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A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis

Nature Medicine volume 17, pages 1261–1268 (2011)Cite this article

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Abstract

We report the involvement of an evolutionarily conserved set of mycobacterial genes, the esx-3 region, in evasion of bacterial killing by innate immunity. Whereas high-dose intravenous infections of mice with the rapidly growing mycobacterial species Mycobacterium smegmatis bearing an intact esx-3 locus were rapidly lethal, infection with an M. smegmatis Δesx-3 mutant (here designated as the IKE strain) was controlled and cleared by a MyD88-dependent bactericidal immune response. Introduction of the orthologous Mycobacterium tuberculosis esx-3 genes into the IKE strain resulted in a strain, designated IKEPLUS, that remained susceptible to innate immune killing and was highly attenuated in mice but had a marked ability to stimulate bactericidal immunity against challenge with virulent M. tuberculosis. Analysis of these adaptive immune responses indicated that the highly protective bactericidal immunity elicited by IKEPLUS was dependent on CD4+ memory T cells and involved a distinct shift in the pattern of cytokine responses by CD4+ cells. Our results establish a role for the esx-3 locus in promoting mycobacterial virulence and also identify the IKE strain as a potentially powerful candidate vaccine vector for eliciting protective immunity to M. tuberculosis.

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Figure 1: Role of the esx-3 region in evasion of innate immunity by M. smegmatis in a high-dose infection model.
Figure 2: Characterization of innate immune responses against the Msmeg IKEPLUS strain.
Figure 3: Bactericidal immunity against Mtb in mice vaccinated with IKEPLUS.
Figure 4: Improvement of histopathology in IKEPLUS-immunized mice during resolution of Mtb infection.
Figure 5: Protection from Mtb challenge by subcutaneous immunization with IKEPLUS.
Figure 6: Role of CD4+ T cells in IKEPLUS-induced protective immunity.

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References

  1. Dye, C., Scheele, S., Dolin, P., Pathania, V. & Raviglione, M.C. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. J. Am. Med. Assoc. 282, 677–686 (1999).

    Article  CAS  Google Scholar 

  2. Gandhi, N.R. et al. Extensively drug-resistant tuberculosis as a cause of death in patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 368, 1575–1580 (2006).

    Article  Google Scholar 

  3. Colditz, G.A. et al. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. J. Am. Med. Assoc. 271, 698–702 (1994).

    Article  CAS  Google Scholar 

  4. Corbett, E.L. et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch. Intern. Med. 163, 1009–1021 (2003).

    Article  Google Scholar 

  5. Hesseling, A.C. et al. The risk of disseminated bacille Calmette-Guerin (BCG) disease in HIV-infected children. Vaccine 25, 14–18 (2007).

    Article  Google Scholar 

  6. Hinchey, J. et al. Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis. J. Clin. Invest. 117, 2279–2288 (2007).

    Article  CAS  Google Scholar 

  7. Orme, I.M. Preclinical testing of new vaccines for tuberculosis: a comprehensive review. Vaccine 24, 2–19 (2006).

    Article  Google Scholar 

  8. Skeiky, Y.A. & Sadoff, J.C. Advances in tuberculosis vaccine strategies. Nat. Rev. Microbiol. 4, 469–476 (2006).

    Article  CAS  Google Scholar 

  9. Sander, C.R. et al. Safety and immunogenicity of a new TB vaccine, MVA85A, in M. tuberculosis infected individuals. Am. J. Respir. Crit. Care Med. 179, 724–733 (2009).

    Article  CAS  Google Scholar 

  10. Armstrong, J.A. & Hart, P.D. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J. Exp. Med. 134, 713–740 (1971).

    Article  CAS  Google Scholar 

  11. Fulton, S.A. et al. Inhibition of major histocompatibility complex II expression and antigen processing in murine alveolar macrophages by Mycobacterium bovis BCG and the 19-kilodalton mycobacterial lipoprotein. Infect. Immun. 72, 2101–2110 (2004).

    Article  CAS  Google Scholar 

  12. Pai, R.K. et al. Prolonged toll-like receptor signaling by Mycobacterium tuberculosis and its 19-kilodalton lipoprotein inhibits gamma interferon-induced regulation of selected genes in macrophages. Infect. Immun. 72, 6603–6614 (2004).

    Article  CAS  Google Scholar 

  13. Dao, D.N. et al. Mycolic acid modification by the mmaA4 gene of M. tuberculosis modulates IL-12 production. PLoS Pathog. 4, e1000081 (2008).

    Article  Google Scholar 

  14. Baena, A. & Porcelli, S.A. Evasion and subversion of antigen presentation by Mycobacterium tuberculosis. Tissue Antigens 74, 189–204 (2009).

    Article  CAS  Google Scholar 

  15. Mahairas, G.G., Sabo, P.J., Hickey, M.J., Singh, D.C. & Stover, C.K. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J. Bacteriol. 178, 1274–1282 (1996).

    Article  CAS  Google Scholar 

  16. Behr, M.A. et al. Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 1520–1523 (1999).

    Article  CAS  Google Scholar 

  17. Lewis, K.N. et al. Deletion of RD1 from Mycobacterium tuberculosis mimics bacille Calmette-Guerin attenuation. J. Infect. Dis. 187, 117–123 (2003).

    Article  Google Scholar 

  18. Pym, A.S. et al. Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis. Nat. Med. 9, 533–539 (2003).

    Article  CAS  Google Scholar 

  19. Stanley, S.A., Raghavan, S., Hwang, W.W. & Cox, J.S. Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proc. Natl. Acad. Sci. USA 100, 13001–13006 (2003).

    Article  CAS  Google Scholar 

  20. Hsu, T. et al. The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc. Natl. Acad. Sci. USA 100, 12420–12425 (2003).

    Article  CAS  Google Scholar 

  21. MacGurn, J.A. & Cox, J.S. A genetic screen for Mycobacterium tuberculosis mutants defective for phagosome maturation arrest identifies components of the ESX-1 secretion system. Infect. Immun. 75, 2668–2678 (2007).

    Article  CAS  Google Scholar 

  22. de Jonge, M.I. et al. ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity. J. Bacteriol. 189, 6028–6034 (2007).

    Article  CAS  Google Scholar 

  23. Xu, J. et al. A unique Mycobacterium ESX-1 protein co-secretes with CFP-10/ESAT-6 and is necessary for inhibiting phagosome maturation. Mol. Microbiol. 66, 787–800 (2007).

    Article  CAS  Google Scholar 

  24. Smith, J. et al. Evidence for pore formation in host cell membranes by ESX-1-secreted ESAT-6 and its role in Mycobacterium marinum escape from the vacuole. Infect. Immun. 76, 5478–5487 (2008).

    Article  CAS  Google Scholar 

  25. van der Wel, N. et al. M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129, 1287–1298 (2007).

    Article  CAS  Google Scholar 

  26. Gey Van Pittius, N.C. et al. The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria. Genome Biol. 2, research0044 (2001).

    Article  CAS  Google Scholar 

  27. Stinear, T.P. et al. Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res. 18, 729–741 (2008).

    Article  CAS  Google Scholar 

  28. Maciag, A. et al. Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon. J. Bacteriol. 189, 730–740 (2007).

    Article  CAS  Google Scholar 

  29. Rodriguez, G.M., Voskuil, M.I., Gold, B., Schoolnik, G.K. & Smith, I. ideR, an essential gene in Mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response. Infect. Immun. 70, 3371–3381 (2002).

    Article  CAS  Google Scholar 

  30. Talaat, A.M., Lyons, R., Howard, S.T. & Johnston, S.A. The temporal expression profile of Mycobacterium tuberculosis infection in mice. Proc. Natl. Acad. Sci. USA 101, 4602–4607 (2004).

    Article  CAS  Google Scholar 

  31. Dubnau, E., Chan, J., Mohan, V.P. & Smith, I. Responses of Mycobacterium tuberculosis to growth in the mouse lung. Infect. Immun. 73, 3754–3757 (2005).

    Article  CAS  Google Scholar 

  32. Sassetti, C.M., Boyd, D.H. & Rubin, E.J. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 48, 77–84 (2003).

    Article  CAS  Google Scholar 

  33. Sassetti, C.M. & Rubin, E.J. Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. USA 100, 12989–12994 (2003).

    Article  CAS  Google Scholar 

  34. Siegrist, M.S. et al. Mycobacterial ESX-3 is required for mycobactin-mediated iron acquisition. Proc. Natl. Acad. Sci. USA 106, 18792–18797 (2009).

    Article  Google Scholar 

  35. Flynn, J.L. & Chan, J. Immunology of tuberculosis. Annu. Rev. Immunol. 19, 93–129 (2001).

    Article  CAS  Google Scholar 

  36. Orme, I.M. & Collins, F.M. Protection against Mycobacterium tuberculosis infection by adoptive immunotherapy. Requirement for T cell-deficient recipients. J. Exp. Med. 158, 74–83 (1983).

    Article  CAS  Google Scholar 

  37. Pierre-Audigier, C. et al. Fatal disseminated Mycobacterium smegmatis infection in a child with inherited interferon gamma receptor deficiency. Clin. Infect. Dis. 24, 982–984 (1997).

    Article  CAS  Google Scholar 

  38. Kumar, K.J., Chandra, J., Mandal, R.N., Dutta, R. & Jain, N.K. Fatal pulmonary infection caused by Mycobacterium smegmatis in an infant. Indian J. Pediatr. 62, 619–621 (1995).

    Article  CAS  Google Scholar 

  39. Abdallah, A.M. et al. The ESX-5 secretion system of Mycobacterium marinum modulates the macrophage response. J. Immunol. 181, 7166–7175 (2008).

    Article  CAS  Google Scholar 

  40. Raghavan, S., Manzanillo, P., Chan, K., Dovey, C. & Cox, J.S. Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454, 717–721 (2008).

    Article  CAS  Google Scholar 

  41. Yang, C. et al. GLS/IL-12-modified Mycobacterium smegmatis as a novel anti-tuberculosis immunotherapeutic vaccine. Int. J. Tuberc. Lung Dis. 13, 1360–1366 (2009).

    CAS  Google Scholar 

  42. Yi, Z. et al. Recombinant M. smegmatis vaccine targeted delivering IL-12/GLS into macrophages can induce specific cellular immunity against M. tuberculosis in BALB/c mice. Vaccine 25, 638–648 (2007).

    Article  CAS  Google Scholar 

  43. Yeremeev, V.V. et al. The 19-kD antigen and protective immunity in a murine model of tuberculosis. Clin. Exp. Immunol. 120, 274–279 (2000).

    Article  CAS  Google Scholar 

  44. Sun, R. et al. Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization, safety and protection against challenge with Mycobacterium tuberculosis. Vaccine 27, 4412–4423 (2009).

    Article  CAS  Google Scholar 

  45. Kita, Y. et al. Novel recombinant BCG and DNA-vaccination against tuberculosis in a cynomolgus monkey model. Vaccine 23, 2132–2135 (2005).

    Article  CAS  Google Scholar 

  46. van Dissel, J.T. et al. Ag85B-ESAT-6 adjuvanted with IC31 promotes strong and long-lived Mycobacterium tuberculosis specific T cell responses in naive human volunteers. Vaccine 28, 3571–3581 (2010).

    Article  CAS  Google Scholar 

  47. Williams, A. et al. Boosting with poxviruses enhances Mycobacterium bovis BCG efficacy against tuberculosis in guinea pigs. Infect. Immun. 73, 3814–3816 (2005).

    Article  CAS  Google Scholar 

  48. Whelan, K.T. et al. Safety and immunogenicity of boosting BCG vaccinated subjects with BCG: comparison with boosting with a new TB vaccine, MVA85A. PLoS ONE 4, e5934 (2009).

    Article  Google Scholar 

  49. Vordermeier, H.M. et al. Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis. Infect. Immun. 77, 3364–3373 (2009).

    Article  CAS  Google Scholar 

  50. Bendelac, A., Bonneville, M. & Kearney, J.F. Autoreactivity by design: innate B and T lymphocytes. Nat. Rev. Immunol. 1, 177–186 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge the support of US National Institutes of Health (NIH) grants AI063537, AI093649, AI092448, AI26170 and AI051519 (Einstein Center for AIDS Research), and the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery. K.A.S. acknowledges support from the Albert Einstein College of Medicine's Institutional AIDS Training Grant T32-AI007501. We thank C. Harding, L. Ramachandra, G. Lauvau and S. Morris for advice and suggestions, as well as C. Colon-Berezin and S. Tiwari for helpful editing of the manuscript. Flow cytometry studies were carried out using core facilities supported by the Einstein Cancer Center (NIH/National Cancer Institute CA013330) and the Einstein Center for AIDS Research (NIH AI051519).

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Author notes

  1. Dee N Dao and Michael F Goldberg: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, New York, USA

    Kari A Sweeney, Dee N Dao, Tsungda Hsu, Paras Jain, Bing Chen, Mei Chen, John Kim, Regy Lukose & William R Jacobs Jr

  2. Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, USA

    Kari A Sweeney, Dee N Dao, Michael F Goldberg, Tsungda Hsu, Manjunatha M Venkataswamy, Paras Jain, Bing Chen, Mei Chen, John Kim, Regy Lukose, John Chan, Steven A Porcelli & William R Jacobs Jr

  3. Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA

    Marcela Henao-Tamayo, Diane Ordway & Ian M Orme

  4. Department of Pathology, Albert Einstein College of Medicine, Bronx, New York, USA

    Rani S Sellers

  5. Department of Medicine, Albert Einstein College of Medicine, Bronx, New York, USA

    John Chan, Steven A Porcelli & William R Jacobs Jr

Authors
  1. Kari A Sweeney
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Contributions

K.A.S. constructed bacterial strains, performed or contributed to the design of most experiments, and analyzed and interpreted data. D.N.D. carried out portions of the infection and challenge experiments. M.F.G. contributed to the T cell adoptive transfer studies and designed, performed and analyzed all flow cytometry analyses. T.H. participated in design and construction of bacterial strains and in the performance of mouse infection and challenge experiments. P.J. contributed to construction of bacterial strains. M.M.V. and M.H.-T. assisted with experiments analyzing responding T cell populations. R.S.S. analyzed and scored the histopathology samples. B.C., M.C., J.K. and R.L. carried out mouse infections, organ harvesting and quantification of bacilli in tissues. D.O., J.C., I.M.O., S.A.P. and W.R.J. Jr. designed and interpreted experiments. K.A.S., S.A.P. and W.R.J. Jr. wrote the manuscript.

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Correspondence to William R Jacobs Jr.

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Sweeney, K., Dao, D., Goldberg, M. et al. A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis. Nat Med 17, 1261–1268 (2011). https://doi.org/10.1038/nm.2420

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