OD 0.5) were used for oral infection experiments. Female flies (5–6 days old) were orally infected with Ecc15WT or Ecc15D4 (5x109 CFUs) together with E. coli pSB401 (5x109 CFUs) for 1.5 h. Bioluminescence imaging of live animals was obtained using NightOWL II LB 983 imaging system (Berthold Technologies, UK). Measurement of Host Developmental Rate Gnotobiotic animals were generated by adding 2x107 CFUs of A. pomorum, Ecc15 or Ecc15 mutants to germ-free embryo. Formation of pupa was monitored every 12 h. Body size of gnotobiotic larvae was measured at the 108 h of development after egg laying. Measurement of Pyocyanin Production The amount of pyocyanin in P. aeruginosa culture supernatants was measured as described previously (Smith et al., 2003). P. aeruginosa was grown for 12 h at 37 C in LB medium. A 5 mL culture supernatant was extracted with 3 mL of chloroform and then re-extracted with 1 mL of 0.2 N HCl. The absorbance of extracted solution was measured at 380 nm. Measurement of Elastase Activity Elastase B activity of P. aeruginosa was measured as described previously (Smith et al., 2003). P. aeruginosa was grown for 8.5 h at 37 C in M9 medium supplemented with 1 mM uridine. Filtered culture supernatant was incubated with elastin–congo red solution for 12 h at 37 C with 200 rpm shaking, and then the elastase activity was measured at 495 nm.

e5 Cell Host & Microbe 27, 1–13.e1–e6, March 11, 2020

Please cite this article in press as: Kim et al., Bacterial Nucleoside Catabolism Controls Quorum Sensing and Commensal-to-Pathogen Transition in the Drosophila Gut, Cell Host & Microbe (2020), https://doi.org/10.1016/j.chom.2020.01.025

QUANTIFICATION AND STATISTICAL ANALYSIS GraphPad Prism 8.2.0 software was used for all analyses. All error bars indicate standard deviation of at least three independent experiments. Comparisons of two samples and multiple samples were performed by Student’s t test and one-way ANOVA (with Dunnett’s multiple comparison post-test), respectively. The log rank test of the Kaplan-Meier was used for the statistical analysis of fly survival experiments. P values of less than 0.05 were considered statistically significant. For each figure, the number of experimental replicates or samples as well as other information relevant for the statistical analysis are included in the accompanying legend. DATA AND CODE AVAILABILITY Data from this study have been deposited in the NCBI Gene Expression Omnibus (GEO) database under the following accession identifier: RNA-seq data (GEO: GSE140194). The accession number for the Ecc15 genome data reported in this paper is DDBJ/ ENA/GenBank: WNLC00000000.

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