The Journal of Membrane Biology

, Volume 247, Issue 3, pp 281–288 | Cite as

Interaction of Phospholipase A of the E. coli Outer Membrane with the Inhibitors of Eucaryotic Phospholipases A2 and Their Effect on the Ca2+-Induced Permeabilization of the Bacterial Membrane

  • Konstantin N. BelosludtsevEmail author
  • Natalia V. Belosludtseva
  • Maxim S. Kondratyev
  • Alexey V. Agafonov
  • Yuriy A. Purtov


Phospholipase A of the bacterial outer membrane (OMPLA) is a β-barrel membrane protein which is activated under various stress conditions. The current study examines interaction of inhibitors of eucaryotic phospholipases A2—palmitoyl trifluoromethyl ketone (PACOCF3) and aristolochic acid (AA)—with OMPLA and considers a possible involvement of the enzyme in the Ca2+-dependent permeabilization of the outer membrane of Escherichia coli. Using the method of molecular docking, it has been predicted that PACOCF3 and AA bind to OMPLA at the same site and with the same affinity as the OMPLA inhibitors, hexadecanesulfonylfluoride and bromophenacyl bromide, and the substrate of the enzyme palmitoyl oleoyl phosphatidylethanolamine. It has also been shown that PACOCF3, AA, and bromophenacyl bromide inhibit the Ca2+-induced temperature-dependent changes in the permeability of the bacterial membrane for the fluorescent probe propidium iodide and suppressed the transformation of E. coli cells with plasmid DNA induced by Ca2+ and heat shock. The cell viability was not affected by the eucaryotic phospholipases A2 inhibitors. The study discusses a possible involvement of OMPLA in the mechanisms of bacterial transmembrane transport based on the permeabilization of the bacterial outer membrane.


OMPLA Escherichia coli Calcium Permeabilization 



We are grateful to Prof. Galina D. Mironova for fruitful discussion. This work was supported by a grant from the Russian Foundation for Basic Research to K.N. Belosludtsev (12-04-00430a) and RF Presidential program for Support of Young Russian Scientists to K.N. Belosludtsev (MK-145.2012.4).


  1. Agafonov A, Gritsenko E, Belosludtsev K, Kovalev A, Gateau-Roesch O, Saris N-EL, Mironova GD (2003) A permeability transition in liposomes induced by the formation of Ca2+/palmitic acid complexes. Biochim Biophys Acta 1609:153–160CrossRefPubMedGoogle Scholar
  2. Belosludtsev KN, Belosludtseva NV, Mironova GD (2005) Possible mechanism for formation and regulation of the palmitate-induced cyclosporin A-insensitive mitochondrial pore. Biochemistry 70:815–821PubMedGoogle Scholar
  3. Belosludtsev KN, Trudovishnikov AS, Belosludtseva NV, Agafonov AV, Mironova GD (2010) Palmitic acid induces the opening of a Ca2+-dependent pore in the plasma membrane of red blood cells: the possible role of the pore in erythrocyte lysis. J Membr Biol 237(1):13–19CrossRefPubMedGoogle Scholar
  4. Bishop RE (2008) Structural biology of membrane-intrinsic β-barrel enzymes: sentinels of the bacterial outer membrane. Biochim Biophys Acta 1778(9):1881–1896CrossRefPubMedGoogle Scholar
  5. Bos MP, Tefsen B, Voet P, Weynants V, van Putten JP, Tommassen J (2005) Function of neisserial outer membrane phospholipase a in autolysis and assessment of its vaccine potential. Infect Immun 73(4):2222–2231PubMedCentralCrossRefPubMedGoogle Scholar
  6. Cohen S, Chang A, Hsu L (1972) Nonchromosomal antibiotic resistance in bacteria. Proc Natl Acad Sci USA 69:2110–2114PubMedCentralCrossRefPubMedGoogle Scholar
  7. Cronan JE (1968) Phospholipid alterations during growth of Escherichia coli. J Bacteriol 95(6):2054–2061PubMedCentralPubMedGoogle Scholar
  8. De Siervo AJ (1969) Alterations in the Phospholipid Composition of Escherichia coli B during Growth at Different Temperatures. J Bacteriol 100(3):1342–1349PubMedCentralPubMedGoogle Scholar
  9. Dekker N (2000) Outer-membrane phospholipase A: known structure, unknown biological function. Mol Microbiol 35:711–717CrossRefPubMedGoogle Scholar
  10. Dekker N, Tommassen J, Verheij HM (1999) Bacteriocin release protein triggers dimerization of outer membrane phospholipase A in vivo. J Bacteriol 181(10):3281–3283PubMedCentralPubMedGoogle Scholar
  11. Dela Vega AL, Delcour AH (1996) Polyamines decrease Escherichia coli outer membrane permeability. J Bacteriol 178(13):3715PubMedCentralPubMedGoogle Scholar
  12. Fleming PJ, Freites JA, Moon CP, Tobias DJ, Fleming KG (2012) Outer membrane phospholipase A in phospholipid bilayers: a model system for concerted computational and experimental investigations of amino acid side chain partitioning into lipid bilayers. Biochim Biophys Acta 2:126–134CrossRefGoogle Scholar
  13. Homma H, Kobayashi T, Chiba N, Karasawa K, Mizushima H, Kudo I, Inoue K, Ikeda H, Sekiguchi M, Nojima S (1984) The DNA sequence encoding pldA gene, the structural gene for detergent-resistant phospholipase A of E. coli. J Biochem 96(6):1655–1664PubMedGoogle Scholar
  14. Horrevoets AJG, Verheij HM, de Haas GH (1991) Inactivation of Escherichia coli outer-membrane phospholipase. A by the affinity label hexadecanesulfonyl fluoride. Evidence foran active site serine. Eur J Biochem 198:247–253CrossRefPubMedGoogle Scholar
  15. Kingma RL, Fragiathaki M, Snijder HJ, Dijkstra BW, Verheij HM, Dekker N, Egmond MR (2000) Unusual catalytic triad of Escherichia coli outer membrane phospholipase A. Biochemistry 39(33):10017–10022CrossRefPubMedGoogle Scholar
  16. Mandel M, Higa A (1970) Calcium dependent bacteriophage DNA infection. J Mol Biol 53:159–162CrossRefPubMedGoogle Scholar
  17. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ (2009) Autodock4 and AutoDockTools4: automated docking with selective receptor flexiblity. J Computational Chem 16:2785–2791CrossRefGoogle Scholar
  18. Nelson ET, Buller CS (1974) Phospholipase activity in bacteriophage-infected Escherichia coli. I. Demonstration of a T4 bacteriophage-associated phospholipase. J Virol 14(3):479–484PubMedCentralPubMedGoogle Scholar
  19. Pagan R, Mackey B (2000) Relationship between membrane damage and cell death in pressure-treated Escherichia coli cells: differences between exponential- and stationary-phase cells and variation among strains. Appl Environ Microbiol 66:2829–2834PubMedCentralCrossRefPubMedGoogle Scholar
  20. Panja S, Aich P, Jana B, Basu T (2008) How does plasmid DNA penetrate cell membranes inartificial transformation process of Escherichia coli? Mol Membr Biol 25(5):411–422CrossRefPubMedGoogle Scholar
  21. Sabelnikov AG, Domaradsky IV (1979) Proton conductor vs. cold in induction of Ca(2 +)-dependent competence in Escherichia coli. Mol Gen Genet 172(3):313–317CrossRefPubMedGoogle Scholar
  22. Scandella CJ, Kornberg A (1971) A membrane-bound phospholipase A1 purified from Escherichia coli. Biochemistry 10(24):4447–4456CrossRefPubMedGoogle Scholar
  23. Shi L, Gunther S, Hubshmann T, Wick LY, Harms H, Muller S (2007) Limits of Propidium Iodide as a Cell Viability Indicator for Environmental Bacteria. Cytometry A 71(8):592–598CrossRefPubMedGoogle Scholar
  24. Snijder HJ, Ubarretxena-Belandia I, Blaauw M, Kalk KH, Verheij HM, Egmond MR, Dekker N, Dijkstra BW (1999) Structural evidence for dimerization regulated activity of an integral membrane phospholipase. Nature 401:717–721CrossRefPubMedGoogle Scholar
  25. Stanley AM, Chuawong P, Hendrickson TL, Fleming KG (2006) Energetics of outer membrane phospholipase A (OMPLA) dimerization. J Mol Biol 358(1):120–131CrossRefPubMedGoogle Scholar
  26. Stanley AM, Treubrodt AM, Chuawong P, Hendrickson TL, Fleming KG (2007) Lipid chain selectivity by outer membrane phospholipase A. J Mol Biol 366(2):461–468CrossRefPubMedGoogle Scholar
  27. Swords WE (2003) Chemical Transformation of E. coli. In: Casali N, Preston A (eds) E. coli Plasmid Vectors: Methods and Applications. Methods in Molecular Biology. Humana Press Inc, Totowa, pp 49–53CrossRefGoogle Scholar
  28. Tarahovsky YS, Khusainov AA, Daugelavichus R, Bakene E (1995) Structural changes in Escherichia coli membranes induced by bacteriophage T4 at different temperatures. Biophys J 68(1):157–163PubMedCentralCrossRefPubMedGoogle Scholar
  29. Tarahovsky YS, Deev AA, Masulis IS, Ivanitsky GR (1998) Structural organization and phase behavior of DNA-calcium-dipalmitoylphosphatidylcholine complex. Biochemistry (Mosc) 63(10):1126–1131Google Scholar
  30. Trott O, Olson AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J Computational Chem 31:455–461Google Scholar
  31. Yoshida N, Sato M (2009) Plasmid uptake by bacteria: a comparison of methods and efficiencies. Appl Microbiol Biotechnol 83:791–798CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Konstantin N. Belosludtsev
    • 1
    Email author
  • Natalia V. Belosludtseva
    • 1
  • Maxim S. Kondratyev
    • 2
  • Alexey V. Agafonov
    • 1
  • Yuriy A. Purtov
    • 2
  1. 1.Institute of Theoretical and Experimental Biophysics RASMoscow RegionRussia
  2. 2.Institute of Cell Biophysics RASMoscow RegionRussia

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