Skip to main content

Advertisement

SpringerLink
Log in

Antimycobacterial activity of Pichia pastoris-derived mature bovine neutrophil β-defensins 5

  • Article
  • Published:
European Journal of Clinical Microbiology & Infectious Diseases Aims and scope Submit manuscript

Abstract

Tuberculosis (TB) is an ongoing threat to global health, and the lack of effective therapies for treating it is also a global problem. Previous studies have shown that human cathelicidin and defensins have effective antimicrobial activity against Mycobacterium spp. To our knowledge, there are no reports on the antimycobacterial effects of bovine neutrophil β-defensins so far. Here, we identified the antimicrobial effect of mature bovine neutrophil β-defensins (mBNBD) 5 against Mycobacterium infection both in vitro and in vivo. The mBNBD5 protein was expressed in Pichia pastoris. To increase the yield of β-defensins, a purification method was employed by adding a 6-His·tag to the C-terminus of the mBNBD5 gene. Our results indicated that recombinant mBNBD5 protein was successfully expressed and purified from Pichia pastoris with intact antimicrobial activity. The recombinant protein exhibited potent bactericidal activity in vitro against M. smegmatis and M. bovis, with a dose-dependent manner and a time-dependent manner. The electron microscope results showed that the bacterial cell wall of M. bovis was disrupted when incubated with mBNBD5 for 72 h. Our data also indicated that the exogenous addition of mBNBD5 could reduce the survival of Mycobacterium spp., especially M. tuberculosis and M. bovis in RAW 264.7 macrophages. These results provide foundations for the development of mBNBD5 as a potential new therapeutic agent for TB treatment.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. O’Reilly LM, Daborn CJ (1995) The epidemiology of Mycobacterium bovis infections in animals and man: a review. Tuber Lung Dis 76(Suppl 1):1–46

    Article  PubMed  Google Scholar 

  2. Grange JM (2001) Mycobacterium bovis infection in human beings. Tuberculosis (Edinb) 81(1–2):71–77. doi:10.1054/tube.2000.0263

    Article  CAS  Google Scholar 

  3. de la Rua-Domenech R (2006) Human Mycobacterium bovis infection in the United Kingdom: incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis. Tuberculosis (Edinb) 86(2):77–109. doi:10.1016/j.tube.2005.05.002

    Article  Google Scholar 

  4. Thoen C, Lobue P, de Kantor I (2006) The importance of Mycobacterium bovis as a zoonosis. Vet Microbiol 112(2–4):339–345. doi:10.1016/j.vetmic.2005.11.047

    Article  PubMed  Google Scholar 

  5. [No authors listed] (1994) Zoonotic tuberculosis (Mycobacterium bovis): memorandum from a WHO meeting (with the participation of FAO). Bull World Health Organ 72(6):851–857

  6. Müller B, Dürr S, Alonso S, Hattendorf J, Laisse CJ, Parsons SD, van Helden PD, Zinsstag J (2013) Zoonotic Mycobacterium bovis-induced tuberculosis in humans. Emerg Infect Dis 19(6):899–908. doi:10.3201/eid1906.120543

    Article  PubMed  Google Scholar 

  7. Cleaveland S, Shaw DJ, Mfinanga SG, Shirima G, Kazwala RR, Eblate E, Sharp M (2007) Mycobacterium bovis in rural Tanzania: risk factors for infection in human and cattle populations. Tuberculosis (Edinb) 87(1):30–43. doi:10.1016/j.tube.2006.03.001

    Article  Google Scholar 

  8. Pinsky BA, Banaei N (2008) Multiplex real-time PCR assay for rapid identification of Mycobacterium tuberculosis complex members to the species level. J Clin Microbiol 46(7):2241–2246. doi:10.1128/JCM.00347-08

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. Cosivi O, Grange JM, Daborn CJ, Raviglione MC, Fujikura T, Cousins D, Robinson RA, Huchzermeyer HF, de Kantor I, Meslin FX (1998) Zoonotic tuberculosis due to Mycobacterium bovis in developing countries. Emerg Infect Dis 4(1):59–70

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  10. Shah NP, Singhal A, Jain A, Kumar P, Uppal SS, Srivatsava MV, Prasad HK (2006) Occurrence of overlooked zoonotic tuberculosis: detection of Mycobacterium bovis in human cerebrospinal fluid. J Clin Microbiol 44(4):1352–1358. doi:10.1128/JCM.44.4.1352-1358.2006

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Hancock RE, Nijnik A, Philpott DJ (2012) Modulating immunity as a therapy for bacterial infections. Nat Rev Microbiol 10(4):243–254. doi:10.1038/nrmicro2745

    Article  PubMed  CAS  Google Scholar 

  12. Peschel A, Sahl HG (2006) The co-evolution of host cationic antimicrobial peptides and microbial resistance. Nat Rev Microbiol 4(7):529–536. doi:10.1038/nrmicro1441

    Article  PubMed  CAS  Google Scholar 

  13. Marr AK, Gooderham WJ, Hancock RE (2006) Antibacterial peptides for therapeutic use: obstacles and realistic outlook. Curr Opin Pharmacol 6(5):468–472. doi:10.1016/j.coph.2006.04.006

    Article  PubMed  CAS  Google Scholar 

  14. Fjell CD, Hiss JA, Hancock RE, Schneider G (2011) Designing antimicrobial peptides: form follows function. Nat Rev Drug Discov 11(1):37–51. doi:10.1038/nrd3591

    PubMed  Google Scholar 

  15. Ganz T, Selsted ME, Szklarek D, Harwig SSL, Daher K, Bainton DF, Lehrer RI (1985) Defensins. Natural peptide antibiotics of human neutrophils. J Clin Invest 76(4):1427–1435

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  16. Jarczak J, Kościuczuk EM, Lisowski P, Strzałkowska N, Jóźwik A, Horbańczuk J, Krzyżewski J, Zwierzchowski L, Bagnicka E (2013) Defensins: natural component of human innate immunity. Hum Immunol 74(9):1069–1079. doi:10.1016/j.humimm.2013.05.008

    Article  PubMed  CAS  Google Scholar 

  17. Menendez A, Brett Finlay B (2007) Defensins in the immunology of bacterial infections. Curr Opin Immunol 19(4):385–391. doi:10.1016/j.coi.2007.06.008

    Article  PubMed  CAS  Google Scholar 

  18. Méndez-Samperio P, Alba L, Trejo A (2007) Mycobacterium bovis-mediated induction of human beta-defensin-2 in epithelial cells is controlled by intracellular calcium and p38MAPK. J Infect 54(5):469–474. doi:10.1016/j.jinf.2006.08.009

    Article  PubMed  Google Scholar 

  19. Raj PA, Dentino AR (2002) Current status of defensins and their role in innate and adaptive immunity. FEMS Microbiol Lett 206(1):9–18

    Article  PubMed  CAS  Google Scholar 

  20. Rivas-Santiago B, Schwander SK, Sarabia C, Diamond G, Klein-Patel ME, Hernandez-Pando R, Ellner JJ, Sada E (2005) Human {beta}-defensin 2 is expressed and associated with Mycobacterium tuberculosis during infection of human alveolar epithelial cells. Infect Immun 73(8):4505–4511. doi:10.1128/IAI.73.8.4505-4511.2005

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Selsted ME, Tang YQ, Morris WL, McGuire PA, Novotny MJ, Smith W, Henschen AH, Cullor JS (1993) Purification, primary structures, and antibacterial activities of beta-defensins, a new family of antimicrobial peptides from bovine neutrophils. J Biol Chem 268(9):6641–6648

    PubMed  CAS  Google Scholar 

  22. Ogata K, Linzer BA, Zuberi RI, Ganz T, Lehrer RI, Catanzaro A (1992) Activity of defensins from human neutrophilic granulocytes against Mycobacterium avium–Mycobacterium intracellulare. Infect Immun 60(11):4720–4725

    PubMed  CAS  PubMed Central  Google Scholar 

  23. Ryan LK, Rhodes J, Bhat M, Diamond G (1998) Expression of beta-defensin genes in bovine alveolar macrophages. Infect Immun 66(2):878–881

    PubMed  CAS  PubMed Central  Google Scholar 

  24. Méndez-Samperio P (2008) Role of antimicrobial peptides in host defense against mycobacterial infections. Peptides 29(10):1836–1841. doi:10.1016/j.peptides.2008.05.024

    Article  PubMed  Google Scholar 

  25. Sonawane A, Santos JC, Mishra BB, Jena P, Progida C, Sorensen OE, Gallo R, Appelberg R, Griffiths G (2011) Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages. Cell Microbiol 13(10):1601–1617. doi:10.1111/j.1462-5822.2011.01644.x

    Article  PubMed  CAS  Google Scholar 

  26. Rivas-Santiago B, Castañeda-Delgado JE, Rivas Santiago CE, Waldbrook M, González-Curiel I, León-Contreras JC, Enciso-Moreno JA, del Villar V, Mendez-Ramos J, Hancock REW, Hernandez-Pando R (2013) Ability of innate defence regulator peptides IDR-1002, IDR-HH2 and IDR-1018 to protect against Mycobacterium tuberculosis infections in animal models. PLoS One 8(3):e59119. doi:10.1371/journal.pone.0059119

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Ganz T, Lehrer RI (1998) Antimicrobial peptides of vertebrates. Curr Opin Immunol 10(1):41–44

    Article  PubMed  CAS  Google Scholar 

  28. Diamond G, Zasloff M, Eck H, Brasseur M, Maloy WL, Bevins CL (1991) Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: peptide isolation and cloning of a cDNA. Proc Natl Acad Sci U S A 88(9):3952–3956

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Cabral KM, Almeida MS, Valente AP, Almeida FC, Kurtenbach E (2003) Production of the active antifungal Pisum sativum defensin 1 (Psd1) in Pichia pastoris: overcoming the inefficiency of the STE13 protease. Protein Expr Purif 31(1):115–122. doi:10.1016/s1046-5928(03)00136-0

    Article  PubMed  CAS  Google Scholar 

  30. Zhao P, Cao G (2012) Production of bioactive sheep beta-defensin-1 in Pichia pastoris. J Ind Microbiol Biotechnol 39(1):11–17. doi:10.1007/s10295-011-0992-x

    Article  PubMed  Google Scholar 

  31. Shi X, Karkut T, Chamankhah M, Alting-Mees M, Hemmingsen SM, Hegedus D (2003) Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris. Protein Expr Purif 28(2):321–330. doi:10.1016/s1046-5928(02)00706-4

    Article  PubMed  CAS  Google Scholar 

  32. Huang Y, Zhou X, Bai Y, Yang L, Yin X, Wang Z, Zhao D (2012) Phagolysosome maturation of macrophages was reduced by PE_PGRS 62 protein expressing in Mycobacterium smegmatis and induced in IFN-gamma priming. Vet Microbiol 160(1–2):117–125. doi:10.1016/j.vetmic.2012.05.011

    Article  PubMed  CAS  Google Scholar 

  33. Schägger H (2006) Tricine-SDS-PAGE. Nat Protoc 1(1):16–22. doi:10.1038/nprot.2006.4

    Article  PubMed  Google Scholar 

  34. Mohammadzadeh T, Sadjjadi S, Habibi P, Sarkari B (2012) Comparison of agar dilution, broth dilution, cylinder plate and disk diffusion methods for evaluation of anti-leishmanial drugs on leishmania promastigotes. Iran J Parasitol 7(3):43–47

    PubMed  CAS  PubMed Central  Google Scholar 

  35. Zeya HI, Spitznagel JK (1968) Arginine-rich proteins of polymorphonuclear leukocyte lysosomes. Antimicrobial specificity and biochemical heterogeneity. J Exp Med 127(5):927–941

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  36. Ganz T, Lehrer RI (1995) Defensins. Pharmacol Ther 66(2):191–205

    Article  PubMed  CAS  Google Scholar 

  37. Harder J, Bartels J, Christophers E, Schroder JM (2001) Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic. J Biol Chem 276(8):5707–5713. doi:10.1074/jbc.M008557200

    Article  PubMed  CAS  Google Scholar 

  38. Si L-G, Liu X-C, Lu Y-Y, Wang G-Y, Li W-M (2007) Soluble expression of active human β-defensin-3 in Escherichia coli and its effects on the growth of host cells. Chin Med J (Engl) 120(8):708–713

    CAS  Google Scholar 

  39. Li Y (2011) Recombinant production of antimicrobial peptides in Escherichia coli: a review. Protein Expr Purif 80(2):260–267. doi:10.1016/j.pep.2011.08.001

    Article  PubMed  CAS  Google Scholar 

  40. Bommarius B, Jenssen H, Elliott M, Kindrachuk J, Pasupuleti M, Gieren H, Jaeger KE, Hancock RE, Kalman D (2010) Cost-effective expression and purification of antimicrobial and host defense peptides in Escherichia coli. Peptides 31(11):1957–1965. doi:10.1016/j.peptides.2010.08.008

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  41. Zorko M, Japelj B, Hafner-Bratkovic I, Jerala R (2009) Expression, purification and structural studies of a short antimicrobial peptide. Biochim Biophys Acta 1788(2):314–323. doi:10.1016/j.bbamem.2008.10.015

    Article  PubMed  CAS  Google Scholar 

  42. Corrales-Garcia L, Ortiz E, Castañeda-Delgado J, Rivas-Santiago B, Corzo G (2013) Bacterial expression and antibiotic activities of recombinant variants of human beta-defensins on pathogenic bacteria and M. tuberculosis. Protein Expr Purif 89(1):33–43. doi:10.1016/j.pep.2013.02.007

    Article  PubMed  CAS  Google Scholar 

  43. Kisich KO, Heifets L, Higgins M, Diamond G (2001) Antimycobacterial agent based on mRNA encoding human beta-defensin 2 enables primary macrophages to restrict growth of Mycobacterium tuberculosis. Infect Immun 69(4):2692–2699. doi:10.1128/IAI.69.4.2692-2699.2001

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  44. Nickel D, Busch M, Mayer D, Hagemann B, Knoll V, Stenger S (2012) Hypoxia triggers the expression of human beta defensin 2 and antimicrobial activity against Mycobacterium tuberculosis in human macrophages. J Immunol 188(8):4001–4007. doi:10.4049/jimmunol.1100976

    Article  PubMed  CAS  Google Scholar 

  45. Gonzalez-Curiel I, Castañeda-Delgado J, Lopez-Lopez N, Araujo Z, Hernandez-Pando R, Gandara-Jasso B, Macias-Segura N, Enciso-Moreno A, Rivas-Santiago B (2011) Differential expression of antimicrobial peptides in active and latent tuberculosis and its relationship with diabetes mellitus. Hum Immunol 72(8):656–662. doi:10.1016/j.humimm.2011.03.027

    Article  PubMed  CAS  Google Scholar 

  46. Rivas-Santiago B, Cervantes-Villagrana A, Sada E, Hernández-Pando R (2012) Expression of beta defensin 2 in experimental pulmonary tuberculosis: tentative approach for vaccine development. Arch Med Res 43(4):324–328. doi:10.1016/j.arcmed.2012.06.005

    Article  PubMed  CAS  Google Scholar 

  47. Rivas-Santiago CE, Rivas-Santiago B, León DA, Castañeda-Delgado J, Hernández Pando R (2011) Induction of beta-defensins by l-isoleucine as novel immunotherapy in experimental murine tuberculosis. Clin Exp Immunol 164(1):80–89. doi:10.1111/j.1365-2249.2010.04313.x

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  48. Kisich KO, Higgins M, Diamond G, Heifets L (2002) Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils. Infect Immun 70(8):4591–4599. doi:10.1128/IAI.70.8.4591-4599.2002

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  49. Liu PT, Stenger S, Tang DH, Modlin RL (2007) Cutting edge: vitamin D-mediated human antimicrobial activity against Mycobacterium tuberculosis is dependent on the induction of cathelicidin. J Immunol 179(4):2060–2063

    Article  PubMed  CAS  Google Scholar 

  50. Yount NY, Yeaman MR (2005) Immunocontinuum: perspectives in antimicrobial peptide mechanisms of action and resistance. Protein Pept Lett 12(1):49–67

    Article  PubMed  CAS  Google Scholar 

  51. Yount NY, Bayer AS, Xiong YQ, Yeaman MR (2006) Advances in antimicrobial peptide immunobiology. Biopolymers 84(5):435–458. doi:10.1002/bip.20543

    Article  PubMed  CAS  Google Scholar 

  52. Sharma S, Verma I, Khuller GK (1999) Biochemical interaction of human neutrophil peptide-1 with Mycobacterium tuberculosis H37Ra. Arch Microbiol 171(5):338–342

    Article  PubMed  CAS  Google Scholar 

  53. Anes E, Kühnel MP, Bos E, Moniz-Pereira J, Habermann A, Griffiths G (2003) Selected lipids activate phagosome actin assembly and maturation resulting in killing of pathogenic mycobacteria. Nat Cell Biol 5(9):793–802. doi:10.1038/ncb1036

    Article  PubMed  CAS  Google Scholar 

  54. Mittal M, Chakravarti S, Sharma V, Sanjeeth BS, Churamani CP, Kanwar NS (2014) Evidence of presence of Mycobacterium tuberculosis in bovine tissue samples by multiplex PCR: possible relevance to reverse zoonosis. Transbound Emerg Dis 61(2):97–104. doi:10.1111/tbed.12203

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National High Technology Research and Development Program of China (863 Program, Project No. 2012AA101302), MoSTRCUK international cooperation project (Project No. 2013DFG32500), Chinese Universities Scientific Fund (Project No. 2013QT004), and CAU Foreign Experts Major Projects (Project No: 2013z018). We would also like to thank China Agricultural University for providing access to the BSL-3 Laboratories for experiments using M. bovis culture and infection.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. The State Key Lab of Agrobiotechnology; Key Lab of Animal Epidemiology and Zoonosis, Ministry of Agriculture; National Animal Transmissible Spongiform Encephalopathy Laboratory; College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People’s Republic of China

    J. Kang, D. Zhao, Y. Lyu, L. Tian, X. Yin, L. Yang, K. Teng & X. Zhou

  2. College of Wildlife Resource, Northeast Forestry University, Harbin, 150030, People’s Republic of China

    L. Tian

Authors
  1. J. Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D. Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Y. Lyu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. L. Tian
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. X. Yin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. L. Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. K. Teng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. X. Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to X. Zhou.

Additional information

Jingjing Kang and Deming Zhao contributed to this work equally.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kang, J., Zhao, D., Lyu, Y. et al. Antimycobacterial activity of Pichia pastoris-derived mature bovine neutrophil β-defensins 5. Eur J Clin Microbiol Infect Dis 33, 1823–1834 (2014). https://doi.org/10.1007/s10096-014-2152-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10096-014-2152-5

Keywords

Search

Navigation