Letter | Published:

Interception of host angiogenic signalling limits mycobacterial growth

Nature volume 517, pages 612615 (29 January 2015) | Download Citation

Abstract

Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis1,2. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish–Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis2. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations3. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimens are largely ineffective.

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Acknowledgements

We thank D. Sisk and J. Saelens for technical assistance, L. Ramakrishnan, P. Edelstein and C. Kontos for helpful discussions, L. Ramakrishnan, W. Britton and J. Coers for critical review of the manuscript, and J. Fuller, C. Gallione, E. Linney, H. Mao, S. Lee, D. Marchuk, H. Matsunami, A. Nixon, J. Perfect, J. Rawls, D. Silver, K. Smith, K. Takaki, J. Tenor and B. Uy for reagents and equipment. This work was funded by an Australian National Health and Medical Research Council CJ Martin Early Career Fellowship (S.H.O.); an American Cancer Society Postdoctoral Fellowship PF-13-223-01-MPC (M.R.C.); the Duke Summer Research Opportunities Program (N.R.S.); a Malaysian Ministry of Science and Technology and Innovation scholarship (K.S.O.); a New Zealand Ministry of Science and Innovation grant UOAX0813 (P.S.C.); the Duke University Center for AIDS Research (CFAR), a National Institutes of Health (NIH)-funded program (5P30 AI064518), and by a Mallinckrodt Scholar Award, a Searle Scholar Award, a Vallee Foundation Young Investigator Award and an NIH Director’s New Innovator Award 1DP2-OD008614 (D.M.T.).

Author information

Affiliations

  1. Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University Medical Center, Durham, North Carolina 27710, USA

    • Stefan H. Oehlers
    • , Mark R. Cronan
    • , Ninecia R. Scott
    • , Monica I. Thomas
    • , Eric M. Walton
    • , Rebecca W. Beerman
    •  & David M. Tobin
  2. Department of Molecular Medicine and Pathology, The University of Auckland, Auckland 1023, New Zealand

    • Kazuhide S. Okuda
    •  & Philip S. Crosier

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Contributions

S.H.O. and D.M.T. designed the experiments and wrote the paper. S.H.O., N.R.S., M.I.T. and K.S.O. performed and analysed the experiments. M.R.C., E.M.W. and R.W.B. generated transgenic zebrafish lines. S.H.O., P.S.C. and D.M.T. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David M. Tobin.

Extended data

Supplementary information

Videos

  1. 1.

    Dissemination of M. marinum-tomato into vascular rich areas of the larva including the intestine and CHT

    Video depicting the dissemination of M. marinum-tomato into vascular rich areas of the larva including the intestine and CHT. Vascular recruitment in this larva is depicted in Supplementary Video 6.

  2. 2.

    Interstitial dissemination of M. marinum-cerulean within a macrophage from a trunk granuloma in a Tg(flk1:EGFP, mpeg1:tomato-caaxtd3) double transgenic larva

    Video depicting interstitial dissemination of M. marinum-cerulean within a macrophage from a trunk granuloma in a Tg(flk1:EGFP, mpeg1:tomato-caaxtd3) double transgenic larva where bacterial are blue labelled, blood vessels are green labelled and macrophages are red labelled.

  3. 3.

    Transfer of M. marinum-cerulean between trunk granulomas in a Tg(flk1:EGFP) larva.

    Video depicting transfer of M. marinum-cerulean between trunk granulomas in a Tg(flk1:EGFP) larva.

  4. 4.

    Coalescence of M. marinum-tomato from a distal site in the trunk to a central trunk granuloma in a Tg(flk1:EGFP) larva

    Video depicting coalescence of M. marinum-tomato from a distal site in the trunk to a central trunk granuloma in a Tg(flk1:EGFP) larva.

  5. 5.

    M. marinum-cerulean dissemination from an established granuloma into the adjacent intersegmental vessel in a Tg(flk1:EGFP, mpeg1:tomato-caaxtd3) double transgenic larva

    Video depicting M. marinum-cerulean dissemination from an established granuloma into the adjacent intersegmental vessel in a Tg(flk1:EGFP, mpeg1:tomato-caaxtd3) double transgenic larva. Stills of this Video are displayed in Figure 1D.

  6. 6.

    M. marinum-tomato and blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi

    Video depicting M. marinum-tomato and blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi. Red arrows indicate sites of vessel sprouting. Supplementary Video 7 depicts only green channel.

  7. 7.

    Blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi

    Video depicting blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi. Red arrows in indicate sites of vessel sprouting. Supplementary Video 6 depicts both red and green channels. Vessel was traced from initial sprouting through to connection with other vasculature in Extended Data 1Ci.

  8. 8.

    M. marinum-tomato and blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi

    Video depicting M. marinum-tomato and blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi. Red arrow indicates the origin of a vessel that sprouts to the bottom right of the field of view before retreating during the course of the Video. Supplementary Video 9 depicts only green channel.

  9. 9.

    Blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi

    Video depicting blood vessel growth in a Tg(flk1:EGFP) larva from 4 dpi. Red arrow indicates the origin of a vessel that sprouts to the bottom right of the field of view before retreating during the course of the Video. Supplementary Video 8 depicts both red and green channels. Vessel was traced from initial sprouting through to the end of the recording period in Extended Data 1Ciii.

  10. 10.

    M. marinum-cerulean growth and endothelial cell division in a Tg(fli1a:nlsEGFPy7, flk1:mCherryis5) larva

    Video depicting M. marinum-cerulean growth and endothelial cell division in a Tg(fli1a:nlsEGFPy7, flk1:mCherryis5) larva. Red arrow indicates previously sprouted endothelial cell nucleus in the somite that divides during the course of the Video.

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DOI

https://doi.org/10.1038/nature13967

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