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Putative roles of a proline–glutamic acid-rich protein (PE3) in intracellular survival and as a candidate for subunit vaccine against Mycobacterium tuberculosis

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The proline–glutamic acid (PE) protein family of Mycobacterium tuberculosis (Mtb) plays diverse roles in the pathogenesis and modulation of host immune responses. The uniqueness of conserved regions of PE proteins may be useful to test and validate their corresponding functions. Hence, the present study has been undertaken to demonstrate the role of PE3 (Rv0159c) for persistence, host immune response and immunoprophylaxis. We have expressed Mtb-specific PE3 gene in M. smegmatis (MS) and used the strain to infect J774A.1 macrophage cells and BALB/c mice. It was observed that during the infection, the MS expressing PE3 showed higher bacterial load when compared to infection with wild-type MS. In hypoxic condition, the expression level of PE3 gene was induced in Mtb, which further showed its relevance in the cell survival during hypoxia-induced persistence. The expression level of PE3 in Mtb was markedly induced during chronic stage of murine infection, which reiterated its importance in mycobacterial persistence in the host. The immunization of mice with recombinant PE3 protein stimulated the secretion of TNF, IL-6 and IL-2 cytokines and generated strong protective immunity against challenge with live mycobacteria, which was evidenced by decreased viable bacilli in the lungs, histopathological changes and increased survival of PE3 immunized mice. Conclusively, the results indicated that PE3 plays significant roles in mycobacterial persistence during infection, modulate host immune response and hence could be a prospective candidate for the development of subunit vaccine against tuberculosis.

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  1. 1.

    Mukhopadhyay S, Nair S, Ghosh S (2012) Pathogenesis in tuberculosis: transcriptomic approaches to unraveling virulence mechanisms and finding new drug targets. FEMS Microbiol Rev 36:463–485

  2. 2.

    Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE III, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544

  3. 3.

    Chaturvedi R, Bansal K, Narayana Y, Kapoor N, Sukumar N, Togarsimalemath SK, Chandra N, Mishra S, Ajitkumar P, Joshi B, Katoch VM, Patil SA, Balaji KN (2010) The multifunctional PE_PGRS11 protein from Mycobacterium tuberculosis plays a role in regulating resistance to oxidative stress. J Biol Chem 285:30389–30403

  4. 4.

    Adindla S, Guruprasad L (2003) Sequence analysis corresponding to the PPE and PE proteins in Mycobacterium tuberculosis and other genomes. J Biosci 28:169–179

  5. 5.

    Koh KW, Lehming N, Seah GT (2009) Degradation-resistant protein domains limit host cell processing and immune detection of mycobacteria. Mol Immunol 46:1312–1318

  6. 6.

    Schnappinger D, Ehrt S, Voskuil MI, Liu Y, Mangan JA, Monahan IM, Dolganov G, Efron B, Butcher PD, Nathan C, Schoolnik GK (2003) Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: insights into the phagosomal environment. J Exp Med 198:693–704

  7. 7.

    Delogu G, Pusceddu C, Bua A, Fadda G, Brennan MJ, Zanetti S (2004) Rv1818c-encoded PE_PGRS protein of Mycobacterium tuberculosis is surface exposed and influences bacterial cell structure. Mol Microbiol 52:725–733

  8. 8.

    Dheenadhayalan V, Delogu G, Brennan MJ (2006) Expression of the PE_PGRS 33 protein in Mycobacterium smegmatis triggers necrosis in macrophages and enhanced mycobacterial survival. Microbes Infect 8:262–272

  9. 9.

    Karboul A, Mazza A, Gey van Pittius NC, Ho JL, Brousseau R, Mardassi H (2008) Frequent homologous recombination events in Mycobacterium tuberculosis PE/PPE multigene families: potential role in antigenic variability. J Bacteriol 190:7838–7846

  10. 10.

    Poulet S, Cole ST (1995) Characterization of the highly abundant polymorphic GC-rich-repetitive sequence (PGRS) present in Mycobacterium tuberculosis. Arch Microbiol 163:87–95

  11. 11.

    Beatty WL, Russell DG (2000) Identification of mycobacterial surface proteins released into subcellular compartments of infected macrophages. Infect Immun 68:6997–7002

  12. 12.

    Beatty WL, Ullrich HJ, Russell DG (2001) Mycobacterial surface moieties are released from infected macrophages by a constitutive exocytic event. Eur J Cell Biol 80:31–40

  13. 13.

    Sampson SL (2011) Mycobacterial PE/PPE proteins at the host-pathogen interface. Clin Dev Immunol 2011:497203

  14. 14.

    Dong D, Wang D, Li M, Wang H, Yu J, Wang C, Liu J, Gao Q (2012) PPE38 modulates the innate immune response and is required for Mycobacterium marinum virulence. Infect Immun 80:43–54

  15. 15.

    Van Crevel R, Ottenhoff TH, van der Meer JW (2002) Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 15:294–309

  16. 16.

    Sultana R, Tanneeru K, Guruprasad L (2011) The PE-PPE domain in mycobacterium reveals a serine alpha/beta hydrolase fold and function: an in silico analysis. PLoS ONE 6:e16745

  17. 17.

    Li Y, Zeng J, Shi J, Wang M, Rao M, Xue C, Du Y, He ZG (2010) A proteome-scale identification of novel antigenic proteins in Mycobacterium tuberculosis toward diagnostic and vaccine development. J Proteome Res 9:4812–4822

  18. 18.

    Chaurasiya SK, Srivastava KK (2009) Downregulation of protein kinase C-alpha enhances intracellular survival of Mycobacteria: role of PknG. BMC Microbiol 9:271

  19. 19.

    Kolaskar AS, Tongaonkar PC (1990) A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276:172–174

  20. 20.

    Iantomasi R, Sali M, Cascioferro A, Palucci I, Zumbo A, Soldini S, Rocca S, Greco E, Maulucci G, De Spirito M, Fraziano M, Fadda G, Manganelli R, Delogu G (2012) PE_PGRS30 is required for the full virulence of Mycobacterium tuberculosis. Cell Microbiol 14:356–367

  21. 21.

    Wayne LG, Hayes LG (1996) An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect Immun 64:2062–2069

  22. 22.

    Sweeney KA, Dao DN, Goldberg MF, Hsu T, Venkataswamy MM, Henao-Tamayo M, Ordway D, Sellers RS, Jain P, Chen B, Chen M, Kim J, Lukose R, Chan J, Orme IM, Porcelli SA, Jacobs WR Jr (2011) A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis. Nat Med 17:1261–1268

  23. 23.

    Delogu G, Sanguinetti M, Posteraro B, Rocca S, Zanetti S, Fadda G (2006) The hbhA gene of Mycobacterium tuberculosis is specifically upregulated in the lungs but not in the spleens of aerogenically infected mice. Infect Immun 74:3006–3011

  24. 24.

    Banu S, Honore N, Saint-Joanis B, Philpott D, Prevost MC, Cole ST (2002) Are the PE-PGRS proteins of Mycobacterium tuberculosis variable surface antigens? Mol Microbiol 44:9–19

  25. 25.

    Brennan MJ, Delogu G (2002) The PE multigene family: a ‘molecular mantra’ for mycobacteria. Trends Microbiol 10:246–249

  26. 26.

    Singh SK, Tripathi DK, Singh PK, Sharma S, Srivastava KK (2012) Protective and survival efficacies of Rv0160c protein in murine model of Mycobacterium tuberculosis. Appl Microbiol Biotechnol. doi:10.1007/s00253-012-4493-2

  27. 27.

    Lun S, Bishai WR (2007) Characterization of a novel cell wall-anchored protein with carboxylesterase activity required for virulence in Mycobacterium tuberculosis. J Biol Chem 282:18348–18356

  28. 28.

    Nag JK, Shrivastava N, Gupta J, Misra Bhattacharya S (2013) Recombinant translation initiation factor-1 of Wolbachia is an immunogenic excretory secretory protein that elicits Th2 mediated immune protection against Brugia malayi. Comp Immunol Microbiol Infect Dis 36:25–38

  29. 29.

    Brennan MJ, Delogu G, Chen Y, Bardarov S, Kriakov J, Alavi M, Jacobs WR Jr (2001) Evidence that mycobacterial PE_PGRS proteins are cell surface constituents that influence interactions with other cells. Infect Immun 69:7326–7333

  30. 30.

    Tiwari BM, Kannan N, Vemu L, Raghunand TR (2012) The Mycobacterium tuberculosis PE Proteins Rv0285 and Rv1386 modulate innate immunity and mediate bacillary survival in macrophages. PLoS ONE 7:e51686

  31. 31.

    Ramakrishnan L, Federspiel NA, Falkow S (2000) Granuloma-specific expression of mycobacterium virulence proteins from the glycine-rich PE-PGRS family. Science 288:1436–1439

  32. 32.

    Voskuil MI, Schnappinger D, Rutherford R, Liu Y, Schoolnik GK (2004) Regulation of the Mycobacterium tuberculosis PE/PPE genes. Tuberculosis (Edinb) 84:256–262

  33. 33.

    Kindler V, Sappino AP, Grau GE, Piguet PF, Vassalli P (1989) The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 56:731–740

  34. 34.

    Senaldi G, Yin S, Shaklee CL, Piguet PF, Mak TW, Ulich TR (1996) Corynebacterium parvum-and Mycobacterium bovis bacillus Calmette-Guerin-induced granuloma formation is inhibited in TNF receptor I (TNF-RI) knockout mice and by treatment with soluble TNF-RI. J Immunol 157:5022–5026

  35. 35.

    Flynn JL, Goldstein MM, Chan J, Triebold KJ, Pfeffer K, Lowenstein CJ, Schreiber R, Mak TW, Bloom BR (1995) Tumor necrosis factor-alpha is required in the protective immune response against Mycobacterium tuberculosis in mice. Immunity 2:561–572

  36. 36.

    Mohan VP, Scanga CA, Yu K, Scott HM, Tanaka KE, Tsang E, Tsai MM, Flynn JL, Chan J (2001) Effects of tumor necrosis factor alpha on host immune response in chronic persistent tuberculosis: possible role for limiting pathology. Infect Immun 69:1847–1855

  37. 37.

    VanHeyningen TK, Collins HL, Russell DG (1997) IL-6 produced by macrophages infected with mycobacterium species suppresses T cell responses. J Immunol 158:330–337

  38. 38.

    Okamura H, Kashiwamura S, Tsutsui H, Yoshimoto T, Nakanishi K (1998) Regulation of interferon-gamma production by IL-12 and IL-18. Curr Opin Immunol 10:259–264

  39. 39.

    Lima KM, dos Santos SA, Santos RR, Brandão IT, Rodrigues JM Jr, Silva CL (2003) Efficacy of DNA-hsp65 vaccination for tuberculosis varies with method of DNA introduction in vivo. Vaccine 22:49–56

  40. 40.

    Skeiky YA, Alderson MR, Ovendale PJ, Guderian JA, Brandt L, Dillon DC, Campos-Neto A, Lobet Y, Dalemans W, Orme IM, Reed SG (2004) Differential immune responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine, Mtb72F, delivered as naked DNA or recombinant protein. J Immunol 172:7618–7628

  41. 41.

    Lindenstrøm T, Agger EM, Korsholm KS, Darrah PA, Aagaard C, Seder RA, Rosenkrands I, Andersen P (2009) Tuberculosis subunit vaccination provides long-term protective immunity characterized by multifunctional CD4 memory T cells. J Immunol 182:8047–8055

  42. 42.

    Skeiky YA, Ovendale PJ, Jen S, Alderson MR, Dillon DC, Smith S, Wilson CB, Orme IM, Reed SG, Campos-Neto A (2000) T cell expression cloning of a Mycobacterium tuberculosis gene encoding a protective antigen associated with the early control of infection. J Immunol 165:7140–7149

  43. 43.

    Delogu G, Brennan MJ (2001) Comparative immune response to PE and PE_PGRS antigens of Mycobacterium tuberculosis. Infect Immun 69:5606–5611

  44. 44.

    Vipond J, Vipond R, Allen-Vercoe E, Clark SO, Hatch GJ, Gooch KE, Bacon J, Hampshire T, Shuttleworth H, Minton NP, Blake K, Williams A, Marsh PD (2006) Selection of novel TB vaccine candidates and their evaluation as DNA vaccines against aerosol challenge. Vaccine 24:6340–6350

  45. 45.

    Romano M, Rindi L, Korf H, Bonanni D, Adnet PY, Jurion F, Garzelli C, Huygen K (2008) Immunogenicity and protective efficacy of tuberculosis subunit vaccines expressing PPE44 (Rv2770c). Vaccine 26:6053–6639

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We thank Director, CSIR-CDRI for his encouragement and support. Technical assistance by D. K. Tripathi is appreciated. Financial supports by CSIR-UNDO and CSIR-INDEPTH are acknowledged. The CSIR-CDRI communication number alloted to this manuscript is 8458.

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Correspondence to Kishore K. Srivastava.

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Singh, S.K., Kumari, R., Singh, D.K. et al. Putative roles of a proline–glutamic acid-rich protein (PE3) in intracellular survival and as a candidate for subunit vaccine against Mycobacterium tuberculosis . Med Microbiol Immunol 202, 365–377 (2013).

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  • Immunology
  • Mycobacterium tuberculosis
  • Pathogenesis
  • Protein expression
  • Vaccines
  • PE3