Advertisement

Overexpressed recombinant quorum quenching lactonase reduces the virulence, motility and biofilm formation of multidrug-resistant Pseudomonas aeruginosa clinical isolates

  • Masarra M. Sakr
  • Khaled M. Aboshanab
  • Walid F. Elkhatib
  • Mahmoud A. Yassien
  • Nadia A. Hassouna
Applied genetics and molecular biotechnology

Abstract

The increasing occurrence of resistance among Pseudomonas aeruginosa clinical isolates necessitates finding alternatives to antibiotics for controlling the infection of such pathogenic bacteria. In this study, lactonase gene ahl-1 from Bacillus weihenstephanensis isolate-P65 was successfully cloned and expressed in Escherichia coli BL21 (DE3) under the control of T7 promoter for utilizing its quorum quenching activity against three multidrug-resistant (MDR) P. aeruginosa clinical isolates. The biological activity of the overexpressed lactonase enzyme (Ahl-1), tested using a synthetic signal and Chromobacterium violaceum CV026 as a biosensor, displayed good catalytic activity using hexanoyl homoserine lactone (HHL) as a substrate and Chromobacterium violaceum (CV026) as a biosensor (77.2 and 133 nm min−1 for the crude and the purified Ahl-lactonase enzymes, respectively). Upon challenging its ability to inhibit the virulence of three MDR P. aeruginosa clinical isolates, recombinant Ahl-1 successfully prevented the accumulation of acylhomoserine lactone signals resulting in a significant reduction in the investigated virulence determinants; protease (from 40 up to 75.5%), pyocyanin (48–75.9%), and rhamnolipids (52.7–63.4%) (P value < 0.05). Ahl-1 also displayed significant inhibitory activities on the swarming motility and biofilm formation of the three tested MDR P. aeruginosa clinical isolates (P value < 0.05). Consequently, Ahl-1 lactonase enzyme in this study is considered a promising therapeutic agent to inhibit P. aeruginosa pathogenicity with no fear of emergence of resistance.

Keywords

Recombinant lactonase Ahl P. aeruginosa Quorum quenching Bacillus weihenstephanensis 

Notes

Acknowledgements

We hereby acknowledge Department of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University, for providing us with all facilities and support required to perform the practical work.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. Aybey A, Demirkan E (2016) Inhibition of quorum sensing-controlled virulence factors in Pseudomonas aeruginosa by human serum paraoxonase. J Med Microbiol 65:105–113.  https://doi.org/10.1099/jmm.0.000206 CrossRefPubMedGoogle Scholar
  2. Cao Y, He S, Zhou Z, Zhang M, Mao W, Zhang H, Yaoa B (2012) Orally administered thermostable N-acyl homoserine lactonase from Bacillus sp. strain AI96 attenuates Aeromonas hydrophila infection in zebrafish. Appl Environ Microbiol 78:1899–1908.  https://doi.org/10.1128/AEM.06139-11 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Chastre J, Fagon JY (2002) Ventilator-associated pneumonia. Am J Respir Crit Care Med 165:867–903.  https://doi.org/10.1164/ajrccm.165.7.2105078 CrossRefPubMedGoogle Scholar
  4. Christiaen SE, Matthijs N, Zhang XH, Nelis HJ, Bossier P, Coenye T (2014) Bacteria that inhibit quorum sensing decrease biofilm formation, virulence in Pseudomonas aeruginosa PAO1. Pathog Dis 70:271–279.  https://doi.org/10.1111/2049-632X.12124 CrossRefPubMedGoogle Scholar
  5. Dong YH, Wang LH, Xu JL, Zhang HB, Zhang XF, Zhang LH (2001) Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase. Nature 411:813–817.  https://doi.org/10.1038/35081101 CrossRefPubMedGoogle Scholar
  6. Essar DW, Eberly L (1990) Evolutionary differences in chromosomal locations of four early genes of tryptophan pathway in fluorescent Pseudomonas: DNA sequences and characterization of Pseudomonas putida trpE and trpGDC. J Bacteriol 172:876–883CrossRefGoogle Scholar
  7. Giamarellou H (2002) Prescribing guidelines for severe Pseudomonas infections. J Antimicrob Chemother 49:229–233CrossRefGoogle Scholar
  8. Guendouze A, Plener L, Bzdrenga J, Pauline J, Rémy B, Elias M, Lavigne J, Daudé D, Chabrière E (2017) Effect of quorum quenching lactonase in clinical isolates of Pseudomonas aeruginosa and comparison with quorum sensing inhibitors. Front Microbiol 8:227.  https://doi.org/10.3389/fmicb.2017.00227 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Hanahan D (1983) Studies on transformation of Escherichia coli with plasmids. J Mol Biol 166:557–580CrossRefGoogle Scholar
  10. Hancock RE (1998) Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria. Clin Infect Dis 27:93–99CrossRefGoogle Scholar
  11. Leadbetter JR (2001) Quieting the raucous crowd. Nature 411:748–749.  https://doi.org/10.1038/35081216 CrossRefPubMedGoogle Scholar
  12. Lee J, Zhang L (2015) The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 6(1):26–41.  https://doi.org/10.1007/s13238-014-0100-x CrossRefPubMedGoogle Scholar
  13. Loeffl J, Stevens DA (2003) Antifungal drug resistance. Clin Infect Dis 36:31–41.  https://doi.org/10.1086/344658 CrossRefGoogle Scholar
  14. Lowry H, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  15. Maschmeyer G, Braveny I (2000) Review of the incidence and prognosis of Pseudomonas aeruginosa infections in cancer patients in the 1990s. Eur J Clin Microbiol Infect Dis 19:915–925CrossRefGoogle Scholar
  16. Mesaros N, Nordmann P, Ple’siat P, Roussel-Delvallez M, Eldere JV, Glupczynski Y, Laethem YV, Jacobs F, Lebecque P, Malfroot A, Tulkens PM, Bambeke FV (2007) Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium. Clin Microbiol Infect 13(6):560–578.  https://doi.org/10.1111/j.1469-0691.2007.01681.x CrossRefPubMedGoogle Scholar
  17. Morens DM, Folkers GK, Fauci AS (2004) The challenge of emerging and re-emerging infectious diseases. Nature 430:242–249.  https://doi.org/10.1038/nature02759 CrossRefPubMedGoogle Scholar
  18. Morikawa M, Hirata Y, Imanaka T (2000) A study on the structure-function relationship of lipopeptide biosurfactants. Biochim Biophys Acta 1488(3):211–218CrossRefGoogle Scholar
  19. Nicodème M, Grill J, JL G (2005) Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions. J Appl Microbiol 99(3):641–648.  https://doi.org/10.1111/j.1365-2672.2005.02634.x CrossRefPubMedGoogle Scholar
  20. Obritsch MD, Fish DN, MacLaren R, Jung R (2005) Nosocomial infections due to MDR Pseudomonas aeruginosa: epidemiology and treatment options. Pharmacotherapy 25:1353–1364.  https://doi.org/10.1592/phco.2005.25.10.1353 CrossRefPubMedGoogle Scholar
  21. Osama D, Elkhatib W, Tawfeik A, Aboulwafa M, Hassouna N (2017) Antimicrobial, antibiofilm and immunomodulatory activities of Lactobacillus rhamnosus and Lactobacillus gasseri against some bacterial pathogens. Int J Biotechnol Wellness Ind 6(1):12–21.  https://doi.org/10.6000/1927-3037.2017.06.01.2 CrossRefGoogle Scholar
  22. Overhage J, Bains M, Brazas MD, Hancock RE (2008) Swarming of Pseudomonas aeruginosa is a complex adaptation leading to increased production of virulence factors and antibiotic resistance. J Bacteriol 190(8):2671–2679.  https://doi.org/10.1128/JB.01659-07 CrossRefPubMedPubMedCentralGoogle Scholar
  23. Rabin N, Zheng Y, Opoku-Temeng C, Du Y, Bonsu E, Sintim HO (2015) Agents that inhibit bacterial biofilm formation. Future Med Chem 7(5):647–671.  https://doi.org/10.4155/fmc.15.7 CrossRefPubMedGoogle Scholar
  24. Rashid MH, Kornberg A (2000) Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 97(9):4885–4890.  https://doi.org/10.1073/pnas.060030097 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Ravn L, Christensen AB, Molin S, Givskov M, Gram L (2001) Methods for detecting acylated homoserine lactones produced by gram-negative bacteria and their application in studies of AHL-production kinetics. J Microbiol Methods 44(3):239–251CrossRefGoogle Scholar
  26. Sakr MM, Aboshanab K, Aboulwafa MM, Hassouna NA (2013) Characterization and complete sequence of lactonase enzyme from Bacillus weihenstephanensis isolate P65 with potential activity against acyl homoserine lactone signal molecules. Biomed Res Int 2013:1–10.  https://doi.org/10.1155/2013/192589 CrossRefGoogle Scholar
  27. Sakr MM, Aboulwafa MM, Aboshanab K, Hassouna NA (2014) Screening and preliminary characterization of the quorum quenching activity of some Bacillus isolates from soil against clinically isolated Pseudomonas aeruginosa homoserine lactones. Malays J Microbiol 10(2):80–91Google Scholar
  28. Sambrook J (2000) SDS—plyacrylamide gel electrophoresis of proteins. Cold Harbour Press, Cold harbourGoogle Scholar
  29. Sambrook J, Russell DW (2001) The Inoue method for preparation and transformation of competent E. coli: “ultra-competent” cells. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  30. Schuster M, Greenberg EP (2006) A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. J Med Microbiol 296:73–81.  https://doi.org/10.1016/j.ijmm.2006.01.036 CrossRefGoogle Scholar
  31. Sio CF, Otten LG, Cool RH, Diggle SP, Braun PG, Bos R, Daykin M, Camara M, Williams P, Quax WJ (2006) Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1. Infect Immun 74(3):1673–1682.  https://doi.org/10.1128/IAI.74.3.1673-1682.2006 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Sligl W, Taylor G, Brindley PG (2006) Five years of nosocomial gram-negative bacteremia in a general intensive care unit: epidemiology, antimicrobial susceptibility patterns, and outcomes. Int J Infect Dis 10:320–325.  https://doi.org/10.1016/j.ijid.2005.07.003 CrossRefPubMedGoogle Scholar
  33. Stefani S, Campana S, Cariani L, Carnovale V, Colombo C, Lleo M, Lula V, Minicucci L, Morelli P, Pizzamiglio G, Taccetti G (2017) Relevance of multidrug-resistant Pseudomonas aeruginosa infections in cystic fibrosis. Int J Med Microbiol 307(6):353–362.  https://doi.org/10.1016/j.ijmm.2017.07.004 CrossRefPubMedGoogle Scholar
  34. Stepanovic S, Vukovic D, Hola V, Bonaventura GD, Djukic S, Cirkovic C, Ruzicka F (2007) Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by Staphylococci. APMIS 115(8):891–899.  https://doi.org/10.1111/j.1600-0463.2007.apm_630.x CrossRefPubMedGoogle Scholar
  35. Studier F, Rosenberg A, Dunn J, Dubendorff J (1990) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. Methods Enzymol 185:60–89CrossRefGoogle Scholar
  36. Torres M, Uroz S, Salto R, Fauchery L, Quesada E, Llamas I (2017) HqiA, a novel quorum-quenching enzyme which expands the AHL lactonase family. Sci Rep 7:943.  https://doi.org/10.1038/s41598-017-01176-7 CrossRefPubMedPubMedCentralGoogle Scholar
  37. Vaisvila R, Morgan RD, Posfai J, Raleigh EA (2001) Discovery and distribution of super-integrons among pseudomonads. Mol Microbiol 42:587–601CrossRefGoogle Scholar
  38. Viretta AU, Fussenegger M (2004) Modeling the quorum sensing regulatory network of human-pathogenic Pseudomonas aeruginosa. Biotechnol Prog 20(3):670–678.  https://doi.org/10.1021/bp034323l CrossRefPubMedGoogle Scholar
  39. Wang Y, Yue D, Yong Z, YangBo H, BaoYu Y, ShiYun C (2007) Effects of quorum sensing autoinducer degradation gene on virulence and biofilm formation of Pseudomonas aeruginosa. Sci China C Life Sci 50(3):385–391.  https://doi.org/10.1007/s11427-007-0044-y CrossRefGoogle Scholar
  40. WHO (2017) Global priority list of antibiotic-resistant bacteria to guide research, discovery and development of new antibiotics. WHO Essential medicines and health products1–7Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Microbiology and Immunology, Faculty of PharmacyAin Shams UniversityCairoEgypt

Personalised recommendations