Folia Microbiologica

, Volume 58, Issue 2, pp 111–121

Immunomodulatory properties of subcellular fractions of a G+ bacterium, Bacillus firmus

  • Dana Cechova
  • Michaela Novakova
  • Karel Mikulik
  • Olga Novotna
  • Jaroslav Julak
  • Peter Zanvit
  • Ludmila Prokesova
Article
  • 197 Downloads

Abstract

Mucosal immunization with non-living antigens usually requires the use of an adjuvant. The adjuvant activity of Bacillus firmus in the mucosal immunization of mice was described by our laboratory previously. In the present study, subcellular localization of B. firmus activities was followed. After mechanical disintegration, subcellular components of bacterium were fractionated by differential centrifugation and salting out. Bacterial cell walls, cytoplasmic membrane fraction, soluble cytoplasmic proteins, and ribosomal fractions were isolated. Their effect on the mouse immune system was studied. Lymphocyte proliferation and immunoglobulin formation in vitro were stimulated by bacterial cell wall (BCW), cytoplasmic membrane (CMF), and ribosomal fractions. BCW and CMF increased antibody formation after intratracheal immunization of mice with influenza A and B viruses, and increased protection against subsequent infection with influenza virus. The BCW fraction even induced intersubtypic cross-protection: Mice immunized with A/California/7/04 (H3N2) + BCW were resistant to the infection by the highly pathogenic A/PR/8/34 (H1N1) virus.

Abbreviations

BAL

Bronchoalveolar lavage

BCW

Bacterial cell wall fraction

CMF

Cytoplasmic membrane fraction

ConA

Concavalin A

EID50

50% egg infective dose

FTS

Fetal calf serum

HT

Hemagglutination titer

iNOS

Inducible NO synthase

NAL

Nasal cavity lavage

PWM

Pokeweed mitogen

RiPF

Purified ribosomal fraction

RiCF

Crude ribosomal fraction

SCPF

Fraction of soluble cytoplasmic proteins

References

  1. Ahmad N, Deeba F, Faisal SM, Khan A, Agrewala JN, Dwivedi V, Owais M (2006) Role of fusogenic non-PC liposomes in elicitation of protective immune response against experimental murine salmonellosis. Biochimie 88:1391–1400PubMedCrossRefGoogle Scholar
  2. Barnes AG, Cerovic V, Hobson PS, Klavinskis LS (2007) Bacillus subtilis spores: a novel microparticle adjuvant which can instruct a balanced Th1 and Th2 immune response to specific antigen. Eur J Immunol 37:1538–1547PubMedCrossRefGoogle Scholar
  3. Bellanti J, Olivieri D, Serrano E (2003) Ribosomal immunostimulation: assessment of studies evaluating its clinical relevance in the prevention of upper and lower respiratory tract infections in children and adults. BioDrugs 17:335–367CrossRefGoogle Scholar
  4. Couch RB, Keitel WA, Cate TR (1997) Improvement of inactivated influenza virus vaccines. J Infect Dis 176(Suppl 1):S38–S44PubMedCrossRefGoogle Scholar
  5. Draing C, Sigel S, Deininger S, Traub S, Munke R, Mayer C, Hareng L, Hartung T, von Hermann ASC (2008) Cytokine induction by gram-positive bacteria. Immunobiology 213:285–296PubMedCrossRefGoogle Scholar
  6. Freedman HH, Sultzer BM (1962) Dissociation of the biological properties of bacterial endotoxin by chemical modification of the molecule. J Exp Med 116:929–942PubMedCrossRefGoogle Scholar
  7. Girvan RC, Knight DA, O’Loughlin CJ, Hayman CM, Hermans IF, Webster GA (2011) MIS416, a non-toxic microparticle adjuvant derived from Propionibacterium acnes comprising immunostimulatory muramyl dipeptide and bacterial DNA promotes cross-priming and Th1 immunity. Vaccine 29(3):545–557PubMedCrossRefGoogle Scholar
  8. Havlickova M, Prokesova L, Zanvit P, Tacner J, Limberkova R (2006) Adjuvant effect of Bacillus firmus in intranasal immunization of guinea pigs with inactivated type B influenza virus. Folia Microbiol (Praha) 51:154–156CrossRefGoogle Scholar
  9. Huang JM, La Ragione RM, Nunez A, Cutting SM (2008) Immunostimulatory activity of Bacillus spores. FEMS Immunol Med Microbiol 53:195–203PubMedCrossRefGoogle Scholar
  10. Jensen GS, Benson KF, Carter SG, Endres JR (2010) GanedenBC30 cell wall and metabolites: anti-inflammatory and immune modulating effects in vitro. BMC Immunol 11:15PubMedCrossRefGoogle Scholar
  11. Lomakova I, Petraskova P, Sterzl I, Prokesova L (2006) Immunomodulatory effects of Bacillus firmus on mouse peritoneal cells in vitro. Folia Microbiol (Praha) 51:243–247CrossRefGoogle Scholar
  12. Lycke N, Benmark M (2010) Mucosal adjuvants and long-term memory development with special focus on CTA1-DD and other ADP-ribosylating toxins. Mucosal Immunol 3:556–566PubMedCrossRefGoogle Scholar
  13. Mara M, Ocenaskova J, Novakova M, Julak J, Mencikova E (1994) Resistance to infection and activation of the monocyto-macrophage system caused by Bacillus firmus and its fractions. Folia Microbiol (Praha) 39:147–151CrossRefGoogle Scholar
  14. Minne A, Louahed J, Mehauden S, Baras B, Renauld JC, Vanbever R (2007) The delivery site of a monovalent influenza vaccine within the respiratory tract impacts on the immune response. Immunology 122:316–325PubMedCrossRefGoogle Scholar
  15. Mlckova P, Cechova D, Chalupna P, Novotna O, Prokesova L (2001) Enhanced systemic and mucosal antibody responses to a model protein antigen after intranasal and intratracheal immunisation using Bacillus firmus as an adjuvant. Immunol Lett 77:39–45PubMedCrossRefGoogle Scholar
  16. Morrison DC, Lei MG, Kirikae T, Chen TY (1993) Endotoxin receptors on mammalian cells. Immunobiology 187:212–226PubMedCrossRefGoogle Scholar
  17. Munford RS (2008) Sensing gram-negative bacterial lipopolysaccharides: a human disease determinant? Infect Immun 76:454–465PubMedCrossRefGoogle Scholar
  18. Mutsch M, Zhou W, Rhodes P, Bopp M, Chen RT, Linder T, Spyr C, Steffen R (2004) Use of the inactivated intranasal influenza vaccine and the risk of Bell’s palsy in Switzerland. N Engl J Med 350:896–903PubMedCrossRefGoogle Scholar
  19. Neumann B, Pospiech A, Schairer HU (1992) Rapid isolation of genomic DNA from gram-negative bacteria. Trends Genet 8:332–333PubMedGoogle Scholar
  20. Nichol KL, Mendelman PM, Mallon KP et al (1999) Effectiveness of live attenuated intranasal influenza virus vaccine in healthy working adults: a randomized controlled trial. Jama 2825(2):137–144CrossRefGoogle Scholar
  21. Ogra PL, Fishaut M, Gallagher MR (1980) Viral vaccination via the mucosal routes. Rev Infect Dis 2:352–369PubMedCrossRefGoogle Scholar
  22. Poltorak A, He X, Smirnova I, Liu MY, Van HC, Du X, Birdwell D, Alejos E, Silva M, Galanos C, Freudenberg M, Ricciardi-Castagnoli P, Layton B, Beutler B (1998) Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282:2085–2088PubMedCrossRefGoogle Scholar
  23. Portales P, Clot J (2006) Immunostimulants revisited: focus on the pharmacology of Ribomunyl. BioDrugs 20:81–84PubMedCrossRefGoogle Scholar
  24. Prokesova L, Mlckova P, Stankova I, Chloubova A, Novotna V, Ladmanova P, Chalupna P, Mara M (1998) Effect of Bacillus firmus on antibody formation after mucosal and parenteral immunization in mice. Immunol Lett 64:161–166PubMedCrossRefGoogle Scholar
  25. Prokesova L, Mlckova P, Stankova I, Ladmanova P, Jezkova J, Chalupna P, Novotna O, Cechova D, Julak J (2002) Immunostimulatory effect of Bacillus firmus on mouse lymphocytes. Folia Microbiol (Praha) 47:193–197CrossRefGoogle Scholar
  26. Prokesova L, Zanvit P, Havlickova M, Tacner J, Jirkovska M, Petraskova P, Novotna O, Cechova D, Julak J (2009) Stimulation of protective and cross-protective immunity against influenza B virus after adjuvant mucosal immunization of mice. Folia Microbiol (Praha) 54:549–552CrossRefGoogle Scholar
  27. Rodriguez-Monroy MA, Moreno-Fierros L (2010) Striking activation of NALT and nasal passages lymphocytes induced by intranasal immunization with Cry1Ac protoxin. Scand J Immunol 71:159–168PubMedCrossRefGoogle Scholar
  28. Ronaghy A, Prakken BJ, Takabayashi K, Firestein GS, Boyle D et al (2002) Immunostimulatory DNA sequences influence the course of adjuvant arthritis. J Immunol 168:51–56PubMedGoogle Scholar
  29. Ryu YH, Baik JE, Yang JS, Kang SS, Im J, Yun CH, Kim DW, Lee K, Chung DK, Ju HR, Han SH (2009) Differential immunostimulatory effects of Gram-positive bacteria due to their lipoteichoic acids. Int Immunopharmacol 9:127–133PubMedCrossRefGoogle Scholar
  30. Selvam R, Maheswari P, Kavitha P, Ravichandran M, Sas B, Ramchand CN (2009) Effect of Bacillus subtilis PB6, a natural probiotic on colon mucosal inflammation and plasma cytokines levels in inflammatory bowel disease. Indian J Biochem Biophys 46:79–85PubMedGoogle Scholar
  31. Smith DJ, Bot S, Dellamary L, Bot A (2003) Evaluation of novel aerosol formulations designed for mucosal vaccination against influenza virus. Vaccine 21:2805–2812PubMedCrossRefGoogle Scholar
  32. Sprott GD, Dicaire CJ, Gurnani K, Deschatelets LA, Krishnan L (2004) Liposome adjuvants prepared from the total polar lipids of Haloferax volcanii, Planococcus spp. and Bacillus firmus differ in ability to elicit and sustain immune responses. Vaccine 22:2154–2162PubMedCrossRefGoogle Scholar
  33. Sugimoto K, Ohata M, Miyoshi J, Ishizaki H, Tsuboi N, Masuda A et al (2004) A serine/threonine kinase, Cot/Tpl2, modulates bacterial DNA-induced IL-12 production and Th cell differentiation. J Clin Invest 114:857–866PubMedGoogle Scholar
  34. Tamura S, Ito Y, Asanuma H, Hirabayashi Y, Suzuki Y, Nagamine T, Aizawa C, Kurata T (1992) Cross-protection against influenza virus infection afforded by trivalent inactivated vaccines inoculated intranasally with cholera toxin B subunit. J Immunol 149:981–988PubMedGoogle Scholar
  35. Tamura S, Yamanaka A, Shimohara M, Tomita T, Komase K, Tsuda Y et al (1994) Synergistic action of cholera toxin B subunit (and Escherichia coli heat-labile toxin B subunit) and a trace amount of cholera whole toxin as an adjuvant for nasal influenza vaccine. Vaccine 12:419–426PubMedCrossRefGoogle Scholar
  36. Uhlmann E, Vollmer J (2003) Recent advances in the development of immunostimulatory oligonucleotides. Curr Opin Drug Discov Devel 6:204–217PubMedGoogle Scholar
  37. Van Roosmalen M, Kanninga R, El Khattabi M, Neef J, Audouy S, Bosma T et al (2006) Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria. Methods 38:144–149PubMedCrossRefGoogle Scholar
  38. Youngner JS, Treanor JJ, Betts RF, Whitaker-Dowling P (1994) Effect of simultaneous administration of cold-adapted and wild-type influenza A viruses on experimental wild-type influenza infection in humans. J Clin Microbiol 32:750–754PubMedGoogle Scholar
  39. Zanvit P, Havlickova M, Tacner J, Novotna O, Jirkovska M, Cechova D, Julak J, Sterzl I, Prokesova L (2008) Protective and cross-protective mucosal immunization of mice by influenza virus type A with bacterial adjuvant. Immunol Lett 115:144–152PubMedCrossRefGoogle Scholar
  40. Zanvit P, Tichopad A, Havlickova M, Novotna O, Jirkovska M, Kolostova K, Cechova D, Julak J, Sterzl I, Prokesova L (2010) Adjuvant effect of Bacillus firmus on the expression of cytokines and toll-like receptors in mouse nasopharynx-associated lymphoid tissue (NALT) after intranasal immunization with inactivated influenza virus type A. Immunol Lett 134:26–34PubMedCrossRefGoogle Scholar
  41. Zidek Z, Tuckova L, Mara M, Barot-Ciorbaru R, Prokesova L, Tlaskalova-Hogenova H (1998) Stimulation of macrophages by Bacillus firmus: production of nitric oxide and cytokines. Int J Immunopharmacol 20:359–368PubMedCrossRefGoogle Scholar

Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2012

Authors and Affiliations

  • Dana Cechova
    • 1
  • Michaela Novakova
    • 1
  • Karel Mikulik
    • 2
  • Olga Novotna
    • 1
  • Jaroslav Julak
    • 1
  • Peter Zanvit
    • 1
  • Ludmila Prokesova
    • 1
  1. 1.First Faculty of Medicine, Institute of Immunology and MicrobiologyCharles University in PraguePrague 2Czech Republic
  2. 2.Institute of MicrobiologyAcademy of Sciences of the Czech RepublicPragueCzech Republic

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