Abstract

Human pathogenic Legionella replicate in alveolar macrophages and cause a potentially lethal form of pneumonia known as Legionnaires' disease1. Here, we have identified a host-directed therapeutic approach to eliminate intracellular Legionella infections. We demonstrate that the genetic deletion, or pharmacological inhibition, of the host cell pro-survival protein BCL-XL induces intrinsic apoptosis of macrophages infected with virulent Legionella strains, thereby abrogating Legionella replication. BCL-XL is essential for the survival of Legionella-infected macrophages due to bacterial inhibition of host-cell protein synthesis, resulting in reduced levels of the short-lived, related BCL-2 pro-survival family member, MCL-1. Consequently, a single dose of a BCL-XL-targeted BH3-mimetic therapy, or myeloid cell-restricted deletion of BCL-XL, limits Legionella replication and prevents lethal lung infections in mice. These results indicate that repurposing BH3-mimetic compounds, originally developed to induce cancer cell apoptosis, may have efficacy in treating Legionnaires' and other diseases caused by intracellular microbes.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    , & Legionnaires’ disease. Lancet (2015).

  2. 2.

    , , & Molecular pathogenesis of infections caused by Legionella pneumophila. Clin. Microbiol. Rev. 23, 274–298 (2010).

  3. 3.

    , , & Aggravating genetic interactions allow a solution to redundancy in a bacterial pathogen. Science 338, 1440–1444 (2012).

  4. 4.

    et al. Public health and economic costs of investigating a suspected outbreak of Legionnaires’ disease. Epidemiol. Infect. 136, 1306–1314 (2008).

  5. 5.

    & NF-κB translocation prevents host cell death after low-dose challenge by Legionella pneumophila. J. Exp. Med. 203, 2177–2189 (2006).

  6. 6.

    et al. Anti-apoptotic signalling by the Dot/Icm secretion system of L. pneumophila. Cell Microbiol. 9, 246–264 (2007).

  7. 7.

    , & Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J. 30, 3667–3683 (2011).

  8. 8.

    , & BCL-2 family antagonists for cancer therapy. Nature Rev. Drug Discov. 7, 989–1000 (2008).

  9. 9.

    , , , & Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity. PLoS Pathog. 2, e18 (2006).

  10. 10.

    et al. Cytosolic recognition of flagellin by mouse macrophages restricts Legionella pneumophila infection. J. Exp. Med. 203, 1093–1104 (2006).

  11. 11.

    et al. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized. Cancer Cell 10, 389–399 (2006).

  12. 12.

    et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nature Med. 19, 202–208 (2013).

  13. 13.

    et al. Discovery of a potent and selective BCL-XL inhibitor with in vivo activity. ACS Med. Chem. Lett. 5, 1088–1093 (2014).

  14. 14.

    et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727–730 (2001).

  15. 15.

    et al. Rapid pathogen-induced apoptosis: a mechanism used by dendritic cells to limit intracellular replication of Legionella pneumophila. PLoS Pathog. 5, e1000478 (2009).

  16. 16.

    et al. Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol. Cell 17, 393–403 (2005).

  17. 17.

    , , & MCL-1S, a splicing variant of the antiapoptotic BCL-2 family member MCL-1, encodes a proapoptotic protein possessing only the BH3 domain. J. Biol. Chem. 275, 25255–25261 (2000).

  18. 18.

    & Rapid turnover of MCL-1 couples translation to cell survival and apoptosis. J. Biol. Chem. 282, 6192–6200 (2007).

  19. 19.

    et al. Enhanced stability of MCL1, a prosurvival BCL2 relative, blunts stress-induced apoptosis, causes male sterility, and promotes tumorigenesis. Proc. Natl Acad. Sci. USA 111, 261–266 (2014).

  20. 20.

    et al. Secreted bacterial effectors that inhibit host protein synthesis are critical for induction of the innate immune response to virulent Legionella pneumophila. PLoS Pathog. 7, e1001289 (2011).

  21. 21.

    & Pathogen signatures activate a ubiquitination pathway that modulates the function of the metabolic checkpoint kinase mTOR. Nature Immunol. 14, 1219–1228 (2013).

  22. 22.

    & The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation. PLoS Pathog. 3, e188 (2007).

  23. 23.

    et al. Targeting eEF1A by a Legionella pneumophila effector leads to inhibition of protein synthesis and induction of host stress response. Cell Microbiol. 11, 911–926 (2009).

  24. 24.

    et al. Legionella pneumophila glucosyltransferase inhibits host elongation factor 1A. Proc. Natl Acad. Sci. USA 103, 16953–16958 (2006).

  25. 25.

    & Host translation at the nexus of infection and immunity. Cell Host Microbe 12, 470–483 (2012).

  26. 26.

    , & Host translational inhibition by Pseudomonas aeruginosa exotoxin A triggers an immune response in Caenorhabditis elegans. Cell Host Microbe 11, 364–374 (2012).

  27. 27.

    , , , & Experimental Legionella longbeachae infection in intratracheally inoculated mice. J. Med. Microbiol. 58, 723–730 (2009).

  28. 28.

    , , & Comparative and functional genomics of Legionella identified eukaryotic like proteins as key players in host–pathogen interactions. Front. Microbiol. 2, 208 (2011).

  29. 29.

    et al. ABT-263: a potent and orally bioavailable Bcl-2 family inhibitor. Cancer Res. 68, 3421–3428 (2008).

  30. 30.

    et al. Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis. Proc. Natl Acad. Sci. USA 112, 5803–5808 (2015).

  31. 31.

    Control of Legionella in hospitals. J. Hosp. Infect. 8, 109–115 (1986).

  32. 32.

    et al. The Dot/Icm effector SdhA is necessary for virulence of Legionella pneumophila in Galleria mellonella and A/J mice. Infect. Immun. 81, 2598–2605 (2013).

  33. 33.

    et al. Analysis of the Legionella longbeachae genome and transcriptome uncovers unique strategies to cause Legionnaires’ disease. PLoS Genet. 6, e1000851 (2010).

  34. 34.

    et al. Cutting edge: NF-κB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J. Immunol. 183, 787–791 (2009).

  35. 35.

    et al. Fas-mediated neutrophil apoptosis is accelerated by Bid, Bak, and Bax and inhibited by Bcl-2 and Mcl-1. Proc. Natl Acad. Sci. USA 108, 13135–13140 (2011).

  36. 36.

    et al. Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy. Sci. Transl. Med. 7, 279ra40 (2015).

Download references

Acknowledgements

The authors thank members of the Monash Micro Imaging facility for their technical support, D. Newman (Monash University) for the statistical analysis, E. Latz (University of Bonn) for immortalized C57Bl/6 macrophages and D. Vaux, J. Silke, W. Alexander, S. Masters, L. Lindqvist and P. Bouillet (Walter and Eliza Hall Institute of Medical Research) for providing mice or reagents. This study was funded by grants and fellowships from the NHMRC (Canberra, Australia), programme grants 606788 (T.L. and E.L.H.) and 1016701 (A.S.), project grants 1024839 (T.N. and J.E.V.), 1051235 (S.P.G.) and 1009145 (L.O.R.), CDF1 fellowships 1052598 (J.E.V.) and 1020363 (A.S.), an NHMRC infrastructure grant, Independent Research Institutes Infrastructure Support Scheme grant 361646, the Victorian State Government (OIS grant) and the Leukemia and Lymphoma Society (SCOR grants 7413 and 7001-13).

Author information

Author notes

    • James E. Vince
    •  & Thomas Naderer

    These authors contributed equally to this work.

Affiliations

  1. Department of Biochemistry and Molecular Biology and the Biomedicine Discovery Institute, Monash University, Clayton 3800, Victoria, Australia

    • Mary Speir
    • , Gilu Abraham
    • , Seong Chow
    • , Adam Vogrin
    •  & Thomas Naderer
  2. Walter and Eliza Hall Institute of Medical Research, Parkville 3052, Victoria, Australia

    • Kate E. Lawlor
    • , Stefan P. Glaser
    • , Lorraine A. O'Reilly
    • , Andreas Strasser
    • , Guillaume Lessene
    • , David C. S. Huang
    •  & James E. Vince
  3. Department of Medical Biology, The University of Melbourne, Parkville 3010, Victoria, Australia

    • Kate E. Lawlor
    • , Stefan P. Glaser
    • , Lorraine A. O'Reilly
    • , Andreas Strasser
    • , Guillaume Lessene
    • , David C. S. Huang
    •  & James E. Vince
  4. Monash Micro Imaging, Monash University, Clayton 3800, Victoria, Australia

    • Keith E. Schulze
  5. Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, 3000, Victoria, Australia

    • Ralf Schuelein
    •  & Elizabeth L. Hartland
  6. Department of Medicine, The University of Melbourne, Parkville 3010, Victoria, Australia

    • Kylie Mason
  7. Department of Microbiology and the Biomedicine Discovery Institute, Monash University, Clayton 3800, Victoria, Australia

    • Trevor Lithgow

Authors

  1. Search for Mary Speir in:

  2. Search for Kate E. Lawlor in:

  3. Search for Stefan P. Glaser in:

  4. Search for Gilu Abraham in:

  5. Search for Seong Chow in:

  6. Search for Adam Vogrin in:

  7. Search for Keith E. Schulze in:

  8. Search for Ralf Schuelein in:

  9. Search for Lorraine A. O'Reilly in:

  10. Search for Kylie Mason in:

  11. Search for Elizabeth L. Hartland in:

  12. Search for Trevor Lithgow in:

  13. Search for Andreas Strasser in:

  14. Search for Guillaume Lessene in:

  15. Search for David C. S. Huang in:

  16. Search for James E. Vince in:

  17. Search for Thomas Naderer in:

Contributions

M.S., K.E.L., S.P.G., S.H., K.E.S., E.L.H., T.L., A.S., G.L., D.C.S.H., J.E.V. and T.N. designed the research and carried out analysis. M.S., K.E.L., G.A., S.H., A.V., J.E.V. and T.N. performed experiments. K.E.S. wrote ImageJ and Metamorph scripts. L.A.O.R. and K.M. generated knockout mice. R.S. and E.L.H. designed and generated GFP-expressing and mutant Legionella. G.L. and D.C.S.H. generated and analysed BH3 mimetic compounds. M.S., K.E.L., A.S., J.E.V. and T.N. wrote the manuscript.

Competing interests

The authors declare that S.P.G., L.A.O.R., A.S., G.L., D.S.C.H. and J.E.V. are employees of The Walter and Eliza Hall Medical Institute, which receives milestone payments from Genentech and AbbVie for the development of ABT-199 for cancer therapy.

Corresponding authors

Correspondence to James E. Vince or Thomas Naderer.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1–5.

Videos

  1. 1.

    Supplementary Video 1

    Time-lapse video microscopy of L. pneumophila infected macrophages.

  2. 2.

    Supplementary Video 2

    Time-lapse video microscopy of ABT-737 treated macrophages infected with L. pneumophila.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmicrobiol.2015.34