The Serotonin Neurotransmitter Modulates Virulence of Enteric Pathogens

•Enterohemorrhagic E. coli (EHEC) and C. rodentium sense serotonin in the gut•Bacterial CpxA acts as a serotonin sensor•Serotonin decreases virulence of EHEC in vitro and C. rodentium during murine infection•Genetic and pharmacological changes in gut serotonin modulate C. rodentium disease The gut-brain axis is crucial to microbial-host interactions. The neurotransmitter serotonin is primarily synthesized in the gastrointestinal (GI) tract, where it is secreted into the lumen and subsequently removed by the serotonin transporter, SERT. Here, we show that serotonin decreases virulence gene expression by enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium, a murine model for EHEC. The membrane-bound histidine sensor kinase, CpxA, is a bacterial serotonin receptor. Serotonin induces dephosphorylation of CpxA, which inactivates the transcriptional factor CpxR controlling expression of virulence genes, notably those within the locus of enterocyte effacement (LEE). Increasing intestinal serotonin by genetically or pharmacologically inhibiting SERT decreases LEE expression and reduces C. rodentium loads. Conversely, inhibiting serotonin synthesis increases pathogenesis and decreases host survival. As other enteric bacteria contain CpxA, this signal exploitation may be engaged by other pathogens. Additionally, repurposing serotonin agonists to inhibit CpxA may represent a potential therapeutic intervention for enteric bacteria. The gut-brain axis is crucial to microbial-host interactions. The neurotransmitter serotonin is primarily synthesized in the gastrointestinal (GI) tract, where it is secreted into the lumen and subsequently removed by the serotonin transporter, SERT. Here, we show that serotonin decreases virulence gene expression by enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium, a murine model for EHEC. The membrane-bound histidine sensor kinase, CpxA, is a bacterial serotonin receptor. Serotonin induces dephosphorylation of CpxA, which inactivates the transcriptional factor CpxR controlling expression of virulence genes, notably those within the locus of enterocyte effacement (LEE). Increasing intestinal serotonin by genetically or pharmacologically inhibiting SERT decreases LEE expression and reduces C. rodentium loads. Conversely, inhibiting serotonin synthesis increases pathogenesis and decreases host survival. As other enteric bacteria contain CpxA, this signal exploitation may be engaged by other pathogens. Additionally, repurposing serotonin agonists to inhibit CpxA may represent a potential therapeutic intervention for enteric bacteria. The gastrointestinal (GI) tract is highly innervated, and neurotransmitters are prominent in this environment. Neurotransmitters and the microbiota are intrinsically connected. Several studies link the composition of the microbiota with changes in behavior and neurological diseases, such as autism, as well as influencing the levels of active neurotransmitters in the GI tract (Hsiao et al., 2013Hsiao E.Y. McBride S.W. Hsien S. Sharon G. Hyde E.R. McCue T. Codelli J.A. Chow J. Reisman S.E. Petrosino J.F. et al.Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders.Cell. 2013; 155: 1451-1463Abstract Full Text Full Text PDF PubMed Scopus (1919) Google Scholar, Reigstad et al., 2015Reigstad C.S. Salmonson C.E. Rainey 3rd, J.F. Szurszewski J.H. Linden D.R. Sonnenburg J.L. Farrugia G. Kashyap P.C. Gut microbes promote colonic serotonin production through an effect of short-chain fatty acids on enterochromaffin cells.FASEB J. 2015; 29: 1395-1403Crossref PubMed Scopus (539) Google Scholar, Sampson and Mazmanian, 2015Sampson T.R. Mazmanian S.K. Control of brain development, function, and behavior by the microbiome.Cell Host Microbe. 2015; 17: 565-576Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). Moreover, neurotransmitters also modify bacterial behavior, and impact virulence of many pathogens (Kendall and Sperandio, 2016Kendall M.M. Sperandio V. What a dinner party! Mechanisms and functions of interkingdom signaling in host-pathogen associations.mBio. 2016; 7: e01748Crossref PubMed Scopus (65) Google Scholar). Neurotransmitters alter gut physiology by modulating intestinal smooth muscle contraction, submucosal blood flow, barrier function, immune responses, and chloride and potassium secretion (Hörger et al., 1998Hörger S. Schultheiss G. Diener M. Segment-specific effects of epinephrine on ion transport in the colon of the rat.Am. J. Physiol. 1998; 275: G1367-G1376PubMed Google Scholar). The microbiota induces biosynthesis of serotonin (Yano et al., 2015Yano J.M. Yu K. Donaldson G.P. Shastri G.G. 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Microbiol. 2006; 8: 646-660Crossref PubMed Scopus (63) Google Scholar), the effect that serotonin has on bacterial pathogenesis remains elusive. This is particularly significant for enteric pathogens because 90% of serotonin is synthesized in the GI tract (Camilleri, 2009Camilleri M. Serotonin in the gastrointestinal tract.Curr. Opin. Endocrinol. Diabetes Obes. 2009; 16: 53-59Crossref PubMed Scopus (108) Google Scholar). The mammalian gut has a rich chemistry landscape, with metabolites, neurotransmitters, and signals derived from both the host and the microbiota influencing the biogeography of the GI tract. Enteric bacterial pathogens sense and respond to these info-chemicals in their environment in a manner that culminates in the most spatiotemporal-efficient expression of their virulence genes (Bäumler and Sperandio, 2016Bäumler A.J. Sperandio V. Interactions between the microbiota and pathogenic bacteria in the gut.Nature. 2016; 535: 85-93Crossref PubMed Scopus (599) Google Scholar). The colon contains tryptophan derivatives, including the host-derived neurotransmitter serotonin (Esmaili et al., 2009Esmaili A. Nazir S.F. Borthakur A. Yu D. Turner J.R. Saksena S. Singla A. Hecht G.A. Alrefai W.A. Gill R.K. Enteropathogenic Escherichia coli infection inhibits intestinal serotonin transporter function and expression.Gastroenterology. 2009; 137: 2074-2083Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, Grøndahl et al., 1998Grøndahl M.L. Jensen G.M. Nielsen C.G. Skadhauge E. Olsen J.E. Hansen M.B. Secretory pathways in Salmonella Typhimurium-induced fluid accumulation in the porcine small intestine.J. Med. Microbiol. 1998; 47: 151-157Crossref PubMed Scopus (40) Google Scholar, Kordasti et al., 2004Kordasti S. Sjövall H. Lundgren O. Svensson L. Serotonin and vasoactive intestinal peptide antagonists attenuate rotavirus diarrhoea.Gut. 2004; 53: 952-957Crossref PubMed Scopus (64) Google Scholar, O'Hara et al., 2006O'Hara J.R. Skinn A.C. MacNaughton W.K. Sherman P.M. Sharkey K.A. Consequences of Citrobacter rodentium infection on enteroendocrine cells and the enteric nervous system in the mouse colon.Cell. Microbiol. 2006; 8: 646-660Crossref PubMed Scopus (63) Google Scholar) (Figure 1A) and the microbiota-derived indole (Kumar and Sperandio, 2019Kumar A. Sperandio V. Indole signaling at the host-microbiota-pathogen interface.mBio. 2019; 10 (e01031-19)Crossref Scopus (66) Google Scholar). Indole is absorbed by host cells and strengthens the integrity of the intestinal barrier, being regarded as a beneficial chemical cue within microbial and/or host interactions (Bansal et al., 2010Bansal T. Alaniz R.C. Wood T.K. Jayaraman A. The bacterial signal indole increases epithelial-cell tight-junction resistance and attenuates indicators of inflammation.Proc. Natl. Acad. Sci. USA. 2010; 107: 228-233Crossref PubMed Scopus (460) Google Scholar). Indole also influences the gut-brain axis. Intestinal indole produced by the microbiota can accumulate in the brain leading to behavioral changes (Jaglin et al., 2018Jaglin M. Rhimi M. Philippe C. Pons N. Bruneau A. Goustard B. Daugé V. Maguin E. Naudon L. Rabot S. Indole, a signaling molecule produced by the gut microbiota, negatively impacts emotional behaviors in rats.Front. Neurosci. 2018; 12: 216Crossref PubMed Scopus (96) Google Scholar). Serotonin is synthesized by enterochromaffin cells by the enzyme tryptophan hydroxylase (TpH1). Upon its synthesis, serotonin is released into the lamina propria and is also secreted into the lumen. Serotonin signaling in the intestinal mucosa is terminated by its removal by SERT, which is expressed by enterocytes (Camilleri, 2009Camilleri M. Serotonin in the gastrointestinal tract.Curr. Opin. Endocrinol. Diabetes Obes. 2009; 16: 53-59Crossref PubMed Scopus (108) Google Scholar). The neurotransmitters epinephrine and norepinephrine have been previously shown to be sensed by bacterial membrane-bound histidine sensor kinases (HKs) (Clarke et al., 2006Clarke M.B. Hughes D.T. Zhu C. Boedeker E.C. Sperandio V. The QseC sensor kinase: a bacterial adrenergic receptor.Proc. Natl. Acad. Sci. USA. 2006; 103: 10420-10425Crossref PubMed Scopus (427) Google Scholar, Reading et al., 2009Reading N.C. Rasko D.A. Torres A.G. Sperandio V. The two-component system QseEF and the membrane protein QseG link adrenergic and stress sensing to bacterial pathogenesis.Proc. Natl. Acad. Sci. USA. 2009; 106: 5889-5894Crossref PubMed Scopus (113) Google Scholar). HKs are the main sensory receptors in bacteria. They have both kinase and phosphatase activity. Upon sensing their signals, they can either be activated (kinase state) or deactivated (phosphatase state). As kinases they phosphorylate their cognate response regulators (RRs), the majority of which are transcriptional factors. Phosphorylation of RRs leads to conformational changes that increase their affinity to their DNA targets enhancing DNA binding. Termination of signaling occurs by auto-dephosphorylation of the HK, leading to dephosphorylation of the RR (Jung et al., 2012Jung K. Fried L. Behr S. Heermann R. Histidine kinases and response regulators in networks.Curr. Opin. Microbiol. 2012; 15: 118-124Crossref PubMed Scopus (162) Google Scholar). Here, we show that serotonin decreases virulence gene expression of the human enteric pathogen enterohemorrhagic E. coli (EHEC), as well as the murine pathogen C. rodentium, which is extensively employed as a surrogate infection model for EHEC (Borenshtein et al., 2008Borenshtein D. McBee M.E. Schauer D.B. Utility of the Citrobacter rodentium infection model in laboratory mice.Curr. Opin. Gastroenterol. 2008; 24: 32-37Crossref PubMed Scopus (104) Google Scholar). Transcriptomic, genetic, and biochemical studies identified the HK CpxA as the serotonin receptor. CpxA also senses the bacterial signal indole, which is chemically similar to serotonin, comprising another example of inter-kingdom chemical signaling (Kumar and Sperandio, 2019Kumar A. Sperandio V. Indole signaling at the host-microbiota-pathogen interface.mBio. 2019; 10 (e01031-19)Crossref Scopus (66) Google Scholar). Utilizing knock out animals and pharmacological inhibitors for TpH1 and SERT, we also demonstrated that elevated intestinal levels of serotonin suppress virulence of C. rodentium, while lowered levels promote pathogenesis. It has been reported that the levels of serotonin vary within different GI compartments (Camilleri, 2009Camilleri M. Serotonin in the gastrointestinal tract.Curr. Opin. Endocrinol. Diabetes Obes. 2009; 16: 53-59Crossref PubMed Scopus (108) Google Scholar). Moreover, the microbiota itself induces the synthesis of serotonin (Fung et al., 2019Fung T.C. Vuong H.E. Luna C.D.G. Pronovost G.N. Aleksandrova A.A. Riley N.G. Vavilina A. McGinn J. Rendon T. Forrest L.R. Hsiao E.Y. Intestinal serotonin and fluoxetine exposure modulate bacterial colonization in the gut.Nat. Microbiol. 2019; 4: 2064-2073Crossref PubMed Scopus (128) Google Scholar, Hsiao et al., 2013Hsiao E.Y. McBride S.W. Hsien S. Sharon G. Hyde E.R. McCue T. Codelli J.A. Chow J. Reisman S.E. Petrosino J.F. et al.Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders.Cell. 2013; 155: 1451-1463Abstract Full Text Full Text PDF PubMed Scopus (1919) Google Scholar). Here, we show that the levels of serotonin in the colon tissues of mice remains similar at a mean concentration of 30 μM, whether or not they had their microbiota depleted through antibiotic treatment (Figure 1A). In contrast depletion of the microbiota has a profound effect on the luminal levels of serotonin, which decrease form a mean concentration of 5 μM in microbiota replete to 500 nM in microbiota-depleted animals (Figure 1A). Because serotonin is structurally similar to indole, and indole decreases expression of virulence genes in EHEC (Kumar and Sperandio, 2019Kumar A. Sperandio V. Indole signaling at the host-microbiota-pathogen interface.mBio. 2019; 10 (e01031-19)Crossref Scopus (66) Google Scholar), we investigated whether serotonin would have a similar effect. EHEC colonizes the human colon leading to outbreaks of bloody diarrhea and hemolytic uremic syndrome (HUS) worldwide (Kaper et al., 2004Kaper J.B. Nataro J.P. Mobley H.L. Pathogenic Escherichia coli.Nat. Rev. Microbiol. 2004; 2: 123-140Crossref PubMed Scopus (3157) Google Scholar). EHEC virulence determinants include the production of the potent Shiga toxin (Stx) that causes HUS, and genes necessary for the attaching and effacing (AE) lesion formation on enterocytes. AE lesion formation requires genes contained within the locus of enterocyte effacement (LEE) pathogenicity island (PI) (McDaniel et al., 1995McDaniel T.K. Jarvis K.G. Donnenberg M.S. Kaper J.B. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens.Proc. Natl. Acad. Sci. USA. 1995; 92: 1664-1668Crossref PubMed Scopus (1016) Google Scholar) (Figure 1B). The LEE region contains five major operons: LEE1–5 (Mellies et al., 1999Mellies J.L. Elliott S.J. Sperandio V. Donnenberg M.S. Kaper J.B. The per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler).Mol. Microbiol. 1999; 33: 296-306Crossref PubMed Scopus (322) Google Scholar), which encode the Ler transcriptional activator of all LEE genes, a type III secretion system (T3SS) that is a molecular syringe that injects bacterial effectors within the host cell, an adhesin (intimin that is encoded by the eae gene), and its receptor Tir that is itself translocated by the T3SS into the host cells (Stevens and Frankel, 2014Stevens M.P. Frankel G.M. The locus of enterocyte Effacement and associated virulence factors of enterohemorrhagic Escherichia coli.Microbiol. Spectr. 2014; 2 (EHEC–0007)Crossref PubMed Scopus (68) Google Scholar). Expression of the LEE genes are decreased in the presence of physiological concentrations of serotonin (10 μM and 100 nM) as investigated through quantitative real-time PCR (Figures 1C–1E), western (Figure 1F) and northern blots (Figure 1G). Because serotonin decreases expression of the ler gene (Figure 1F) that encodes the transcription activator of all other LEE genes, it is congruent that expression of espA, espB, eae, and tir are also decreased by serotonin (Figures 1C–1G). Given that LEE expression is necessary for AE lesion formation, as expected, serotonin also decreases formation of these lesions by EHEC on epithelial cells (Figures 1H and 1I). To investigate the impact of serotonin in the EHEC transcriptome, RNA-seq was performed. Serotonin treatment changed the expression of 68 genes, with 55 being downregulated and 13 upregulated (GSE144795). Within the downregulated genes are the LEE genes (Figures 2A and S1). Notably, these small number of genes altered by serotonin in the EHEC transcriptome did not impact its growth (Figures S2A and S2B), or the expression of the flagella genes and motility (Figures S2C and S2D), suggesting that it is mostly involved in regulation of virulence gene expression. Previous studies have linked neurotransmitter sensing by bacterial pathogens to membrane-bound HKs (Clarke et al., 2006Clarke M.B. Hughes D.T. Zhu C. Boedeker E.C. Sperandio V. The QseC sensor kinase: a bacterial adrenergic receptor.Proc. Natl. Acad. Sci. USA. 2006; 103: 10420-10425Crossref PubMed Scopus (427) Google Scholar, Moreira et al., 2010Moreira C.G. Weinshenker D. Sperandio V. QseC mediates Salmonella enterica serovar typhimurium virulence in vitro and in vivo.Infect. Immun. 2010; 78: 914-926Crossref PubMed Scopus (119) Google Scholar, Reading et al., 2009Reading N.C. Rasko D.A. Torres A.G. Sperandio V. The two-component system QseEF and the membrane protein QseG link adrenergic and stress sensing to bacterial pathogenesis.Proc. Natl. Acad. Sci. USA. 2009; 106: 5889-5894Crossref PubMed Scopus (113) Google Scholar). HKs have both kinase and phosphatase activities, and can either increase or decrease their phosphorylation upon sensing a signal (Jung et al., 2012Jung K. Fried L. Behr S. Heermann R. Histidine kinases and response regulators in networks.Curr. Opin. Microbiol. 2012; 15: 118-124Crossref PubMed Scopus (162) Google Scholar). QseC and QseE, for example, have been shown to sense epinephrine and norepinephrine (Clarke et al., 2006Clarke M.B. Hughes D.T. Zhu C. Boedeker E.C. Sperandio V. The QseC sensor kinase: a bacterial adrenergic receptor.Proc. Natl. Acad. Sci. USA. 2006; 103: 10420-10425Crossref PubMed Scopus (427) Google Scholar, Moreira et al., 2010Moreira C.G. Weinshenker D. Sperandio V. QseC mediates Salmonella enterica serovar typhimurium virulence in vitro and in vivo.Infect. Immun. 2010; 78: 914-926Crossref PubMed Scopus (119) Google Scholar, Reading et al., 2009Reading N.C. Rasko D.A. Torres A.G. Sperandio V. The two-component system QseEF and the membrane protein QseG link adrenergic and stress sensing to bacterial pathogenesis.Proc. Natl. Acad. Sci. USA. 2009; 106: 5889-5894Crossref PubMed Scopus (113) Google Scholar). Small molecules that are sensed by bacteria for differential gene regulation also tend to differentially regulate expression of their HK (Clarke et al., 2006Clarke M.B. Hughes D.T. Zhu C. Boedeker E.C. Sperandio V. The QseC sensor kinase: a bacterial adrenergic receptor.Proc. Natl. Acad. Sci. USA. 2006; 103: 10420-10425Crossref PubMed Scopus (427) Google Scholar). Using the same concept, we searched the serotonin transcriptome for HKs that were differentially regulated by this neurotransmitter. The only HK whose expression was significantly regulated by serotonin was CpxA (Figures 2A, 2B, and S1B). CpxA senses indole (Kumar and Sperandio, 2019Kumar A. Sperandio V. Indole signaling at the host-microbiota-pathogen interface.mBio. 2019; 10 (e01031-19)Crossref Scopus (66) Google Scholar), a tryptophan derived bacterial signal (Wang et al., 2001Wang D. Ding X. Rather P.N. Indole can act as an extracellular signal in Escherichia coli.J. Bacteriol. 2001; 183: 4210-4216Crossref PubMed Scopus (192) Google Scholar) that is structurally similar to serotonin, and has also been shown to decrease LEE gene expression in EHEC (Kumar and Sperandio, 2019Kumar A. Sperandio V. Indole signaling at the host-microbiota-pathogen interface.mBio. 2019; 10 (e01031-19)Crossref Scopus (66) Google Scholar). Using genetic and biochemical phenotypic analyses, we showed that CpxA also senses the mammalian neurotransmitter serotonin (Figures 2C–2H). Transcription of espA is decreased by serotonin in EHEC WT but is not altered in ΔcpxA (Figure 2C). Moreover, serotonin, but not tryptophan, decreases CpxA autophosphorylation (Figures 2D and 2E). Tritiated serotonin binds to CpxA reconstituted in liposomes (Figure 2F), while tryptophan does not bind to CpxA (Figure 2G). CpxA is part of the CpxRA two-component system, where CpxA is a HK that upon autophosphorylation, phosphotransfers its phosphate to the RR CpxR, which is a transcriptional factor that regulates gene expression. HKs can phosphorylate and dephosphorylate their RRs, and in its phosphorylated state CpxR binds to its DNA targets. Dephosphorylation of RRs inhibits their function (Jung et al., 2012Jung K. Fried L. Behr S. Heermann R. Histidine kinases and response regulators in networks.Curr. Opin. Microbiol. 2012; 15: 118-124Crossref PubMed Scopus (162) Google Scholar). In its active state, the CpxRA system activates transcription of the LEE genes, with transcription of espA being decreased in ΔcpxA and ΔcpxR, and restored upon complementation (Figure 2H). EspA expression is needed for AE lesion formation, and congruently ΔcpxA is defective for AE lesion formation (Figure 2I). CpxRA regulation occurs at the level of ler (the first gene within the LEE1 operon) transcription (Figures 2J and S3A–S3D), consequently, impacting transcription of all LEE genes, inasmuch as Ler is needed for their expression (Mellies et al., 1999Mellies J.L. Elliott S.J. Sperandio V. Donnenberg M.S. Kaper J.B. The per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler).Mol. Microbiol. 1999; 33: 296-306Crossref PubMed Scopus (322) Google Scholar). In its phosphorylated state, CpxR directly binds to and activates transcription of ler (LEE1) (Figures 2K and S3C). Dephosphorylation of RRs inhibits their function. Upon serotonin-dependent dephosphorylation of CpxA (Figures 2D and 2E), CpxR is also dephosphorylated, and the dephosphorylated RR can no longer bind to the ler regulatory region to activate its transcription. Consequently, by blocking activation of CpxA, serotonin inhibits LEE gene expression in EHEC (Figures 2B–2L). To gain insights into the structure-activity relationship (SAR) of serotonin signaling to EHEC, we also investigated whether other synthetic serotonin analogs decreased LEE gene expression. All of the analogs tested (tryptamine, 3-indole acetonitrile, 5-hydroxyindole acetic acid, 3-indole acetic acid, and 5-hydroxyindole) decreased LEE transcription (Figure S4), suggesting that minor chemical changes within the structure of serotonin does not ablate serotonin’s effect on LEE gene expression. This is congruent with CpxA also sensing indole (Kumar and Sperandio, 2019Kumar A. Sperandio V. Indole signaling at the host-microbiota-pathogen interface.mBio. 2019; 10 (e01031-19)Crossref Scopus (66) Google Scholar). These data show that CpxA biochemically converge inter-kingdom chemical signaling, with one receptor sensing both bacterial and host-derived chemically related signals. Serotonin also decreases expression of the LEE genes in C. rodentium in a CpxA-dependent manner (Figure S5A). C. rodentium is a natural murine pathogen that also harbors the LEE genes, colonizes the colon of mice leading to disease, and is extensively used as a surrogate murine infection model for EHEC, given that EHEC is unable to cause disease in mice (Luperchio and Schauer, 2001Luperchio S.A. Schauer D.B. Molecular pathogenesis of Citrobacter rodentium and transmissible murine colonic hyperplasia.Microbes Infect. 2001; 3: 333-340Crossref PubMed Scopus (212) Google Scholar). The utilization of the C. rodentium model capitalizes in merging the powerful genetically tractability of host and pathogen to unravel the mechanisms involved in host recognition and infection. However, a C. rodentium cpxRA deletion strain is heavily attenuated and cannot colonize mice (Thomassin et al., 2015Thomassin J.L. Giannakopoulou N. Zhu L. Gross J. Salmon K. Leclerc J.M. Daigle F. Le Moual H. 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