Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1’s role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.
References
[1]
Suez J, Korem T, Zeevi D, Zilberman-Schapira G, Thaiss CA, Maza O, et al. Artificial sweeteners induce glucose intolerance by altering the gut microbiota. Nature. 2014;514: 181–6. doi: 10.1038/nature13793. pmid:25231862
[2]
Spasova DS, Surh CD. Blowing on embers: commensal microbiota and our immune system. Front Immunol. 2014;5: 318. doi: 10.3389/fimmu.2014.00318. pmid:25120539
[3]
Moreno-Indias I, Cardona F, Tinahones FJ, Queipo-Ortu？o MI. Impact of the gut microbiota on the development of obesity and type 2 diabetes mellitus. Front Microbiol. 2014;5: 190. doi: 10.3389/fmicb.2014.00190. pmid:24808896
[4]
DuPont HL. Adverse Effect of Lomotil Therapy in Shigellosis. JAMA J Am Med Assoc. American Medical Association; 1973;226: 1525. doi: 10.1001/jama.1973.03230130013006.
[5]
Lee W- J, Brey PT. How microbiomes influence metazoan development: insights from history and Drosophila modeling of gut-microbe interactions. Annu Rev Cell Dev Biol. 2013;29: 571–92. doi: 10.1146/annurev-cellbio-101512-122333. pmid:23808845
[6]
Kim S-H, Lee W-J. Role of DUOX in gut inflammation: lessons from Drosophila model of gut-microbiota interactions. Front Cell Infect Microbiol. 2014;3: 116. doi: 10.3389/fcimb.2013.00116. pmid:24455491
[7]
Buchon N, Broderick N a, Poidevin M, Pradervand S, Lemaitre B. Drosophila intestinal response to bacterial infection: activation of host defense and stem cell proliferation. Cell Host Microbe. Elsevier Ltd; 2009;5: 200–11. doi: 10.1016/j.chom.2009.01.003.
[8]
Lee K-A, Kim S-H, Kim E-K, Ha E-M, You H, Kim B, et al. Bacterial-derived uracil as a modulator of mucosal immunity and gut-microbe homeostasis in Drosophila. Cell. Elsevier Inc.; 2013;153: 797–811. doi: 10.1016/j.cell.2013.04.009.
[9]
Ha E-M, Lee K-A, Park SH, Kim S-H, Nam H-J, Lee H-Y, et al. Regulation of DUOX by the Galphaq-phospholipase Cbeta-Ca2+ pathway in Drosophila gut immunity. Dev Cell. 2009;16: 386–97. doi: 10.1016/j.devcel.2008.12.015. pmid:19289084
[10]
Ha E-M, Lee K-A, Seo YY, Kim S-H, Lim J-H, Oh B-H, et al. Coordination of multiple dual oxidase-regulatory pathways in responses to commensal and infectious microbes in drosophila gut. Nat Immunol. Nature Publishing Group; 2009;10: 949–57. doi: 10.1038/ni.1765.
[11]
Ha E-M, Oh C-T, Bae YS, Lee W-J. A direct role for dual oxidase in Drosophila gut immunity. Science (80-). 2005;847: 847–50. doi: 10.1126/science.1117311.
[12]
Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol. 2004;4: 181–9. doi: 10.1038/nri1312. pmid:15039755
[13]
Nozawa K, Kawabata-Shoda E, Doihara H, Kojima R, Okada H, Mochizuki S, et al. TRPA1 regulates gastrointestinal motility through serotonin release from enterochromaffin cells. Proc Natl Acad Sci U S A. 2009;106: 3408–13. doi: 10.1073/pnas.0805323106. pmid:19211797
[14]
Yano JM, Yu K, Donaldson GP, Shastri GG, Ann P, Ma L, et al. Indigenous Bacteria from the Gut Microbiota Regulate Host Serotonin Biosynthesis. Cell. 2015;161: 264–276. doi: 10.1016/j.cell.2015.02.047. pmid:25860609
[15]
LaJeunesse DR, Johnson B, Presnell JS, Catignas KK, Zapotoczny G. Peristalsis in the junction region of the Drosophila larval midgut is modulated by DH31 expressing enteroendocrine cells. BMC Physiol. 2010;10: 14. doi: 10.1186/1472-6793-10-14. pmid:20698983
[16]
Park J- H, Kwon JY. Heterogeneous expression of Drosophila gustatory receptors in enteroendocrine cells. PLoS One. 2011;6: e29022. doi: 10.1371/journal.pone.0029022. pmid:22194978
[17]
Veenstra JA. Peptidergic paracrine and endocrine cells in the midgut of the fruit fly maggot.—PubMed—NCBI. Cell Tissue Res. 2009;336: 309–323. doi: 10.1007/s00441-009-0769-y. pmid:19319573
[18]
Bessac BF, Sivula M, von Hehn CA, Escalera J, Cohn L, Jordt SE. TRPA1 is a major oxidant sensor in murine airway sensory neurons. J Clin Invest. 2008/04/10 ed. 2008;118: 1899–1910. doi: 10.1172/JCI34192. pmid:18398506
[19]
Kang K, Pulver SR, Panzano VC, Chang EC, Griffith LC, Theobald DL, et al. Analysis of Drosophila TRPA1 reveals an ancient origin for human chemical nociception. Nature. Nature Publishing Group; 2010;464: 597–600. doi: 10.1038/nature08848. pmid:20237474
[20]
Hamada FN, Rosenzweig M, Kang K, Pulver SR, Ghezzi A, Jegla TJ, et al. An internal thermal sensor controlling temperature preference in Drosophila. Nature. Macmillan Publishers Limited. All rights reserved; 2008;454: 217–220. doi: 10.1038/nature07001. pmid:18548007
[21]
Viswanath V, Story GM, Peier AM, Petrus MJ, Hwang SW, Patapoutian A, et al. Opposite thermosensor in fruitfly and mouse. Nature. 2003;423: 822–823. doi: 10.1038/423822a. pmid:12815418
[22]
Kang K, Panzano VC, Chang EC, Ni L, Dainis AM, Jenkins AM, et al. Modulation of TRPA1 thermal sensitivity enables sensory discrimination in Drosophila. Nature. Nature Publishing Group; 2012;481: 76–80. doi: 10.1038/nature10715.
[23]
Du EJ, Ahn TJ, Choi MS, Kwon I, Kim H- W, Kwon JY, et al. The Mosquito Repellent Citronellal Directly Potentiates Drosophila TRPA1, Facilitating Feeding Suppression. Mol Cells. 2015;38: 911–7. doi: 10.14348/molcells.2015.0215. pmid:26447139
[24]
Kwon Y, Kim SH, Ronderos DS, Lee Y, Akitake B, Woodward OM, et al. Drosophila TRPA1 channel is required to avoid the naturally occurring insect repellent citronellal. Curr Biol. 2010;20: 1672–8. doi: 10.1016/j.cub.2010.08.016. pmid:20797863
[25]
Zhong L, Bellemer A, Yan H, Ken H, Jessica R, Hwang RY, et al. Thermosensory and non-thermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat sensor domains of a thermoTRP channel. Cell Rep. Tracey et al.; 2012;1: 43–55. doi: 10.1016/j.celrep.2011.11.002.
[26]
Celniker SE, Dillon LAL, Gerstein MB, Gunsalus KC, Henikoff S, Karpen GH, et al. Unlocking the secrets of the genome. Nature. 2009;459: 927–30. doi: 10.1038/459927a. pmid:19536255
[27]
Buchon N, Osman D, David FP a, Fang HY, Boquete J-P, Deplancke B, et al. Morphological and molecular characterization of adult midgut compartmentalization in Drosophila. Cell Rep. The Authors; 2013;3: 1725–38. doi: 10.1016/j.celrep.2013.04.001.
[28]
Rosenzweig M, Kang K, Garrity PA. Distinct TRP channels are required for warm and cool avoidance in Drosophila melanogaster. Proc Natl Acad Sci U S A. 2008;105: 14668–73. doi: 10.1073/pnas.0805041105. pmid:18787131
[29]
Kim SH, Lee Y, Akitake B, Woodward OM, Guggino WB, Montell C. Drosophila TRPA1 channel mediates chemical avoidance in gustatory receptor neurons. Proc Natl Acad Sci U S A. 2010;107: 8440–5. doi: 10.1073/pnas.1001425107. pmid:20404155
[30]
Wayland MT, Defaye A, Rocha J, Jayaram SA, Royet J, Miguel-Aliaga I, et al. Spotting the differences: Probing host/microbiota interactions with a dedicated software tool for the analysis of faecal outputs in Drosophila. J Insect Physiol. Elsevier Ltd; 2014;69: 126–135. doi: 10.1016/j.jinsphys.2014.05.023. pmid:24907675
[31]
Cognigni P, Bailey AP, Miguel-aliaga I. Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis. Cell Metab. Elsevier Inc.; 2011;13: 92–104. doi: 10.1016/j.cmet.2010.12.010. pmid:21195352
[32]
Hinman A, Chuang H-HH, Bautista DM, Julius D. TRP channel activation by reversible covalent modification. Proc Natl Acad Sci U S A. 2006/12/14 ed. 2006;103: 19564–19568. [pii] doi: 10.1073/pnas.0609598103. pmid:17164327
[33]
Chen X, Lee K-A, Ha E-M, Lee KM, Seo YY, Choi HK, et al. A specific and sensitive method for detection of hypochlorous acid for the imaging of microbe-induced HOCl production. Chem Commun (Camb). 2011;47: 4373–5. doi: 10.1039/c1cc10589b.
[34]
Lee K-S, Iijima-Ando K, Iijima K, Lee W-J, Lee JH, Yu K, et al. JNK/FOXO-mediated neuronal expression of fly homologue of peroxiredoxin II reduces oxidative stress and extends life span. J Biol Chem. 2009;284: 29454–61. doi: 10.1074/jbc.M109.028027. pmid:19720829
[35]
Hedengren M, Dushay MS, Ando I, Ekengren S, Wihlborg M, Hultmark D. Relish, a Central Factor in the Control of Humoral but Not Cellular Immunity in Drosophila. Mol Cell. 1999;4: 827–837. doi: 10.1016/S1097-2765(00)80392-5. pmid:10619029
Berg JM, Tymoczko JL, Stryer L. Glycolysis Is an Energy-Conversion Pathway in Many Organisms. W H Freeman; 2002.
[42]
Parker BW, Schwessinger EA, Jakob U, Gray MJ. The RclR protein is a reactive chlorine-specific transcription factor in Escherichia coli. J Biol Chem. 2013;288: 32574–84. doi: 10.1074/jbc.M113.503516. pmid:24078635
[43]
Drazic A, Miura H, Peschek J, Le Y, Bach NC, Kriehuber T, et al. Methionine oxidation activates a transcription factor in response to oxidative stress. Proc Natl Acad Sci U S A. 2013;110: 9493–8. doi: 10.1073/pnas.1300578110. pmid:23690622
[44]
Gray MJ, Wholey W-Y, Jakob U. Bacterial responses to reactive chlorine species. Annu Rev Microbiol. 2013;67: 141–60. doi: 10.1146/annurev-micro-102912-142520. pmid:23768204
[45]
Ha E-M, Oh C-T, Ryu J-H, Bae Y-S, Kang S-W, Jang I-H, et al. An antioxidant system required for host protection against gut infection in Drosophila. Dev Cell. 2005;8: 125–32. doi: 10.1016/j.devcel.2004.11.007. pmid:15621536
[46]
Zhou Y, Suzuki Y, Uchida K, Tominaga M. Identification of a splice variant of mouse TRPA1 that regulates TRPA1 activity. Nat Commun. Nature Publishing Group; 2013;4: 2399. doi: 10.1038/ncomms3399.
[47]
Guntur AR, Gu P, Takle K, Chen J, Xiang Y, Yang C-H. Drosophila TRPA1 isoforms detect UV light via photochemical production of H 2 O 2. Proc Natl Acad Sci. 2015; 201514862. doi: 10.1073/pnas.1514862112.
[48]
Ringel Y, Maharshak N. Intestinal microbiota and immune function in the pathogenesis of irritable bowel syndrome. Am J Physiol Gastrointest Liver Physiol. 2013;305: G529–41. doi: 10.1152/ajpgi.00207.2012. pmid:23886861
[49]
Groth AC, Fish M, Nusse R, Calos MP. Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31. Genetics. 2004;166: 1775–82. pmid:15126397 doi: 10.1534/genetics.166.4.1775
[50]
Markstein M, Pitsouli C, Villalta C, Celniker SE, Perrimon N. Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat Genet. 2008;40: 476–83. doi: 10.1038/ng.101. pmid:18311141
[51]
Ni J-Q, Markstein M, Binari R, Pfeiffer B, Liu L-P, Villalta C, et al. Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster. Nat Methods. Nature Publishing Group; 2008;5: 49–51. doi: 10.1038/nmeth1146. pmid:18084299
[52]
Ja WW, Carvalho GB, Mak EM, de la Rosa NN, Fang AY, Liong JC, et al. Prandiology of Drosophila and the CAFE assay. Proc Natl Acad Sci U S A. 2007;104: 8253–6. doi: 10.1073/pnas.0702726104. pmid:17494737
