Skip to main content

Thank you for visiting nature.com. 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.

  • Technical Report
  • Published:

In vivo imaging and tracking of host–microbiota interactions via metabolic labeling of gut anaerobic bacteria

Nature Medicine volume 21, pages 1091–1100 (2015)Cite this article

Subjects

Abstract

The intestine is densely populated by anaerobic commensal bacteria. These microorganisms shape immune system development, but understanding of host–commensal interactions is hampered by a lack of tools for studying the anaerobic intestinal environment. We applied metabolic oligosaccharide engineering and bioorthogonal click chemistry to label various commensal anaerobes, including Bacteroides fragilis, a common and immunologically important commensal. We studied the dissemination of B. fragilis after acute peritonitis and characterized the interactions of the intact microbe and its polysaccharide components in myeloid and B cell lineages. We were able to assess the distribution and colonization of labeled B. fragilis along the intestine, as well as niche competition after coadministration of multiple species of the microbiota. We also fluorescently labeled nine additional commensals (eight anaerobic and one microaerophilic) from three phyla common in the gut—Bacteroidetes, Firmicutes and Proteobacteria—as well as one aerobic pathogen (Staphylococcus aureus). This strategy permits visualization of the anaerobic microbial niche by various methods, including intravital two-photon microscopy and non-invasive whole-body imaging, and can be used to study microbial colonization and host–microbe interactions in real time.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Generation of fluorescent anaerobic commensal gut bacteria by MOE-BCC.
Figure 2: MOE-BCC labeling preserves the immunoregulatory activity of PSA in B. fragilis.
Figure 3: In vivo uptake of AF647-labeled B. fragilis after acute infection.
Figure 4: Dissemination of AF647-labeled B. fragilis and PSA to secondary lymphoid organs.
Figure 5: In vivo tracking and visualization of B. fragilis in the intestine.
Figure 6: Labeling of various anaerobic gut commensals and assessment of species competition.

Similar content being viewed by others

References

  1. Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome. Nature 486, 207–214 (2012).

  2. Turnbaugh, P.J. et al. The human microbiome project. Nature 449, 804–810 (2007).

    Article  CAS  Google Scholar 

  3. Hooper, L.V., Littman, D.R. & Macpherson, A.J. Interactions between the microbiota and the immune system. Science 336, 1268–1273 (2012).

    Article  CAS  Google Scholar 

  4. Ivanov, I.I. & Littman, D.R. Modulation of immune homeostasis by commensal bacteria. Curr. Opin. Microbiol. 14, 106–114 (2011).

    Article  CAS  Google Scholar 

  5. Atarashi, K. et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature 500, 232–236 (2013).

    Article  CAS  Google Scholar 

  6. Ahern, P.P., Faith, J.J. & Gordon, J.I. Mining the human gut microbiota for effector strains that shape the immune system. Immunity 40, 815–823 (2014).

    Article  CAS  Google Scholar 

  7. Atarashi, K. et al. Induction of colonic regulatory T cells by indigenous Clostridium species. Science 331, 337–341 (2011).

    Article  CAS  Google Scholar 

  8. Ivanov, I.I. et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485–498, 33 (2009).

    Article  CAS  Google Scholar 

  9. An, D. et al. Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells. Cell 156, 123–133 (2014).

    Article  CAS  Google Scholar 

  10. Dasgupta, S. & Kasper, D.L. Relevance of commensal microbiota in the treatment and prevention of inflammatory bowel disease. Inflamm. Bowel Dis. 19, 2478–2489 (2013).

    Article  Google Scholar 

  11. Yurkovetskiy, L.A., Pickard, J.M. & Chervonsky, A.V. Microbiota and autoimmunity: exploring new avenues. Cell Host Microbe 17, 548–552 (2015).

    Article  CAS  Google Scholar 

  12. Sommer, F. & Backhed, F. The gut microbiota—masters of host development and physiology. Nat. Rev. Microbiol. 11, 227–238 (2013).

    Article  CAS  Google Scholar 

  13. Surana, N.K. & Kasper, D.L. The yin yang of bacterial polysaccharides: lessons learned from B. fragilis PSA. Immunol. Rev. 245, 13–26 (2012).

    Article  CAS  Google Scholar 

  14. Mazmanian, S.K., Liu, C.H., Tzianabos, A.O. & Kasper, D.L. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell 122, 107–118 (2005).

    Article  CAS  Google Scholar 

  15. Krinos, C.M. et al. Extensive surface diversity of a commensal microorganism by multiple DNA inversions. Nature 414, 555–558 (2001).

    Article  CAS  Google Scholar 

  16. Coyne, M.J., Chatzidaki-Livanis, M., Paoletti, L.C. & Comstock, L.E. Role of glycan synthesis in colonization of the mammalian gut by the bacterial symbiont Bacteroides fragilis. Proc. Natl. Acad. Sci. USA 105, 13099–13104 (2008).

    Article  CAS  Google Scholar 

  17. Dasgupta, S., Erturk-Hasdemir, D., Ochoa-Reparaz, J., Reinecker, H.C. & Kasper, D.L. Plasmacytoid dendritic cells mediate anti-inflammatory responses to a gut commensal molecule via both innate and adaptive mechanisms. Cell Host Microbe 15, 413–423 (2014).

    Article  CAS  Google Scholar 

  18. Ochoa-Repáraz, J. et al. A polysaccharide from the human commensal Bacteroides fragilis protects against CNS demyelinating disease. Mucosal Immunol. 3, 487–495 (2010).

    Article  Google Scholar 

  19. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  Google Scholar 

  20. Craggs, T.D. Green fluorescent protein: structure, folding and chromophore maturation. Chem. Soc. Rev. 38, 2865–2875 (2009).

    Article  CAS  Google Scholar 

  21. Boyce, M. & Bertozzi, C.R. Bringing chemistry to life. Nat. Methods 8, 638–642 (2011).

    Article  CAS  Google Scholar 

  22. Sletten, E.M. & Bertozzi, C.R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality. Angew. Chem. Int. Edn. Engl. 48, 6974–6998 (2009).

    Article  CAS  Google Scholar 

  23. Dehnert, K.W. et al. Metabolic labeling of fucosylated glycans in developing zebrafish. ACS Chem. Biol. 6, 547–552 (2011).

    Article  CAS  Google Scholar 

  24. Dumont, A., Malleron, A., Awwad, M., Dukan, S. & Vauzeilles, B. Click-mediated labeling of bacterial membranes through metabolic modification of the lipopolysaccharide inner core. Angew. Chem. Int. Edn. Engl. 51, 3143–3146 (2012).

    Article  CAS  Google Scholar 

  25. Kaewsapsak, P., Esonu, O. & Dube, D.H. Recruiting the host's immune system to target Helicobacter pylori's surface glycans. ChemBioChem 14, 721–726 (2013).

    Article  CAS  Google Scholar 

  26. Cobb, B.A. & Kasper, D.L. Characteristics of carbohydrate antigen binding to the presentation protein HLA-DR. Glycobiology 18, 707–718 (2008).

    Article  CAS  Google Scholar 

  27. Hawkins, E.D. et al. Measuring lymphocyte proliferation, survival and differentiation using CFSE time-series data. Nat. Protoc. 2, 2057–2067 (2007).

    Article  CAS  Google Scholar 

  28. Troy, E.B., Carey, V.J., Kasper, D.L. & Comstock, L.E. Orientations of the Bacteroides fragilis capsular polysaccharide biosynthesis locus promoters during symbiosis and infection. J. Bacteriol. 192, 5832–5836 (2010).

    Article  CAS  Google Scholar 

  29. Coyne, M.J. et al. Polysaccharide biosynthesis locus required for virulence of Bacteroides fragilis. Infect. Immun. 69, 4342–4350 (2001).

    Article  CAS  Google Scholar 

  30. Coyne, M.J., Weinacht, K.G., Krinos, C.M. & Comstock, L.E. Mpi recombinase globally modulates the surface architecture of a human commensal bacterium. Proc. Natl. Acad. Sci. USA 100, 10446–10451 (2003).

    Article  CAS  Google Scholar 

  31. Nord, C.E. Incidence and significance of intraperitoneal aerobic and anaerobic bacteria. Clin. Ther. 12, 9–20 (1990).

    PubMed  Google Scholar 

  32. Millet, Y.A. et al. Insights into Vibrio cholerae intestinal colonization from monitoring fluorescently labeled bacteria. PLoS Pathog. 10, e1004405 (2014).

    Article  Google Scholar 

  33. Farache, J., Zigmond, E., Shakhar, G. & Jung, S. Contributions of dendritic cells and macrophages to intestinal homeostasis and immune defense. Immunol. Cell Biol. 91, 232–239 (2013).

    Article  CAS  Google Scholar 

  34. Dethlefsen, L., McFall-Ngai, M. & Relman, D.A. An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449, 811–818 (2007).

    Article  CAS  Google Scholar 

  35. Martinez-Pomares, L. & Gordon, S. CD169+ macrophages at the crossroads of antigen presentation. Trends Immunol. 33, 66–70 (2012).

    Article  CAS  Google Scholar 

  36. Cyster, J.G. B cell follicles and antigen encounters of the third kind. Nat. Immunol. 11, 989–996 (2010).

    Article  CAS  Google Scholar 

  37. Vinuesa, C.G., Linterman, M.A., Goodnow, C.C. & Randall, K.L. T cells and follicular dendritic cells in germinal center B-cell formation and selection. Immunol. Rev. 237, 72–89 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Neurobiology Imaging Facility for consultation and instrument availability that supported this work. This facility is supported in part by the Neural Imaging Center as part of NINDS P30 Core Center grant NS072030. We also thank N. Barteneva of the Flow and Imaging Cytometry Resource for help in whole-body imaging on the IVIS Spectrum (funded by grants NIH 1 S10 OD016401 and BCH/PCMM); J. Czupryna and M. Guerra from PerkinElmer for consultation and expert advice; C. Araneo (Division of Immunology Flow Cytometry Core Facility, HMS) and L. Comstock for helpful discussions, comments and reagents; and members of the Kasper and von Andrian labs for helpful discussions. This work was also partly supported by the US National Institutes of Health (grants PO1 AI1112521, RO1 AI111595 (to U.H.v.A.) and 5T32 HL066987 (to D.A.)). Additional support to U.H.v.A. was provided by U19AI095261 and the Ragon Institute at MGH, MIT and Harvard. N.G.-Z. was supported by an EMBO Long-Term Fellowship (ALTF 251-2011), the Human Frontiers Science Program (LT000079/2012), a UNESCO L'Oreal National and International Women in Science Award, a Fulbright Award and the Weizmann Institute of Science–National Postdoctoral Award Program for Advancing Women in Science. J.E.H. was supported by the Cancer Research Institute Irvington Fellowship Program. N.C.R. was supported by a departmental Kirschstein-NRSA training grant.

Author information

Author notes

  1. Naama Geva-Zatorsky and David Alvarez: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts, USA

    Naama Geva-Zatorsky, David Alvarez, Jason E Hudak, Nicola C Reading, Deniz Erturk-Hasdemir, Suryasarathi Dasgupta, Ulrich H von Andrian & Dennis L Kasper

  2. The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA

    Ulrich H von Andrian

Authors
  1. Naama Geva-Zatorsky
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David Alvarez
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jason E Hudak
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nicola C Reading
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Deniz Erturk-Hasdemir
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Suryasarathi Dasgupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ulrich H von Andrian
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dennis L Kasper
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

N.G.-Z. and D.A. designed the experiments, analyzed the data and wrote the manuscript with help from J.E.H. and N.C.R. D.E.-H. and S.D. provided experimental help and expertise. D.L.K. supervised the study, edited the manuscript and provided helpful comments, with assistance from U.H.v.A.

Corresponding author

Correspondence to Dennis L Kasper.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 & Supplementary Table 1 (PDF 7615 kb)

MOE fluorescent labeling of surface glycans of B. fragilis. (MP4 2723 kb)

Phagocytic uptake of B. fragilis by macrophages. (MP4 31252 kb)

Visualization of B. fragilis in the distal small intestine by intravital two-photon microscopy (MP4 13352 kb)

41591_2015_BFnm3929_MOESM36_ESM.mp4

Non-invasive 3D whole-body longitudinal optical fluorescence imaging of live mice after oral administration of labeled B. fragilis (MP4 1612 kb)

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Geva-Zatorsky, N., Alvarez, D., Hudak, J. et al. In vivo imaging and tracking of host–microbiota interactions via metabolic labeling of gut anaerobic bacteria. Nat Med 21, 1091–1100 (2015). https://doi.org/10.1038/nm.3929

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.3929

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

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