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Marine Biotechnology

, Volume 16, Issue 6, pp 707–715 | Cite as

Quorum-Quenching Activity of the AHL-Lactonase from Bacillus licheniformis DAHB1 Inhibits Vibrio Biofilm Formation In Vitro and Reduces Shrimp Intestinal Colonisation and Mortality

  • G. Vinoj
  • B. VaseeharanEmail author
  • S. Thomas
  • A. J. Spiers
  • S. Shanthi
Original Article

Abstract

Vibrio parahaemolyticus is a significant cause of gastroenteritis resulting from the consumption of undercooked sea foods and often cause significant infections in shrimp aquaculture. Vibrio virulence is associated with biofilm formation and is regulated by N-acylated homoserine lactone (AHL)-mediated quorum sensing. In an attempt to reduce vibrio colonisation of shrimps and mortality, we screened native intestinal bacilli from Indian white shrimps (Fenneropenaeus indicus) for an isolate which showed biofilm-inhibitory activity (quorum quenching) against the pathogen V. parahaemolyticus DAHP1. The AHL-lactonase (AiiA) expressed by one of these, Bacillus licheniformis DAHB1, was characterised as having a broad-spectrum AHL substrate specificity and intrinsic resistance to the acid conditions of the shrimp intestine. Purified recombinant AiiA inhibited vibrio biofilm development in a cover slip assay and significantly attenuated infection and mortality in shrimps reared in a recirculation aquaculture system. Investigation of intestinal samples also showed that AiiA treatment also reduced vibrio viable counts and biofilm development as determined by confocal laser scanning microscopy (CLSM) imaging. These findings suggest that the B. licheniformis DAHB1 quorum-quenching AiiA might be developed for use as a prophylactic treatment to inhibit or reduce vibrio colonisation and mortality of shrimps in aquaculture.

Keywords

AHL-lactonase AiiA Bacillus Biofilm Quorum quenching Vibrio parahaemolyticus 

Notes

Acknowledgments

This work was supported by the Indian University Grants Commission (Grant F. no. 36-5/2008 (SR-)). AS was involved in the preparation of the manuscript but not in the experimental work or data analyses.

Supplementary material

10126_2014_9585_Fig5_ESM.gif (54 kb)
Supp. Figure 1

AiiA protein in vitro biofilm inhibition. Shown here are CLSM and light microscopic (40×) images demonstrating the impact of AiiA on biofilm-formation by V. parahaemolyticus DAHP1-GFP after 24 h; control (A & D), with AiiA (C & B). (GIF 54 kb)

10126_2014_9585_MOESM1_ESM.tif (1.4 mb)
High Resolution Image (TIFF 1,387 kb)

References

  1. Bai F, Han Y, Chen J, Zhang XH (2008) Disruption of quorum sensing in Vibrio harveyi by the AiiA protein of Bacillus thuringiensis. Aquac 27:436–40Google Scholar
  2. Bruhn JB, Dalsgaard I, Nielsen KF, Buchholtz C, Larsen JL, Gram L (2005) Quorum sensing signal molecules (acylated homoserine lactones) in gram-negative fish pathogenic bacteria. Dis Aquat Organs 65:43–52CrossRefGoogle Scholar
  3. Cao Y, He S, Zhou Z, Zhang M, Mao W, Zhang H, Yao B (2012) Orally administered thermostable N-Acyl homoserine lactonase from Bacillus sp. strain AI96 attenuates Aeromonas hydrophila infection in Zebrafish. Appl Environ Microbiol 78:899–1908Google Scholar
  4. Chen R, Zhou Z, Cao Y, Bai Y, Yao B (2010) High yield expression of an AHL lactonase from Bacillus sp. B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture. Microb Cell Fact 9:39PubMedCentralCrossRefPubMedGoogle Scholar
  5. Czajkowski R, Jafra S (2009) Quenching of acyl-homoserine lactone-dependent quorum sensing by enzymatic disruption of signal molecules. Acta Biochim Pol 56:1–16PubMedGoogle Scholar
  6. Defoirdt T, Boon N, Sorgeloos P, Verstraete W, Bossier P (2008) Quorum sensing and quorum quenching in Vibrio harveyi: lessons learned from in vivo work. ISME J 2:19–26CrossRefPubMedGoogle Scholar
  7. Defoirdt T, Boon N, Sorgeloos P, Verstraete W, Bossier P (2007) Alternatives to antibiotics to control bacterial infections: luminescent vibriosis in aquaculture as an example. Trends Biotechnol 25:472–479CrossRefPubMedGoogle Scholar
  8. Defoirdt T, Thanh LD, Van Delsen B, De Schryver P, Sorgeloos P, Boon N, Bossier P (2011) N-acylhomoserine lactone-degrading Bacillus strains isolated from aquaculture animals. Aquaculture 311:258–260CrossRefGoogle Scholar
  9. Dong YH, Gusti AR, ZhangQ XJL, Zhang LH (2002) Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species. Appl Environ Microbiol 68:1754–1759PubMedCentralCrossRefPubMedGoogle Scholar
  10. Dong YH, Zhang LH (2005) Quorum sensing and quorum quenching enzymes. J Microbiol 43:101–109PubMedGoogle Scholar
  11. Dong YH, Zhang XF, Xu JL, Zhang LH (2004) Insecticidal Bacillus thuringiensis silences Erwinia carotovora virulence by a new form of microbial antagonism, signal interference. Appl Environ Microbiol 70:954–960PubMedCentralCrossRefPubMedGoogle Scholar
  12. Dunn AK, Millikan DS, Adin DM, Bose JL, Stabb EV (2006) New rfp- and pES213-derived tools for analysing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Appl Environ Microbiol 72:802–810PubMedCentralCrossRefPubMedGoogle Scholar
  13. Fast W, Tipton PA (2012) The enzymes of bacterial census and censorship. Trends Biochem Sci 37:7–14PubMedCentralCrossRefPubMedGoogle Scholar
  14. Fuqua C, Parsek MR (2001) Greenberg EP Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu Rev Genet 35:439–468CrossRefPubMedGoogle Scholar
  15. Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S (2000) Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146:2395–2407CrossRefPubMedGoogle Scholar
  16. Johnson CN (2013) Fitness factors in Vibrios: a mini-review. Microb Ecol 65:826–851Google Scholar
  17. Kamruzzaman M, Udden SM, Cameron DE, Calderwood SB et al (2010) Quorum-regulated biofilms enhance the development of conditionally viable, environmental Vibrio cholera. Proc Natl Acad Sci U S A 107:1588–1593PubMedCentralCrossRefPubMedGoogle Scholar
  18. Leadbetter JR, Greenberg EP (2000) Metabolism of acyl-homoserine lactone quorum-sensing signals by Variovorax paradoxus. J Bacteriol 182:6921–6926PubMedCentralCrossRefPubMedGoogle Scholar
  19. McClean KH, Winson MK, Fish L, Taylor A, Chhabra SR, Camara M, Daykin M, Lamb JH, Swift S, Bycroft BW, Stewart GS, Williams P (1997) Quorum sensing and Chromobacterium violaceum, exploitation of violacein production and inhibition for the detection of N-acylhomoserine lactones. Microbiology 143:3703–3711CrossRefPubMedGoogle Scholar
  20. Morohoshi T, Inaba T, Kato N, Kanai K, Ikeda T (2004) Identification of quorum-sensing signal molecules and the LuxRI homologs in fish pathogen Edwardsiella tarda. J Biosci Bioeng 98:274–281CrossRefPubMedGoogle Scholar
  21. Ng WL, Basslern BL (2009) Bacterial quorum-sensing network architectures. Annu Rev Genet 43:197–222PubMedCentralCrossRefPubMedGoogle Scholar
  22. Nhan DT, Cam V, Wille M, Defoirdt T, Bossier P, Sorgeloos P (2010) Quorum quenching bacteria protects Macrobrachium rosenbergii larvae from Vibrio harveyi infection. J Appl Microbiol 109:1007–1016CrossRefPubMedGoogle Scholar
  23. Ninawe AS, Selvin J (2009) Probiotics in shrimp aquaculture: avenues and challenges. Crit Rev Microbiol 35:43–66CrossRefPubMedGoogle Scholar
  24. Pan J, Huang T, Yao F, Huang Z, Powell CA, Qiu S, Guan X (2008) Expression and characterization of aiiA gene from Bacillus subtilis BS-1. Microbiol Res 163:711–716CrossRefPubMedGoogle Scholar
  25. Pratt LA, Kolter R (1998) Genetic analysis of Escherichia coli biofilm formation: roles of flagella, motility, chemotaxis and type I pili. Mol Microbiol 30:285–293CrossRefPubMedGoogle Scholar
  26. Rasmussen TB, Givskov M (2006) Quorum sensing inhibitors: a bargain of effects. Microbiology 152:895–904CrossRefPubMedGoogle Scholar
  27. Roche DM, Byers JT, Smith DS, Glansdorp FG, Spring DR, Welch M (2004) Communications blackout? Do N-acylhomoserinelactone-degrading enzymes have any role in quorum sensing. Microbiology 150:2023–2028CrossRefPubMedGoogle Scholar
  28. Sarkar BL, Nair GB, Banerjee AK, Pal SC (1985) Seasonal distribution of Vibrio parahaemolyticus in freshwater environs and in association with freshwater fishes in Calcutta. Appl Environ Microbiol 49:132–136PubMedCentralPubMedGoogle Scholar
  29. Su YC, Liu C (2007) Vibrio parahaemolyticus: a concern of seafood safety. Food Microbiol 24:549–558CrossRefPubMedGoogle Scholar
  30. Swift S, Karlyshev AV, Fish L, Durant EL, Winson MK, Chhabra SR, Williams P, Macintyre S, Stewart GS (1997) Quorum sensing in Aeromonas hydrophila and Aeromonas salmonicida: identification of the LuxRI homologs AhyRI and AsaRI and their cognate N-acylhomoserine lactone signal molecules. J Bacteriol 179:5271–5281PubMedCentralPubMedGoogle Scholar
  31. Thompson FL, Lida T, Swings J (2004) Biodiversity of Vibrios. Microbiol Mol Rev 68:403–431CrossRefGoogle Scholar
  32. Vaseeharan B, Shanthi S, Prabhu NM (2011) A novel clip domain serine proteinase (SPs) gene from the haemocytes of Indian white shrimp Fenneropenaeus indicus. Fish and Shellfish Immunology 30(3):980–5.CrossRefPubMedGoogle Scholar
  33. Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703PubMedCentralPubMedGoogle Scholar
  34. Yeung PS, Boor KJ (2004) Epidemiology, pathogenesis, and prevention of foodborne Vibrio parahaemolyticus infections. Foodborne Pathog Dis 1:74–88CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • G. Vinoj
    • 1
  • B. Vaseeharan
    • 1
    Email author
  • S. Thomas
    • 2
  • A. J. Spiers
    • 3
  • S. Shanthi
    • 1
  1. 1.Crustacean Molecular Biology and Genomics Laboratory, Department of Animal Health and ManagementAlagappa UniversityKaraikudiIndia
  2. 2.Cholera and Environmental Microbiology Laboratory, Department of Molecular MicrobiologyRajiv Gandhi Centre for BiotechnologyTrivandrumIndia
  3. 3.SIMBIOS CentreAbertay UniversityDundeeUnited Kingdom

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