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.

Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish

Nature Protocols volume 8, pages 1114–1124 (2013)Cite this article

Subjects

Abstract

Mycobacterium marinum–infected zebrafish are used to study tuberculosis pathogenesis, as well as for antitubercular drug discovery. The small size of zebrafish larvae coupled with their optical transparency allows for rapid analysis of bacterial burdens and host survival in response to genetic and pharmacological manipulations of both mycobacteria and host. Automated fluorescence microscopy and automated plate fluorimetry (APF) are coupled with facile husbandry to facilitate large-scale, repeated analysis of individual infected fish. Both methods allow for in vivo screening of chemical libraries, requiring only 0.1 μmol of drug per fish to assess efficacy; they also permit a more detailed evaluation of the individual stages of tuberculosis pathogenesis. Here we describe a 16-h protocol spanning 22 d, in which zebrafish larvae are infected via the two primary injection sites, the hindbrain ventricle and caudal vein; this is followed by the high-throughput evaluation of pathogenesis and antimicrobial efficacy.

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

$39.95

Learn more

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

Figure 1: Single-cell preparation.
Figure 2: Microinjection sites.
Figure 3: VAMP.
Figure 4: Experimental results.

References

  1. Ramakrishnan, L. Revisiting the role of the granuloma in tuberculosis. Nat. Rev. Immunol. 12, 352–366 (2012).

    Article  CAS  Google Scholar 

  2. Davis, J.M. et al. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity 17, 693–702 (2002).

    Article  CAS  Google Scholar 

  3. Clay, H. et al. Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish. Cell Host Microbe 2, 29–39 (2007).

    Article  CAS  Google Scholar 

  4. Davis, J.M. & Ramakrishnan, L. The role of the granuloma in expansion and dissemination of early tuberculous infection. Cell 136, 37–49 (2009).

    Article  CAS  Google Scholar 

  5. Volkman, H.E. et al. Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant. PLoS Biol. 2, e367 (2004).

    Article  Google Scholar 

  6. Volkman, H.E. et al. Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium. Science 327, 466–469 (2010).

    Article  CAS  Google Scholar 

  7. Cosma, C.L., Klein, K., Kim, R., Beery, D. & Ramakrishnan, L. Mycobacterium marinum Erp is a virulence determinant required for cell wall integrity and intracellular survival. Infect. Immun. 74, 3125–3133 (2006).

    Article  CAS  Google Scholar 

  8. Herbomel, P., Thisse, B. & Thisse, C. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development 126, 3735–3745 (1999).

    CAS  Google Scholar 

  9. Yang, C.T. et al. Neutrophils exert protection in the early tuberculous granuloma by oxidative killing of mycobacteria phagocytosed from infected macrophages. Cell Host Microbe 12, 301–312 (2012).

    Article  CAS  Google Scholar 

  10. Brannon, M.K. et al. Pseudomonas aeruginosa Type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos. Cell Microbiol. 11, 755–768 (2009).

    Article  CAS  Google Scholar 

  11. Neely, M.N., Pfeifer, J.D. & Caparon, M. Streptococcus-zebrafish model of bacterial pathogenesis. Infect. Immun. 70, 3904–3914 (2002).

    Article  CAS  Google Scholar 

  12. Ludwig, M. et al. Whole-body analysis of a viral infection: vascular endothelium is a primary target of infectious hematopoietic necrosis virus in zebrafish larvae. PLoS Pathog. 7, e1001269 (2011).

    Article  CAS  Google Scholar 

  13. Phelan, P.E. et al. Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio). J. Virol. 79, 1842–1852 (2005).

    Article  CAS  Google Scholar 

  14. Lu, M.W. et al. The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model. Mol. Immunol. 45, 1146–1152 (2008).

    Article  CAS  Google Scholar 

  15. Chao, C.C. et al. Zebrafish as a model host for Candida albicans infection. Infect. Immun. 78, 2512–2521 (2010).

    Article  CAS  Google Scholar 

  16. Burgos, J.S., Ripoll-Gomez, J., Alfaro, J.M., Sastre, I. & Valdivieso, F. Zebrafish as a new model for herpes simplex virus type 1 infection. Zebrafish 5, 323–333 (2008).

    Article  CAS  Google Scholar 

  17. Davis, J.M., Haake, D.A. & Ramakrishnan, L. Leptospira interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues. PLoS Negl. Trop. Dis. 3, e463 (2009).

    Article  Google Scholar 

  18. Pham, L.N., Kanther, M., Semova, I. & Rawls, J.F. Methods for generating and colonizing gnotobiotic zebrafish. Nat. Protoc. 3, 1862–1875 (2008).

    Article  CAS  Google Scholar 

  19. Haldi, M., Ton, C., Seng, W.L. & McGrath, P. Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish. Angiogenesis 9, 139–151 (2006).

    Article  Google Scholar 

  20. Eguiara, A. et al. Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification. Cell Cycle 10, 3751–3757 (2011).

    Article  CAS  Google Scholar 

  21. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Danio rerio). University of Oregon Press, 4th edn. (2000).

  22. Ellett, F., Pase, L., Hayman, J.W., Andrianopoulos, A. & Lieschke, G.J. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117, e49–56 (2011).

    Article  CAS  Google Scholar 

  23. Cosma, C.L., Swaim, L.E., Volkman, H., Ramakrishnan, L. & Davis, J.M. Zebrafish and frog models of Mycobacterium marinum infection. Curr. Protoc. Microbiol. 3, 10B.2.1–10B.2.33 (2006).

    Google Scholar 

  24. Takaki, K., Cosma, C.L., Troll, M.A. & Ramakrishnan, L. An in vivo platform for rapid high-throughput antitubercular drug discovery. Cell Rep. 2, 175–184 (2012).

    Article  CAS  Google Scholar 

  25. Tobin, D.M. et al. Host genotype-specific therapies can optimize the inflammatory response to mycobacterial infections. Cell 148, 434–446 (2012).

    Article  CAS  Google Scholar 

  26. Adams, K.N. et al. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism. Cell 145, 39–53 (2011).

    Article  CAS  Google Scholar 

  27. Tobin, D.M. et al. The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans. Cell 140, 717–730 (2010).

    Article  CAS  Google Scholar 

  28. Clay, H., Volkman, H.E. & Ramakrishnan, L. Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death. Immunity 29, 283–294 (2008).

    Article  CAS  Google Scholar 

  29. Wolinsky, E. Nontuberculous mycobacteria and associated diseases. Am. Rev. Respir. Dis. 119, 107–159 (1979).

    CAS  PubMed  Google Scholar 

  30. Ramakrishnan, L. Images in clinical medicine. Mycobacterium marinum infection of the hand. N. Engl. J. Med. 337, 612 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank F. Roca for images of intracellular bacteria and larval illustrations; C.J. Cambier for macrophage recruitment images and movies; and C. Cosma, M. Troll, D. Berry, D. Tobin, J. Cameron and R. Berg for helpful discussion and protocol development.

Author information

Authors and Affiliations

  1. Department of Microbiology, University of Washington, Seattle, Washington, USA

    Kevin Takaki, J Muse Davis, Kathryn Winglee & Lalita Ramakrishnan

  2. Department of Medicine, University of Washington, Seattle, Washington, USA

    Lalita Ramakrishnan

  3. Department of Immunology, University of Washington, Seattle, Washington, USA.

    Lalita Ramakrishnan

Authors
  1. Kevin Takaki
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. J Muse Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kathryn Winglee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lalita Ramakrishnan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

J.M.D. developed the caudal vein injection technique and macrophage recruitment assay. K.T. conceived and developed the single-cell protocol, VAMP, cryo-anesthesia, high-throughput microscopy and the autofluorescence-based survival assay. K.T. developed the fluorescence constructs and APF. K.T. performed the experiments and tutorial videos. K.W. developed FPC. L.R. conceived the larval infection model and FPC and guided the development of all protocols. K.T. and L.R. wrote the manuscript.

Corresponding author

Correspondence to Lalita Ramakrishnan.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

General setup of microinjection station and micromanipulator. (a) Safe and efficient injection of larval zebrafish is achieved with a well-designed injection station. (b) The micromanipulator should be setup at right angles, with each knob giving exclusive control to x, y, and z-planes, and with the needle angled downwards to approximately 30°. After the initial setup, and during injections, the micromanipulator will only be moved in the z-plane by control of the z-knob (red arrow). (PDF 2508 kb)

Supplementary Figure 2

Microinjection Metrics (a) High resolution image of microinjection needle. Outer diameter of needle tip is approximately 10 μm. Note that the point of the needle may be beveled, blunt or irregular. Although some users prefer one type over another, microinjection can be performed with all types of needle points. Scale bar 50 μm. (b) Microinjection into mineral oil corresponding with caudal vein and hindbrain microinjection volumes. Microinjection diameters average at 139 nm with a calculated volume of 1.4 nL. Scale bar 200 μm. (PDF 1281 kb)

Supplementary Method

ImageJ FPC Macro (TXT 0 kb)

Supplementary Video 1

Hindbrain injections (MPG 23972 kb)

Supplementary Video 2

Hindbrain injections via the forebrain (MPG 26782 kb)

Supplementary Video 3

Syringing bacteria (MPG 19126 kb)

Supplementary Video 4

Needle break (MPG 7302 kb)

Supplementary Video 5

Caudal vein injection (MPG 23086 kb)

Supplementary Video 6

Macrophage recruitment (MOV 64864 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Takaki, K., Davis, J., Winglee, K. et al. Evaluation of the pathogenesis and treatment of Mycobacterium marinum infection in zebrafish. Nat Protoc 8, 1114–1124 (2013). https://doi.org/10.1038/nprot.2013.068

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2013.068

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research