Parasites, Hosts and Diseases

The Korea Society for Parasitology and Tropical Medicine (대한기생충학·열대의학회)
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Comparison of specific activity and cytopathic effects of purified 33 kDa serine proteinase from Acanthamoeba strains with different degree of virulence

  • Kim, Won-Tae (Department of Parasitology, Kyungpook National University School of Medicine) ;
  • Kong, Hyun-Hee (Department of Parasitology, Kyungpook National University School of Medicine) ;
  • Ha, Young-Ran (Department of Parasitology, Kyungpook National University School of Medicine) ;
  • Hong, Yeon-Chul (Department of Parasitology, Kyungpook National University School of Medicine) ;
  • Jeong, Hae-Jin (Department of Parasitology, Pusan National University School of Medicine) ;
  • Yu, Hak-Sun (Department of Parasitology, Pusan National University School of Medicine) ;
  • Chung, Dong-Il (Department of Parasitology, Kyungpook National University School of Medicine)
  • Published : 2006.12.10
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Abstract

The pathogenic mechanism of granulomatous amebic encephalitis (GAE) and amebic keratitis (AK) by Acanthamoeba has yet to be clarified. Pretense has been recognized to play an important role in the pathogenesis of GAE and AK. In the present study, we have compared specific activity and cytopathic effects (CPE) of purified 33 kDa serine proteinases from Acanthamoeba strains with different degree of virulence (A. healyi OC-3A, A. lugdunensis KA/E2, and A. castelianii Neff). Trophozoites of the 3 strains revealed different degrees of CPE on human corneal epithelial (HCE) cells. The effect was remarkably reduced by adding phenylmethylsulfonylfluoride (PMSF), a serine proteinase inhibitor. This result indicated that PMSF-susceptible proteinase is the main component causing cytopathy to HCE cells by Acanthamoeba. The purified 33 kDa serine proteinase showed strong activity toward HCE cells and extracellular matrix proteins. The purified proteinase from OC-3A, the most virulent strain, demonstrated the highest enzyme activity compared to KA/E2, an ocular isolate, and Neff, a soil isolate. Polyclonal antibodies against the purified 33 kDa serine proteinase inhibit almost completely the proteolytic activity of culture supernatant of Acanthamoeba. In line with these results, the 33 kDa serine proteinase is suggested to play an important role in pathogenesis and to be the main component of virulence factor of Acanthamoeba.

Keywords

References

  1. Alfieri SC, Correia CE, Motegi SA, Pral EM (2000) Proteinase activities in total extracts and in medium conditioned by Acanthamoeba polyphaga trophozoites. J Parasitol 86: 220-227 https://doi.org/10.1645/0022-3395(2000)086[0220:PAITEA]2.0.CO;2
  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  3. Cao Z, Jefferson DM, Panjwani N (1998) Role of carbohydrate- mediated adherence in cytopathogenic mechanisms of Acanthamoeba. J Biol Chem 273: 15838-15845 https://doi.org/10.1074/jbc.273.25.15838
  4. Cho JH, Na BK, Kim TS, Song CY (2000) Purification and characterization of an extracellular serine proteinase from Acanthamoeba castellanii. IUBMB Life 50: 209-214 https://doi.org/10.1080/152165400300001534
  5. Garate M, Cao Z, Bateman E, Panjwani N (2004) Cloning and characterization of a novel mannose-binding protein of Acanthamoeba. J Biol Chem 279: 29849-29856 https://doi.org/10.1074/jbc.M402334200
  6. Hong YC, Kong HH, Ock MS, Kim IS, Chung DI (2000) Isolation and characterization of a cDNA encoding a subtilisin-like serine proteinase (AhSUB) from Acanthamoeba healyi. Mol Biochem Parasitol 111: 441-446 https://doi.org/10.1016/S0166-6851(00)00326-1
  7. Hong YC, Lee WM, Kong HH, Jeong HJ, Chung DI (2004) Molecular cloning and characterization of a cDNA encoding a laminin-binding protein (AhLBP) from Acanthamoeba healyi. Exp Parasitol 106: 95-102 https://doi.org/10.1016/j.exppara.2004.01.011
  8. Hurt M, Niederkorn J, Alizadeh H (2003) Effects of mannose on Acanthamoeba castellanii proliferation and cytolytic ability to corneal epithelial cells. Invest Ophthalmol Vis Sci 44: 3424-3431 https://doi.org/10.1167/iovs.03-0019
  9. Khan NA (2003) Pathogenesis of Acanthamoeba infections. Microb Pathog 34: 277-285 https://doi.org/10.1016/S0882-4010(03)00061-5
  10. Kim HK, Ha YR, Yu HS, Kong HH, Chung DI (2003) Purification and characterization of a 33 kDa serine protease from Acanthamoeba lugdunensis KA/E2 isolated from a Korean keratitis patient. Korean J Parasitol 41: 189-196 https://doi.org/10.3347/kjp.2003.41.4.189
  11. Kong HH, Kim TH, Chung DI (2000) Purification and characterization of a secretory serine proteinase of Acanthamoeba healyi isolated from GAE. J Parasitol 86: 12-17 https://doi.org/10.1645/0022-3395(2000)086[0012:PACOAS]2.0.CO;2
  12. Leher H, Silvany R, Alizadeh H, Huang J, Niederkorn JY (1998) Mannose induces the release of cytopathic factors from Acanthamoeba castellanii. Infect Immun 66: 5-10
  13. Marciano-Cabral F, Puffenbarger R, Cabral GA (2000) The increasing importance of Acanthamoeba infections. J Eukaryot Microbiol 47: 29-36 https://doi.org/10.1111/j.1550-7408.2000.tb00007.x
  14. Marciano-Cabral F, Cabral G (2003) Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 16: 273-307 https://doi.org/10.1128/CMR.16.2.273-307.2003
  15. Mckerrow JH, Sun E, Rosenthal PJ, Bouvier J (1993) The proteases and pathogenicity of parasitic protozoa. Annu Rev Microbiol 47: 821-853 https://doi.org/10.1146/annurev.mi.47.100193.004133
  16. Mitra MM, Alizadeh H, Gerard RD, Niederkorn JY (1995) Characterization of a plasminogen activator produced by Acanthamoeba castellanii. Mol Biochem Parasitol 73: 157-164 https://doi.org/10.1016/0166-6851(94)00109-Z
  17. Moura H, Wallace S, Visvesvara GS (1992) Acanthamoeba healyi n. sp. and the isoenzyme and immunoblot profiles of Acanthamoeba spp., groups 1 and 3. J Protozool 39: 573-583 https://doi.org/10.1111/j.1550-7408.1992.tb04853.x
  18. Na BK, Kim JC, Song CY (2001) Characterization and pathogenetic role of proteinase from Acanthamoeba castellanii. Microb Pathog 30: 39-48 https://doi.org/10.1006/mpat.2000.0403
  19. Schuster FL, Visvesvara GS (2004) Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Int J Parasitol 34: 1001-1027 https://doi.org/10.1016/j.ijpara.2004.06.004
  20. Yu HS, Kong HH, Kim SY, Hahn YH, Hahn TW, Chung DI (2004) Laboratory investigation of Acanthamoeba lugdunensis from patients with keratitis. Invest Ophthalmol Vis Sci 45: 1418-1426 https://doi.org/10.1167/iovs.03-0433