Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Vibrio cholerae residing in food vacuoles expelled by protozoa are more infectious in vivo


Vibrio cholerae interacts with many organisms in the environment, including heterotrophic protists (protozoa). Several species of protozoa have been reported to release undigested bacteria in expelled food vacuoles (EFVs) when feeding on some pathogens. While the production of EFVs has been reported, their biological role as a vector for the transmission of pathogens remains unknown. Here we report that ciliated protozoa release EFVs containing V. cholerae. The EFVs are stable, the cells inside them are protected from multiple stresses, and large numbers of cells escape when incubated at 37 °C or in the presence of nutrients. We show that OmpU, a major outer membrane protein positively regulated by ToxR, has a role in the production of EFVs. Notably, cells released from EFVs have growth and colonization advantages over planktonic cells both in vitro and in vivo. Our results suggest that EFVs facilitate V. cholerae survival in the environment, enhancing their infectious potential and may contribute to the dissemination of epidemic V. cholerae strains. These results improve our understanding of the mechanisms of persistence and the modes of transmission of V. cholerae and may further apply to other opportunistic pathogens that have been shown to be released by protists in EFVs.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Production of EFVs containing V. cholerae.
Fig. 2: Production of EFVs by different Vibrio spp. and ciliate wild-type strains.
Fig. 3: Number of EFVs produced by different V. cholerae mutants.
Fig. 4: Survival of V. cholerae cells in EFVs under stress and starvation conditions.
Fig. 5: Escape of V. cholerae from EFVs under different nutrient and temperature conditions.
Fig. 6: Competition index for in vitro and in vivo assays of V. cholerae EFVs versus planktonic V. cholerae and working model.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.


  1. Ali, M., Nelson, A. R., Lopez, A. L. & Sack, D. A. Updated global burden of cholera in endemic countries. PLoS Negl. Trop. Dis. 9, e0003832 (2015).

    PubMed  PubMed Central  Google Scholar 

  2. Colwell, R. & Huq, A. Marine ecosystems and cholera. Hydrobiologia 460, 141–145 (2001).

    Google Scholar 

  3. Martinelli Filho, J. E., Lopes, R. M., Rivera, I. N. G. & Colwell, R. R. Vibrio cholerae O1 detection in estuarine and coastal zooplankton. J. Plankton Res. 33, 51–62 (2010).

    Google Scholar 

  4. Nair, G. B. et al. Ecology of Vibrio cholerae in the freshwater environs of Calcutta, India. Microb. Ecol. 15, 203–215 (1988).

    CAS  PubMed  Google Scholar 

  5. Vezzulli, L., Pruzzo, C., Huq, A. & Colwell, R. R. Environmental reservoirs of Vibrio cholerae and their role in cholera. Environ. Microbiol. Rep. 2, 27–33 (2010).

    PubMed  Google Scholar 

  6. Colwell, R. R. et al. Reduction of cholera in Bangladeshi villages by simple filtration. Proc. Natl Acad. Sci. USA 100, 1051–1055 (2003).

    CAS  PubMed  Google Scholar 

  7. Acosta, C. J. et al. Cholera outbreak in southern Tanzania: risk factors and patterns of transmission. Emerg. Infect. Dis. 7, 583–587 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Rabbani, G. H. & Greenough, W. B. III. Food as a vehicle of transmission of cholera. J. Diarrhoeal Dis. Res. 17, 1–9 (1999).

    CAS  PubMed  Google Scholar 

  9. Berk, S. G. et al. Packaging of live Legionella pneumophila into pellets expelled by Tetrahymena spp. does not require bacterial replication and depends on a Dot/Icm-mediated survival mechanism. J. Appl. Environ. Microbiol. 74, 2187–2199 (2008).

    CAS  Google Scholar 

  10. Bouyer, S., Imbert, C., Rodier, M.-H. & Héchard, Y. Long-term survival of Legionella pneumophila associated with Acanthamoeba castellanii vesicles. Environ. Microbiol. 9, 1341–1344 (2007).

    CAS  PubMed  Google Scholar 

  11. Denoncourt, A. M., Paquet, V. E. & Charette, S. J. Potential role of bacteria packaging by protozoa in the persistence and transmission of pathogenic bacteria. Front. Microbiol. 5, 240 (2014).

    PubMed  PubMed Central  Google Scholar 

  12. Gourabathini, P., Brandl, M. T., Redding, K. S., Gunderson, J. H. & Berk, S. G. Interactions between food-borne pathogens and protozoa isolated from lettuce and spinach. Appl. Environ. Microbiol. 74, 2518–2525 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Koubar, M., Rodier, M.-H., Garduño, R. A. & Frère, J. Passage through Tetrahymena tropicalis enhances the resistance to stress and the infectivity of Legionella pneumophila. FEMS Microbiol. Lett. 325, 10–15 (2011).

    CAS  PubMed  Google Scholar 

  14. Marciano-Cabral, F. & Cabral, G. Acanthamoeba spp. as agents of disease in humans. Clin. Microbiol. Rev. 16, 273–307 (2003).

    PubMed  PubMed Central  Google Scholar 

  15. Paquet, V. E. & Charette, S. J. Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium discoideum: social amoebae can also package bacteria. FEMS Microbiol. Ecol. 92, fiw025 (2016).

    PubMed  Google Scholar 

  16. Raghu Nadhanan, R. & Thomas, C. J. Colpoda secrete viable Listeria monocytogenes within faecal pellets. Environ. Microbiol. 16, 396–404 (2013).

    PubMed  Google Scholar 

  17. Trigui, H., Paquet, V. E., Charette, S. J. & Faucher, S. P. Packaging of Campylobacter jejuni into multilamellar bodies by the ciliate Tetrahymena pyriformis. J. Appl. Environ. Microbiol. 82, 2783–2790 (2016).

    CAS  Google Scholar 

  18. Rehfuss, M. Y. M., Parker, C. T. & Brandl, M. T. Salmonella transcriptional signature in Tetrahymena phagosomes and role of acid tolerance in passage through the protist. ISME J. 5, 262–273 (2011).

    CAS  PubMed  Google Scholar 

  19. Berk, S. G., Ting, R. S., Turner, G. W. & Ashburn, R. J. Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl. Environ. Microbiol. 64, 279–286 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Vaitkevicius, K. et al. A Vibrio cholerae protease needed for killing of Caenorhabditis elegans has a role in protection from natural predator grazing. Proc. Natl Acad. Sci. USA 103, 9280–9285 (2006).

    CAS  PubMed  Google Scholar 

  21. Sun, S., Tay, Q. X. M., Kjellberg, S., Rice, S. A. & McDougald, D. Quorum sensing-regulated chitin metabolism provides grazing resistance to Vibrio cholerae biofilms. ISME J. 9, 1812–1820 (2015).

    PubMed  PubMed Central  Google Scholar 

  22. Noorian, P. et al. Pyomelanin produced by Vibrio cholerae confers resistance to predation by Acanthamoeba castellanii. FEMS Microbiol. Ecol. 93, fix147 (2017).

    PubMed Central  Google Scholar 

  23. Sun, S., Kjelleberg, S. & McDougald, D. Relative contributions of Vibrio polysaccharide and quorum sensing to the resistance of Vibrio cholerae to predation by heterotrophic protists. PLoS ONE 8, e56338 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Casper-Lindley, C. & Yildiz, F. H. VpsT is a transcriptional regulator required for expression of vps biosynthesis genes and the development of rugose colonial morphology in Vibrio cholerae O1 El Tor. J. Bacteriol. 186, 1574–1578 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Jobling, M. G. & Holmes, R. K. Characterization of hapR, a positive regulator of the Vibrio cholerae HA/protease gene hap, and its identification as a functional homologue of the Vibrio harveyi luxR gene. Mol. Microbiol. 26, 1023–1034 (1997).

    CAS  PubMed  Google Scholar 

  26. Pratt, J. T., McDonough, E. & Camilli, A. PhoB regulates motility, biofilms, and cyclic di-GMP in Vibrio cholerae. J. Bacteriol. 191, 6632–6642 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Miller, V. L. & Mekalanos, J. J. Synthesis of cholera toxin is positively regulated at the transcriptional level by toxR. Proc. Natl Acad. Sci. USA 81, 3471–3475 (1984).

    CAS  PubMed  Google Scholar 

  28. Chourashi, R. et al. Role of a sensor histidine kinase ChiS of Vibrio cholerae in pathogenesis. Int. J. Med. Microbiol. 306, 657–665 (2016).

    CAS  PubMed  Google Scholar 

  29. Klose, K. E. & Mekalanos, J. J. Differential regulation of multiple flagellins in Vibrio cholerae. J. Bacteriol. 180, 303–316 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Merrell, D. S. & Camilli, A. Regulation of Vibrio cholerae genes required for acid tolerance by a member of the ‘ToxR-like’ family of transcriptional regulators. J. Bacteriol. 182, 5342–5350 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Sperandio, V., Girón, J. A., Silveira, W. D. & Kaper, J. B. The OmpU outer membrane protein, a potential adherence factor of Vibrio cholerae. Infect. Immun. 63, 4433–4438 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Pohlner, J., Meyer, T. F., Jalajakumari, M. B. & Manning, P. A. Nucleotide sequence of ompV, the gene for a major Vibrio cholerae outer membrane protein. Mol. Gen. Genet. 205, 494–500 (1986).

    CAS  PubMed  Google Scholar 

  33. Hankins, J. V., Madsen, J. A., Giles, D. K., Brodbelt, J. S. & Trent, M. S. Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in Gram-positive and Gram-negative bacteria. Proc. Natl Acad. Sci. USA 109, 8722–8727 (2012).

    CAS  PubMed  Google Scholar 

  34. Lin, W. et al. Identification of a Vibrio cholerae RTX toxin gene cluster that is tightly linked to the cholera toxin prophage. Proc. Natl Acad. Sci. USA 96, 1071–1076 (1999).

    CAS  PubMed  Google Scholar 

  35. Toma, C. & Honma, Y. Cloning and genetic analysis of the Vibrio cholerae aminopeptidase gene. Infect. Immun. 64, 4495–4500 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 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).

    CAS  PubMed  Google Scholar 

  37. Waldor, M. K. & Mekalanos, J. J. Lysogenic conversion by a filamentous phage encoding cholera toxin. Science 272, 1910–1914 (1996).

    CAS  PubMed  Google Scholar 

  38. Ishikawa, T., Rompikuntal, P. K., Lindmark, B., Milton, D. L. & Wai, S. N. Quorum sensing regulation of the two hcp alleles in Vibrio cholerae O1 strains. PLoS ONE 4, e6734 (2009).

    Google Scholar 

  39. Lomma, M. et al. The Legionella pneumophila F-box protein Lpp2082 (AnkB) modulates ubiquitination of the host protein parvin B and promotes intracellular replication. Cell Microbiol. 12, 1272–1291 (2010).

    CAS  PubMed  Google Scholar 

  40. Mathur, J. & Waldor, M. K. The Vibrio cholerae ToxR-regulated porin OmpU confers resistance to antimicrobial peptides. Infect. Immun. 72, 3577–3583 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Provenzano, D., Schuhmacher, D. A., Barker, J. L. & Klose, K. E. The virulence regulatory protein ToxR mediates enhanced bile resistance in Vibrio cholerae and other pathogenic Vibrio species. Infect. Immun. 68, 1491–1497 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Merrell, D. S., Bailey, C., Kaper, J. B. & Camilli, A. The ToxR-mediated organic acid tolerance response of Vibrio cholerae requires OmpU. J. Bacteriol. 183, 2746–2754 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Russell, T. L. et al. Upper gastrointestinal pH in seventy-nine healthy, elderly North American men and women. Pharm. Res. 10, 187–196 (1993).

    CAS  PubMed  Google Scholar 

  44. Östling, J. et al. in Starvation in Bacteria (ed. Kjelleberg, S.) 169–174 (Springer, 1993).

  45. Jacobs, M. E. et al. The Tetrahymena thermophila phagosome proteome. Eukaryot. Cell 5, 1990–2000 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Kinchen, J. M. & Ravichandran, K. S. Phagosome maturation: going through the acid test. Nat. Rev. Mol. Cell Biol. 9, 781–795 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Duperthuy, M. et al. The major outer membrane protein OmpU of Vibrio splendidus contributes to host antimicrobial peptide resistance and is required for virulence in the oyster Crassostrea gigas. Environ. Microbiol. 12, 951–963 (2010).

    CAS  PubMed  Google Scholar 

  48. Mathur, J., Davis, B. M. & Waldor, M. K. Antimicrobial peptides activate the Vibrio cholerae σE regulon through an OmpU-dependent signalling pathway. Mol. Microbiol. 63, 848–858 (2007).

    CAS  PubMed  Google Scholar 

  49. Wibbenmeyer, J. A., Provenzano, D., Landry, C. F., Klose, K. E. & Delcour, A. H. Vibrio cholerae OmpU and OmpT porins are differentially affected by bile. Infect. Immun. 70, 121–126 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Duperthuy, M. et al. Use of OmpU porins for attachment and invasion of Crassostrea gigas immune cells by the oyster pathogen Vibrio splendidus. Proc. Natl Acad. Sci. USA 108, 2993–2998 (2011).

    CAS  PubMed  Google Scholar 

  51. Thurman, J., Drinkall, J. & Parry, J. D. Digestion of bacteria by the freshwater ciliate Tetrahymena pyriformis. Aquat. Micro. Ecol. 60, 163–174 (2010).

    Google Scholar 

  52. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. & Pease, L. R. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene 77, 61–68 (1989).

    CAS  PubMed  Google Scholar 

  53. Dalia, A. B., McDonough, E. & Camilli, A. Multiplex genome editing by natural transformation. Proc. Natl Acad. Sci. USA 111, 8937–8942 (2014).

    CAS  PubMed  Google Scholar 

  54. Tischler, A. D. & Camilli, A. Cyclic diguanylate regulates Vibrio cholerae virulence gene expression. Infect. Immun. 73, 5873–5882 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references


The authors thank S. A. Rice, S. Longford and B. Morgan for critical evaluation of the manuscript, I. T. C. Hin for graphic illustrations, K. Li for assistance with TEM imaging and L. Cole for advice on confocal microscopy. This work was supported by Australian Research Council Discovery Project DP170100453, the United States NIH (AI055058), the Pew Latin American Fellows Program in the Biomedical Sciences from PEW Charitable trusts, the CONICYT Becas Chile doctoral (72140329) and postdoctoral fellowships, the ithree Institute and The Microbial Imaging Facility, Faculty of Science, University of Technology Sydney. This project was also partly funded by the Australian Centre for Genomic Epidemiological Microbiology (AusGEM), a collaborative partnership between the New South Wales Department of Primary Industries and the University of Technology Sydney and by the National Research Foundation and Ministry of Education Singapore under its Research Centre of Excellence Programme to the Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University.

Author information

Authors and Affiliations



G.E.-V., P.N., A.C. and D.M. designed the study and planned the experiments. G.E.-V., P.N., C.A.S.-V., B.B.A.R., M.M.H., C.A. and A.C. carried out the experiments. G.E.-V., B.B.A.R. and M.S.J. performed the microscopy experiments. G.E.-V., P.N. and C.A. carried out the biological assays. G.E.-V., C.A.S.-V. and A.C. performed the infection assay. M.P. assisted with TEM imaging. G.E.-V., P.N., S.S., A.C., M.L. and D.M. contributed to interpretation of the results. G.E.-V. and D.M. took the lead in writing the manuscript. A.C., S.K., S.P.D., M.L. and D.M. provided funding. All authors provided critical feedback and helped shape the manuscript.

Corresponding author

Correspondence to Diane McDougald.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Table 1, Supplementary References, Supplementary Figs. 1–5, dataset legends and Supplementary Video legends.

Reporting Summary

Supplementary Dataset 1

Calculation of competition index (CI) for the infant mice in vitro and in vivo experiments.

Supplementary Dataset 2

Numbers of V. cholerae cells in EFVs treated with Live/Dead BacLight. Table showing the number of surfaces (V. cholerae live (green) and dead (red) cells) determined using confocal images of EFVs on Imaris v9.2 (Bitplane).

Supplementary Video 1

Egestion of EFVs containing live V. cholerae from T. pyriformis. Real time video (phase contrast, transmitted light) showing the egestion of one EFV containing viable V. cholerae cells to the extracellular space. Video is representative of three independent experiments.

Supplementary Video 2

Composite video showing T. pyriformis containing V. cholera GFP in food vacuoles. Pictures were taken by confocal microscopy from the top to the bottom of the sample. The nucleus is stained with DAPI (blue), membrane phospholipids were stained with FM 4–64 FX (red) and V. cholerae is GFP-tagged (green). Video is representative of three independent experiments.

Supplementary Video 3

Composite video showing a single EFV containing V. cholera GFP. Pictures were taken by confocal microscopy from the top to the bottom of the sample. FM 4–64 FX was used to stain the EFV membranes (red) and V. cholerae was GFP-tagged (green). Video is representative of three independent experiments.

Supplementary Video 4

Escape of V. cholerae from EFVs in LB broth at 37 °C. Time-lapse video (~3 h) showing the escape of V. cholerae from EFVs in less than 15 min after incubation. Video is representative of three independent experiments.

Supplementary Video 5

Escape of V. cholerae GFP from EFVs in LB broth at 37 °C. Time-lapse video (~3 h) showing the escape of V. cholerae from EFVs. Video is representative of three independent experiments.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Espinoza-Vergara, G., Noorian, P., Silva-Valenzuela, C.A. et al. Vibrio cholerae residing in food vacuoles expelled by protozoa are more infectious in vivo. Nat Microbiol 4, 2466–2474 (2019).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing