Phospholipase Activities in Clinical and Environmental Isolates of Acanthamoeba

  • Matin, Abdul (Institute of Biomedical and Genetic Engineering) ;
  • Jung, Suk-Yul (Department of Biomedical Laboratory Science, Molecular Diagnosis Research Institute, Namseoul University)
  • Received : 2010.09.29
  • Accepted : 2010.12.16
  • Published : 2011.03.15


The pathogenesis and pathophysiology of Acanthamoeba infections remain incompletely understood. Phospholipases are known to cleave phospholipids, suggesting their possible involvement in the host cell plasma membrane disruption leading to host cell penetration and lysis. The aims of the present study were to determine phospholipase activities in Acanthamoeba and to determine their roles in the pathogenesis of Acanthamoeba. Using an encephalitis isolate (T1 genotype), a keratitis isolate (T4 genotype), and an environmental isolate (T7 genotype), we demonstrated that Acanthamoeba exhibited phospholipase $A_2$ (PLA$_2$). and phospholipase D (PLD) activities in a spectrophotometry-based assay. Interestingly, the encephalitis isolates of Acanthamoeba exhibited higher phospholipase activities as compared with the keratitis isolates, but the environmental isolates exhibited the highest phospholipase activities. Moreover, Acanthamoeba isolates exhibited higher PLD activities compared with the PLA$_2$. Acanthamoeba exhibited optimal phospholipase activities at $37^{\circ}C$ and at neutral pH indicating their physiological relevance. The functional role of phospholipases was determined by in vitro assays using human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. We observed that a PLD-specific inhibitor, i.e., compound 48/80, partially inhibited Acanthamoeba encephalitis isolate cytotoxicity of the host cells, while PLA$_2$-specific inhibitor, i.e., cytidine 5'-diphosphocholine, had no effect on parasite-mediated HBMEC cytotoxicity. Overall, the T7 exhibited higher phospholipase activities as compared to the T4. In contract, the T7 exhibited minimal binding to, or cytotoxicity of, HBMEC.


  1. Khan NA. Acanthamoeba: biology and increasing importance in human health. FEMS Microbiol Rev 2006; 30: 564-595.
  2. Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 2003; 16: 273-307.
  3. Schuster FL, Visvesvara GS. Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 2004; 34: 1001-1027.
  4. Jung SY, Alsam S, Kim KS, Khan NA. Pathogen-pathogen interactions: a comparative study of Escherichia coli interactions with the clinical and environmental isolates of Acanthamoeba. World J Microbiol Biotechnol 2008; 24: 2339-2348.
  5. Victoria EJ, Korn ED. Plasma membrane and soluble lysophospholipases of Acanthamoeba castellanii. Arch Biochem Biophys 1975; 171: 255-258.
  6. Victoria EJ, Korn ED. Enzymes of phospholipid metabolism in the plasma membrane of Acanthamoeba castellanii. J Lipid Res 1975; 16: 54-60.
  7. Cursons RTM, Brown TJ, Keys EA. Virulence of pathogenic free-living amoebae. J Parasitol 1978; 64: 744-745.
  8. Ghannoum MA. Potential role of phospholipases in virulence and fungal pathogenesis. Clin Microbiol Rev 2000; 13: 122-143.
  9. Exton JH. Phospholipase D-structure, regulation and function. Rev Physiol Biochem Pharmacol 2002; 144: 1-94.
  10. McDermott M, Wakelam MJO, Morris AJ. Phospholipase D. Biochem Cell Biol 2004; 82: 225-253.
  11. Akiba S, Sato T. Cellular function of calcium-independent phospholipase $A_2$. Biol Pharm Bull 2004; 27: 1174-1178.
  12. Kudo I. Diversity of phospholipase $A_2$ enzymes. Foreword. Biol Pharm Bull 2004; 27: 1157.
  13. Sissons J, Kim KS, Stins M, Jayasekera S, Alsam S, Khan NA. Acanthamoeba castellanii induces host cell death via a phosphatidylinositol 3-kinase-dependent mechanism. Infect Immun 2005; 73: 2704-2708.
  14. Stins MF, Gilles F, Kim KS. Selective expression of adhesion molecules on human brain microvascular endothelial cells. J Neuroimmunol 1997; 76: 81-90.
  15. Alsam S, Kim KS, Stins M, Rivas AO, Sissons J, Khan NA. Acanthamoeba interactions with human brain microvascular endothelial cells. Microb Pathog 2003; 35: 235-241.
  16. Matin A, Stins M, Kim KS, Khan NA. Balamuthia mandrillaris exhibits metalloprotease activities. FEMS Immunol Med Microbiol 2006; 47: 83-91.
  17. Sissons J, Alsam S, Jayasekera S, Khan NA. Ecto-ATPases of clinical and non-clinical isolates of Acanthamoeba. Microb Pathog 2004; 37: 231-239.
  18. Leher H, Silvany R, Alizadeh H, Huang J, Niederkorn JY. Mannose induces the release of cytopathic factors from Acanthamoeba castellanii. Infect Immun 1998; 66: 5-10.
  19. Dennis EA, Rhee SG, Billah MM, Hannun YA. Role of phospholipase in generating lipid second messengers in signal transduction. FASEB J 1991; 5: 2068-2077.
  20. Serhan CN, Haeggström JZ, Leslie CC. Lipid mediator networks in cell signaling: update and impact of cytokines. FASEB J 1996; 10: 1147-1158.
  21. Oishi K, Raynor RL, Charp PA, Kuo JF. Regulation of protein kinase C by lysophospholipids. Potential role in signal transduction. J Biol Chem 1988; 263: 6865-6871.
  22. Bryant AE, Stevens DL. Phospholipase C and perfringolysin O from Clostridium perfringens upregulate endothelial cell-leukocyte adherence molecule 1 and intercellular leukocyte adherence molecule 1 expression and induce interleukin-8 synthesis in cultured human umbilical vein endothelial cells. Infect Immun 1996; 64: 358-362.
  23. Bunting M, Lorant DE, Bryant AE, Zimmerman GA, McIntyre TM, Stevens DL, Prescott SM. Alpha toxin from Clostridium perfringens induces proinflammatory changes in endothelial cells. J Clin Invest 1997; 100: 565-574.
  24. Saffer LD, Long Krug SA, Schwartzman JD. The role of phospholipase in host cell penetration by Toxoplasma gondii. Am J Trop Med Hyg 1989; 40: 145-149.
  25. Long-Krug SA, Fischer KJ, Hysmith RM, Ravdin JI. Phospholipase A enzymes of Entamoeba histolytica: description and subcellular localization. J Infect Dis 1985; 152: 536-541.
  26. McNamara PJ, Bradley GA, Songer JG. Targeted mutagenesis of the phospholipase D gene results in decreased virulence of Corynebacterium pseudotuberculosis. Mol Microbiol 1994; 12: 921-930.
  27. Kameyama S, Sato H, Murata R. The role of alpha-toxin of Clostridium perfringens in experimental gas gangrene in guinea pigs. Jpn J Med Sci Biol 1972; 25: 200.
  28. Hanel H, Kirsch R, Schmidts HL, Kottmann H. New systematically active antimycotics from the beta-blocker category. Mycoses 1995; 38: 251-264.