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.

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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.


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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

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.

Correspondence to Diane McDougald.

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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.

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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).

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