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Intracellular offspring released from SFB filaments are flagellated

Abstract

The gut commensal segmented filamentous bacterium (SFB) attaches to the ileal epithelium and potently stimulates the host immune system. Using transmission electron microscopy (TEM), we show that mouse and rat SFB are flagellated above the concave tip at the unicellular intracellular offspring (IO) stage and that flagellation occurs prior to full IO differentiation and release of IOs from SFB filaments. This finding adds a missing link to the SFB life cycle.

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Fig. 1: SFB from mouse and rat are flagellated at the intracellular offspring stage.
Fig. 2: SFB flagellation occurs during in vitro growth and IO development.

Data availability

The data relative to in vivo growth of mSFB shown in this paper are representative of four independent experiments with n = 320 bacteria imaged, including 91 flagellated SFB. Figure 1 shows five representative images, with additional images (n = 3, n = 5 and n = 6) shown in Fig. 2 and Supplementary Figs. 3 and 10, respectively. Data relative to in vivo growth of rSFB shown in this paper are representative of three independent experiments with n = 468 bacteria imaged, including 108 flagellated SFB. Figure 1 shows three representative images, with additional images (n = 8 and n = 4) shown in Supplementary Figs. 4 and 11, respectively. The data relative to in vitro growth of mSFB shown in this paper are representative of four independent experiments with n = 278 bacteria imaged, including 38 flagellated SFB. Figure 2 shows four representative images, with additional images (n = 5 and n = 1) shown in Supplementary Figs. 7 and 9, respectively. Raw data and the summary of the TEM analysis is available in the Supplementary Data, as are the raw data for Figs. 1k, 1l, 2d, 2f and 2k and Supplementary Figs. 3g, 4i, 5a–f, 6b, 7f and 8e, including replicas where appropriate. Any additional images not included in the paper or its Supplementary Information can be made available by the corresponding author upon reasonable request. No custom computer code has been used in this study.

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Acknowledgements

We are grateful to the members of the Centre for Gnotobiology of the Insitut Pasteur (T. Angélique, E. Maranghi, M. Jacob and M.G. Lopez Dieguez) for technical support with the gnotobiotic mice and also thank C. Schmitt and R. Tournebize for technical assistance. We thank the UTechS PBI and UBI (Center for Resources and Research in Technology, Institut Pasteur, Paris), the France-BioImaging infrastructure network supported by the ANR (ANR-10-INSB-04; Investments for the Future) and the Région Ile-de-France (programme DIM-Malinf) for the use of the Zeiss LSM 780 Elyra PS1 microscope. This work was supported by the ERC Advanced Grant DECRYPT (339579) to P.J.S. (a Howard Hughes Medical Institute Foreign Scholar), a Pasteur LabEx IBEID Doctoral fellowship to I.N., an ERC Advanced Grant IMMUNOBIOTA (339407) to N.C.-B. and a Bill and Melinda Gates Foundation Grand Challenge Grant (OPP1141322) to P.S.; O.G., A.S., M.B., M.M.-N., G.N., J.K.-L. and P.J.S. were supported by Pasteur Institute. N.C.-B. and P.S. were supported by Paris Descartes University and N.C.-B., P.J.S. and P.S. were also supported by INSERM.

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Authors and Affiliations

Authors

Contributions

The project was conceived by P.S. and P.J.S. Monocolonizations were overseen by M.B. and V.G.-R. In vitro experiments were carried out by I.N. SFB sample preparations for TEM, scanning electron microscopy (SEM), SIM, fluorescent in situ hyridization (FISH), western blot and TLR5 assays were carried out by I.N. and P.S. TEM imaging was done by O.G. and P.S. and SEM imaging by M.M.N. Ultrastructural work was overseen by J.K.-L. Structured illumination microscopy imaging was carried out by A.C. and A.S., and TEM image analysis, RNA analysis and mass spectrometry preparations by I.N. Mass spectrometry was performed by V.J. and overseen by I.C.G. Western blots and FISH were done by C.M., and TLR5 reporter cell assays by I.N. Technical help was provided by G.N. rSFB was obtained from T.S. and Y.U.; G.P.D. provided technical input for TEM grid preparation and first visualization of flagellated IOs using TEM, with assistance from O.G. Funding for I.N. and A.C. was provided by N.C.-B., for G.P.D. by P.J.S., and for C.M. by P.S.; P.S. prepared the manuscript. The position of G.P.D. in the author list is disputed by G.P.D. The published order of authors has been approved by the Institut Pasteur Comité d’Intégrité Scientifique et de Conciliation, by the INSERM Délégation à l’Intégrité Scientifique and by all authors except G.P.D.

Corresponding author

Correspondence to Pamela Schnupf.

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Ethics statement

Animal experiments were approved by the Ethics Committee #89 (CETEA Institut Pasteur, reference 2013-0030) and performed in compliance with French and European regulations on the care and protection of laboratory animals (EC Directive 2010/63 and French Law 2013- 118, 6 February 2013).

Competing interests

The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–11, Table 1 and data legends.

Reporting Summary

Supplementary Data 1

Raw figure data.

Supplementary Data 2

TEM analysis raw data and summary.

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Nkamba, I., Mulet, C., Dubey, G.P. et al. Intracellular offspring released from SFB filaments are flagellated. Nat Microbiol 5, 34–39 (2020). https://doi.org/10.1038/s41564-019-0608-1

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