Abstract
Vibrio cholerae is a human diarrheal pathogen and an estuarine organism that associates with both terrestrial and aquatic arthropods. Using the model terrestrial arthropod Drosophila melanogaster, we previously showed that V. cholerae adheres to the arthropod intestine and activates the enteroendocrine cell innate immune response to augment expression of the enteroendocrine peptide tachykinin (Tk). Here we show that enteroendocrine innate immune signaling and Tk promote V. cholerae colonization of the arthropod intestine. To investigate this, we measure the impact of TkRNAi on intestinal gene expression. In addition to decreasing expression of antimicrobial peptides and lipases, TkRNAi reduced the expression of chitinases and chitin-binding proteins including the small, secreted chitin-binding protein Peritrophin-15a (Peri-15a). Peri-15a interacts with chitin fibrils in the peritrophic matrix, a protective coating that overlies the arthropod intestinal epithelium. We uncover that Peri-15a is essential for robust V. cholerae colonization of the gut. Homologs of Peri-15a are widespread in both terrestrial and aquatic organisms including marine non-biting midges, marine copepods, rotifers, and cyanobacteria. We propose that V. cholerae activation of the enteroendocrine cell innate immune response and Peri-15a expression represents a strategy to maximize colonization of the arthropod host intestine.
Similar content being viewed by others
Data availability
The RNA-seq data generated in this study have been deposited in the NCBI repository (accession no. GSE294931). Remaining raw data generated are available at https://doi.org/10.5281/zenodo.18624207. Source data are provided with this paper.
References
Hsiao, A. & Zhu, J. Pathogenicity and virulence regulation of Vibrio cholerae at the interface of host-gut microbiome interactions. Virulence 11, 1582–1599 (2020).
Miyoshi, S. I. et al. Isolation of Vibrio cholerae and vibrio vulnificus from estuarine waters, and genotyping of V. vulnificus isolates using loop-mediated isothermal amplification. Microorganisms 12, 877 (2024).
Banerjee, S. K., Rutley, R. & Bussey, J. Diversity and dynamics of the Canadian Coastal vibrio community: an emerging trend detected in the temperate regions. J. Bacteriol. 200, e00787–17 (2018).
Lizarraga-Partida, M. L. et al. Association of Vibrio cholerae with plankton in coastal areas of Mexico. Environ. Microbiol. 11, 201–208 (2009).
de Magny, G. C. et al. Role of Zooplankton diversity in Vibrio cholerae population dynamics and in the incidence of cholera in the Bangladesh Sundarbans. Appl. Environ. Microbiol. 77, 6125–6132 (2011).
Broza, M., Gancz, H. & Kashi, Y. The association between non-biting midges and Vibrio cholerae. Environ. Microbiol. 10, 3193–3200 (2008).
Sela, R., Hammer, B. K. & Halpern, M. Quorum-sensing signaling by chironomid egg masses’ microbiota, affects haemagglutinin/protease (HAP) production by Vibrio cholerae. Mol. Ecol. 30, 1736–1746 (2021).
Huq, A. et al. Ecological relationships between Vibrio cholerae and planktonic crustacean copepods. Appl. Environ. Microbiol. 45, 275–283 (1983).
Pruzzo, C., Vezzulli, L. & Colwell, R. R. Global impact of Vibrio cholerae interactions with chitin. Environ. Microbiol. 10, 1400–1410 (2008).
Chiavelli, D. A., Marsh, J. W. & Taylor, R. K. The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton. Appl. Environ. Microbiol. 67, 3220–3225 (2001).
Hayes, C. A., Dalia, T. N. & Dalia, A. B. Systematic genetic dissection of chitin degradation and uptake in Vibrio cholerae. Environ. Microbiol. 19, 4154–4163 (2017).
Meibom, K. L. et al. The Vibrio cholerae chitin utilization program. Proc. Natl. Acad. Sci. USA 101, 2524–2529 (2004).
Meibom, K. L., Blokesch, M., Dolganov, N. A., Wu, C. Y. & Schoolnik, G. K. Chitin induces natural competence in Vibrio cholerae. Science 310, 1824–1827 (2005).
Blokesch, M. & Schoolnik, G. K. Serogroup conversion of Vibrio cholerae in aquatic reservoirs. PLoS Pathog. 3, e81 (2007).
Kamareddine, L. et al. Activation of Vibrio cholerae quorum sensing promotes survival of an arthropod host. Nat. Microbiol. 3, 243–252 (2018).
Wang, Z., Hang, S., Purdy, A. E. & Watnick, P. I. Mutations in the IMD pathway and mustard counter Vibrio cholerae suppression of intestinal stem cell division in Drosophila. MBio 4, e00337–00313 (2013).
Purdy, A. E. & Watnick, P. I. Spatially selective colonization of the arthropod intestine through activation of Vibrio cholerae biofilm formation. Proc. Natl. Acad. Sci. USA 108, 19737–19742 (2011).
Vanhove, A. S. et al. Vibrio cholerae ensures function of host proteins required for virulence through consumption of luminal methionine sulfoxide. PLoS Pathog. 13, e1006428 (2017).
Jugder, B. E. et al. Vibrio cholerae high cell density quorum sensing activates the host intestinal innate immune response. Cell Rep. 40, 111368 (2022).
Blow, N. S. et al. Vibrio cholerae infection of Drosophila melanogaster mimics the human disease cholera. PLoS Pathog. 1, e8 (2005).
Fast, D., Kostiuk, B., Foley, E. & Pukatzki, S. Commensal pathogen competition impacts host viability. Proc. Natl. Acad. Sci. USA 115, 7099–7104 (2018).
Xu, T., Novotny, A., Zamora-Terol, S., Hamback, P. A. & Winder, M. Dynamics of gut bacteria across different Zooplankton Genera in the Baltic Sea. Microb. Ecol. 87, 48 (2024).
Fast, D. et al. Vibrio cholerae-symbiont interactions inhibit intestinal repair in Drosophila. Cell Rep. 30, 1088–1100 e1085 (2020).
Wong, A. C., Vanhove, A. S. & Watnick, P. I. The interplay between intestinal bacteria and host metabolism in health and disease: lessons from Drosophila melanogaster. Dis. Model Mech. 9, 271–281 (2016).
Muthukrishnan, S., Merzendorfer, H., Arakane, Y. & Yang, Q. Chitin organizing and modifying enzymes and proteins involved in remodeling of the insect cuticle. Adv. Exp. Med. Biol. 1142, 83–114 (2019).
Li, H., Qi, Y. & Jasper, H. Preventing age-related decline of gut compartmentalization limits microbiota dysbiosis and extends lifespan. Cell Host Microbe 19, 240–253 (2016).
Watnick, P. I. & Jugder, B. E. Microbial control of intestinal homeostasis via enteroendocrine cell innate immune signaling. Trends Microbiol. 28, 141–149 (2020).
Jugder, B. E., Kamareddine, L. & Watnick, P. I. Microbiota-derived acetate activates intestinal innate immunity via the Tip60 histone acetyltransferase complex. Immunity 54, 1683–1697 e1683 (2021).
Kamareddine, L., Robins, W. P., Berkey, C. D., Mekalanos, J. J. & Watnick, P. I. The Drosophila immune deficiency pathway modulates enteroendocrine function and host metabolism. Cell Metab. 28, 449–462 e445 (2018).
Dutta, D. et al. Regional cell-specific transcriptome mapping reveals regulatory complexity in the adult Drosophila Midgut. Cell Rep. 12, 346–358 (2015).
Kleino, A. & Silverman, N. The Drosophila IMD pathway in the activation of the humoral immune response. Dev. Comp. Immunol. 42, 25–35 (2014).
Song, W., Veenstra, J. A. & Perrimon, N. Control of lipid metabolism by tachykinin in Drosophila. Cell Rep. 9, 40–47 (2014).
Almada, A. A. & Tarrant, A. M. Vibrio elicits targeted transcriptional responses from copepod hosts. FEMS Microbiol. Ecol. 92, fiw072 (2016).
Varadi, M. et al. AlphaFold Protein Structure Database in 2024: providing structure coverage for over 214 million protein sequences. Nucleic Acids Res. 52, D368–D375 (2024).
Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).
Perkins, L. A. et al. The transgenic RNAi project at Harvard Medical School: resources and validation. Genetics 201, 843–852 (2015).
Hanson, M. A. et al. Synergy and remarkable specificity of antimicrobial peptides in vivo using a systematic knockout approach. Elife 8, e44341 (2019).
Ozturk-Colak, A. et al. FlyBase: updates to the Drosophila genes and genomes database. Genetics 227, iyad211 (2024).
Hegedus, D., Erlandson, M., Gillott, C. & Toprak, U. New insights into peritrophic matrix synthesis, architecture, and function. Annu. Rev. Entomol. 54, 285–302 (2009).
Kuraishi, T., Binggeli, O., Opota, O., Buchon, N. & Lemaitre, B. Genetic evidence for a protective role of the peritrophic matrix against intestinal bacterial infection in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 108, 15966–15971 (2011).
Weiss, B. L., Savage, A. F., Griffith, B. C., Wu, Y. & Aksoy, S. The peritrophic matrix mediates differential infection outcomes in the tsetse fly gut following challenge with commensal, pathogenic, and parasitic microbes. J. Immunol. 193, 773–782 (2014).
Madeira, F. et al. The EMBL-EBI job dispatcher sequence analysis tools framework in 2024. Nucleic Acids Res. 52, W521–W525 (2024).
Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).
Wijffels, G. et al. A novel family of chitin-binding proteins from insect type 2 peritrophic matrix. cDNA sequences, chitin binding activity, and cellular localization. J. Biol. Chem. 276, 15527–15536 (2001).
Hu, D. Q. et al. The effect of group IV chitinase, HaCHT4, on the chitin content of the peritrophic matrix (PM) during larval growth and development of Helicoverpa armigera. Pest Manag. Sci. 78, 1815–1823 (2022).
Leader, D. P., Krause, S. A., Pandit, A., Davies, S. A. & Dow, J. A. T. FlyAtlas 2: a new version of the Drosophila melanogaster expression atlas with RNA-seq, miRNA-seq and sex-specific data. Nucleic Acids Res. 46, D809–D815 (2018).
Sun, S., Tay, Q. X., Kjelleberg, S., Rice, S. A. & McDougald, D. Quorum sensing-regulated chitin metabolism provides grazing resistance to Vibrio cholerae biofilms. ISME J. 9, 1812–1820 (2015).
Wucher, B. R. et al. Vibrio cholerae filamentation promotes chitin surface attachment at the expense of competition in biofilms. Proc. Natl. Acad. Sci. USA 116, 14216–14221 (2019).
Nahar, S. et al. Role of shrimp chitin in the ecology of toxigenic Vibrio cholerae and cholera transmission. Front. Microbiol. 2, 260 (2011).
Li, X. & Roseman, S. The chitinolytic cascade in Vibrios is regulated by chitin oligosaccharides and a two-component chitin catabolic sensor/kinase. Proc. Natl. Acad. Sci. USA 101, 627–631 (2004).
Markov, E. Y., Kulikalova, E. S., Urbanovich, L. Y., Vishnyakov, V. S. & Balakhonov, S. V. Chitin and products of its hydrolysis in Vibrio cholerae ecology. Biochemistry 80, 1109–1116 (2015).
Klancher, C. A., Yamamoto, S., Dalia, T. N. & Dalia, A. B. ChiS is a noncanonical DNA-binding hybrid sensor kinase that directly regulates the chitin utilization program in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 117, 20180–20189 (2020).
Hang, S. et al. The acetate switch of an intestinal pathogen disrupts host insulin signaling and lipid metabolism. Cell Host Microbe 16, 592–604 (2014).
Souza, C. P., Almeida, B. C., Colwell, R. R. & Rivera, I. N. The importance of chitin in the marine environment. Mar Biotechnol. 13, 823–830 (2011).
Kirn, T. J., Jude, B. A. & Taylor, R. K. A colonization factor links Vibrio cholerae environmental survival and human infection. Nature 438, 863–866 (2005).
Lloyd, C. J. et al. A peptide-binding domain shared with an Antarctic bacterium facilitates Vibrio cholerae human cell binding and intestinal colonization. Proc. Natl. Acad. Sci. USA 120, e2308238120 (2023).
Tamplin, M. L., Gauzens, A. L., Huq, A., Sack, D. A. & Colwell, R. R. Attachment of Vibrio cholerae serogroup O1 to zooplankton and phytoplankton of Bangladesh waters. Appl. Environ. Microbiol. 56, 1977–1980 (1990).
Shukla, B. N., Singh, D. V. & Sanyal, S. C. Attachment of non-culturable toxigenic Vibrio cholerae O1 and non-O1 and Aeromonas spp. to the aquatic arthropod Gerris spinolae and plants in the River Ganga, Varanasi. FEMS Immunol. Med. Microbiol. 12, 113–120 (1995).
Rawlings, T. K., Ruiz, G. M. & Colwell, R. R. Association of Vibrio cholerae O1 El Tor and O139 Bengal with the Copepods Acartia tonsa and Eurytemora affinis. Appl. Environ. Microbiol. 73, 7926–7933 (2007).
Mueller, R. S. et al. Vibrio cholerae strains possess multiple strategies for abiotic and biotic surface colonization. J. Bacteriol. 189, 5348–5360 (2007).
Schauer, S. et al. Dynamics of Vibrio cholerae abundance in Austrian saline lakes, assessed with quantitative solid-phase cytometry. Environ. Microbiol. 17, 4366–4378 (2015).
Turner, J. W., Malayil, L., Guadagnoli, D., Cole, D. & Lipp, E. K. Detection of Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae with respect to seasonal fluctuations in temperature and plankton abundance. Environ. Microbiol. 16, 1019–1028 (2014).
Wang, Y. G., Tseng, L. C., Lin, M. & Hwang, J. S. Vertical and geographic distribution of copepod communities at late summer in the Amerasian Basin, Arctic Ocean. PLoS ONE 14, e0219319 (2019).
Gluchowska, M. et al. Variations in the structural and functional diversity of zooplankton over vertical and horizontal environmental gradients en route to the Arctic Ocean through the Fram Strait. PLoS ONE 12, e0171715 (2017).
Zhang, Z. et al. Spatio-temporal variations of zooplankton and correlations with environmental parameters around Tiaowei Island, Fujian, China. Int. J. Environ. Res. Public Health 19, 12731 (2022).
Venkataramana, V., Gawade, L., Bharathi, M. D. & Sarma, V. Role of salinity on zooplankton assemblages in the tropical Indian estuaries during post monsoon. Mar. Pollut. Bull. 190, 114816 (2023).
Boldrocchi, G., Moussa Omar, Y., Rowat, D. & Bettinetti, R. First results on zooplankton community composition and contamination by some persistent organic pollutants in the Gulf of Tadjoura (Djibouti). Sci. Total Environ. 627, 812–821 (2018).
Mrak, P., Bogataj, U., Strus, J. & Znidarsic, N. Cuticle morphogenesis in crustacean embryonic and postembryonic stages. Arthropod. Struct. Dev. 46, 77–95 (2017).
Sochard, M. R., Wilson, D. F., Austin, B. & Colwell, R. R. Bacteria associated with the surface and gut of marine copepods. Appl. Environ. Microbiol. 37, 750–759 (1979).
Moussian, B. The apical plasma membrane of chitin-synthesizing epithelia. Insect Sci. 20, 139–146 (2013).
Rose, C. et al. An investigation into the protein composition of the teneral Glossina morsitans morsitans peritrophic matrix. PLoS Negl. Trop. Dis. 8, e2691 (2014).
Klancher, C. A. et al. The ChiS-family DNA-binding domain contains a cryptic helix-turn-helix variant. mBio 12, e03287–20 (2021).
Wong, E. et al. The Vibrio cholerae colonization factor GbpA possesses a modular structure that governs binding to different host surfaces. PLoS Pathog. 8, e1002373 (2012).
Loose, J. S., Forsberg, Z., Fraaije, M. W., Eijsink, V. G. & Vaaje-Kolstad, G. A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase. FEBS Lett. 588, 3435–3440 (2014).
Syed, K. A. et al. The Vibrio cholerae flagellar regulatory hierarchy controls expression of virulence factors. J. Bacteriol. 191, 6555–6570 (2009).
Song, W., Veenstra, J. A. & Perrimon, N. Control of lipid metabolism by tachykinin in Drosophila. Cell Rep. 30, 2461 (2020).
Haugo, A. J. & Watnick, P. I. Vibrio cholerae CytR is a repressor of biofilm development. Mol. Microbiol. 45, 471–483 (2002).
Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).
Patro, R., Duggal, G., Love, M. I., Irizarry, R. A. & Kingsford, C. Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods 14, 417–419 (2017).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Cornwell, M. et al. VIPER: Visualization Pipeline for RNA-seq, a Snakemake workflow for efficient and complete RNA-seq analysis. BMC Bioinform. 19, 135 (2018).
Acknowledgements
This work was supported by NIH R01AI158247 and NIH R01AI162701 to P.I.W. and NIH F31DK130254 to D. B. Anti-TK antibodies were generously provided by Jan Veenstra and AMP-deficient flies and the corresponding parental strain were generously provided by Bruno Lemaitre. The TK-Gal4 and NP1-Gal4 (Myo1A-Gal4) driver flies were kind gifts from Norbert Perrimon. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. Microscopy images were acquired at the Microscopy Resources on the North Quad (MicRoN) core at Harvard Medical School. We thank Paola Montero Lopis and Praju Vikas Anekal at the MicRoN core for providing expertise with image acquisition and quantification.
Author information
Authors and Affiliations
Contributions
D.B. and P.I.W. conceived the project. D.B., T.F.P. and P.I.W. designed the experiments. D.B. and T.F.P. contributed equally. D.B., T.F.P., L.F. and S. H. performed the experiments and acquired the data. D.B. T.F.P., L.F and P.I.W. analyzed the data and interpreted the results. T.F.P., D. B. and P.I.W. revised the work. D. B, T. F. P., and P.I.W. wrote the manuscript with input from all authors. P.I.W. supervised the project and acquired funding. All authors reviewed and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Peer review
Peer review information
Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Source data
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Barraza, D., Paulo, T.F., Findley, L. et al. A conserved, immune-regulated peritrophin promotes Vibrio cholerae colonization of the arthropod intestine. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70629-3
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41467-026-70629-3
