M. Phagocytic activity in transgenic N. fowleri
V. CONCLUSION
In this study, I cloned and characterized the nf-actin gene to evaluate the role of Nf-actin gene in pathogenic N. fowleri. The nf-Nf-actin gene has the coding sequence of 1.2 kb, produced a 50 kDa recombinant protein (Nf-actin-His-tagged protein) in E.coli. The sequence indentity was 82 % with nonpathogenic N. gruberi, but has no sequence identity with other mammals and human actin gene. The Nf-actin was located on the cytoplasm, pseudopodia and especially food-cup structure in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri cocultured with target cells, Nf-actin strongly expressed on food-cup structure concerning with pahgocytosis. When N. fowleri were treated with cytochalasin D, actin polymerization inhibitor, N. fowleri showed decreasing the number of food-cup structures in comparison with control N. fowleri. To examine the function of Nf-actin and nfa1 gene in pathogenesis of N. fowleri, I constructed the Ubi-pEGFP-C2/nf-actin and Ubi-pEGFP-C2/nfa1 vectors. The nf-actin or nfa1 overexpressed N.
fowleri was identified by Western blot and fluorescence microscopy, and then, cell adhesion, cytotoxicity and phagocytotic activity assays were performed. In addition, to confirm the roles of nf-actin and nfa1, N. fowleri was knock-downed nf-actin and nfa1 gene using anti-sense oligomers. The nf-actin or nfa1 overexpressed N. fowleri showed strongly adhesion to fibronectin and fibrinogen. The cytotoxicity and phagocytic activity of nf-actin or nfa1 overexpressed N. fowleri were significantly increased in comparison with wild-type N.
fowleri. Finally, these results suggest that Nf-actin and Nfa1 play important roles in the cell adhesion, phagocytic activity and pathogenicity of pathogenic N. fowleri.
- 63 -
REFERENCES
1. Ahn KS, Henny HR Jr: An Ancanthamoeba ubiquitin-fusion protein; cDNA and deduced protein sequence. Biochim Biophys Acta 1218:109-11, 1994
2. Aldape K, Huizinga H, Bouvier J, McKerrow J: Naegleria fowleri; characterization of a secreted histolytic cysteine protease. Exp Parasitol 78:230–241, 1994
3. Anderson K, Jamieson A: Primary amoebic meningoencephalitis. Lancet 2:379, 1972 4. Apley J, Clarke SK, Roome AP, Sandry SA, Saygi G, Silk B, Warhurst DC: Primary
amoebic meningoencephalitis in Britain. Br Med J 7:596–599, 1970
5. Barbour SE, Marciano-Cabral F: Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A2. Biochim Biophys Acta 1530:123–
133, 2001
6. Badger JL, Stins MF, Kim KS: Citrobacter freundii invades and replicates in human brain microvascular endothelial cells. Infect Immun 67:4208–4215, 1999
7. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 7:248–254, 1976
8. Bretscher A: Microfilaments structure and function in the cortical cytoskeleton. Annu Rev Cell Biol 7:337–374, 1991
9. Carter RF: Primary amoebic meningoencephalitis: clinical, pathological and epidemiological features of six fatal cases. J Pathol Bacteriol 96:1–25, 1968
10. Carter RF: Description of Naegleira species isolated two cased of primary amoebic
- 64 -
menigoencephalatis and of the experimental pathological changes induced by it. J Pathol 100:217-44, 1970
11. Carter RF: Primary amoebic menigoecncephalitis. An appraisal of present knowledge.
Trans R Soc Trop Med Hyg 66:193-213, 1972
12. Cervantes-Sandoval I, Serrano-Luna JJ, Tapia-Malagón JL, Pacheco-Yépez J, Silva-Olivares A, Galindo-Gómez S, Tsutsumi V, Shibayama M: Characterization of Naegleria fowleri strains isolated from human cases of primary amoebic meningoencephalitis in Mexico. Rev Invest Clin 59:342–347, 2007
13. Cline M, Carchman R, Marciano-Cabral F: Movement of Naegleria fowleri stimulated by mammalian cells in vitro. J Protozool 33:10–13, 1986
14. Cho MS, Jung SY, Park S, Kim KH, Kim HI, Sohn S, Kim HJ, Im KI, Shin HJ:
Immunological characterizations of a cloned 13.1-kilodalton protein from pathogenic Naegleria fowleri. Clin Diagn Lab Immunol 10:954–959, 2003
15. Coxon PY, Summersgill JT, Ramirez JA, Miller RD: Signal transduction during Legionella pneumophila entry into human monocytes. Infect Immun 66(6):2905–291, 1998
16. Culbertson CG: Pathogenic Naegleria and Hartmannella (Acanthamoeba). Ann N Y Acad Sci 74:1018–1022, 1970
17. Cursons RT, Brown TJ: Use of cell cultures as an indicator of pathogenicity of free-living amoeba. J Clin Pathol 31:1–11, 1978
18. Cursons RT, Brown TJ, Keys EA: Effect of disinfectants on pathogenic free-living amoebae; in axenic conditions. Appl Environ Microbiol 40:62-6, 1980
- 65 -
19. De Jonckheere JF, van de Voorde H: Differences in destruction of cysts of pathogenic and nonpathogenic Naegleria and Acanthamoeba by chlorine. Appl Environ Microbiol 31:294-7, 1976
20. De Jonckheere JF: Growth characteristics, cytopathic effect in cell culture, and virulence in mice of 36 type strains belonging to 19 different Acanthamoebae spp.
Appl Environ Microbiol 39:681–685, 1980
21. De Jonckheere JF: Origin and evolution of the worldwide distributed pathogenic amoeboflagellate Naegleria fowleri. Infect Genet Evol 11(7):1520-8, 2011
22. De Lourdes Mun˜oz M, Das P, Tovar R: Entamoeba histolytica trophozoites activated by collagen type I and Ca2+ have a structured cytoskeleton during collagenase secretion. Cell Motil Cytoskeleton 50:45–54, 2001
23. Eleonor TB, Moon EK, Kong HH, Chung DI: Acanthamoeba healyi; Molecular cloning and characterization of a coronin homologue, an actin-related protein. Exp Parasitol 110(2):114-22, 2005
24. F. Rivero, F. Cvrčková: In "Origins and evolution of eukaryotic endomembranes and cytoskeleton". Bioscience 2006
25. Finlay BB, Ruschkowski S: Cytoskeletal rearrangements accompanying Salmonella entry into epithelial cells. J Cell Sci 99:283–296, 1991
26. Goode BL, Eck MJ: Mechanism and function of formins in the control of actin assembly. Annu Rev Biochem 76:593-627, 2007
27. Gordon VR, Asem EK, Vodkin MH, McLaughlin GL: Acanthamoeba binds to extracellular matrix proteins in vitro. Invest Ophthalmol Vis Sci 34:658–662, 1993
- 66 -
28. Gumbiner BM: Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84:345–357, 1996
29. Han KL, Lee HJ, Shin MH, Shin HJ, Im KI, Park SJ: The involvement of an integrin-like protein and protein kinase C in amoebic adhesion to fibronectin and amoebic cytotoxicity. Parasitol Res 94:53–60, 2004
30. Hostos E: Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility. Cell Biology 10(13):4097-104, 1993
31. Hostos E: The coronin family of actin-associated proteins. Trends In Cell Biology (9):345-50, 1999
32. Hysmith RM, Franson RC: Elevated levels of cellular and extrecellular phospholipases from pathogenic Naegleria fowleri. Biochim Biophys Acta 711:26–32, 1982
33. Jensch S: The ubiquitin-conjugation system. Annu Rev Genet 26:179-207, 1992 34. Jeong SY, Kang SY, Lee SC, Song KJ, Im KI, Shin HJ: Decreasing effect of an
anti-Nfa1 polyclonal antibody on the in vitro cytotoxicity of pathogenic Naegleria fowleri.
Korean J Parasitol 42:35–40, 2004
35. Jeong SY, Lee SC, Song KJ, Park S, Kim K, Kwon MH, Im KI, Shin HJ: Expression of the nfa1 gene cloned from pathogenic Naegleria fowleri in nonpathogenic N.
gruberi enhances cytotoxicity against CHO target cells in vitro. Infect Immun 73(7):4098-105, 2005
36. John DT, Cole TB Jr, Marciano-Cabral F: Sucker-like structures on the pathogenic amoeba Naegleria fowleri. Appl Environ Microbiol 47:12–14, 1984
- 67 -
37. Kang SY, Song KJ, Jeong SR, Kim JH, Park S, Kim K, Kwon MH, Shin HJ: Role of the Nfa1 protein in pathogenic Naegleria fowleri co-cultured with CHO target cells.
Clin Diagn Lab Immunol 12:873–876, 2005
38. Khan NA, Wang Y, Kim KJ, Chung JW, Wass CA, Kim KS: Cytotoxic necrotizing factor-1 contributes to Escherichia coli K1 invasion of the central nervous system. J Biol Chem 277(18):15607–15612, 2002
39. Kim JH, Kim D, Shin HJ: Contact-Independent Cell Death of Human Microglial Cells due to Pathogenic Naegleria fowleri Trophozoites. Korean J Parasitol 46(4):217-221, 2008
40. Kim JH, Yang AH, Sohn HJ, Kim D, Song KJ, Shin, HJ: Immunodominant antigens in Naegleria fowleri excretory-secretory proteins were potential pathogenic factors.
Parasitol Research 105 (6):1675-1681, 2009
41. Kollars TM, Willhen WE: The occurrence of antibodies to Nagleria species in wild mammal. J Parasitol 82:73-77, 1996
42. Lazarides E, Weber K: Actin antibody; the specific visualization of actin filaments in non-muscle cells. Proc Natl Acad Sci US 71(6):2268-72, 1974
43. Lee YJ, Kim JH, Jeong SR, Song KJ, Kim K, Park S, Park MS, Shin HJ: Production of Nfa1-specific monoclonal antibodies that influences the in vitro cytotoxicity of Naegleria fowleri trophozoites on microglial cells. Parasitol Res 101:1191–1196, 2007
44. Lee YJ, Kim JH, Sohn HJ, Lee J, Jung SY, Chwae YJ, Kim K, Park S, Shin HJ:
Effects of immunization with the rNfa1 protein on experimental Naegleria
fowleri-- 68 fowleri-- PAM mice. Parasite Immunol 33:382–389, 2011
45. Li E, Yang WG, Zhang T, Stanley SL Jr: Interaction of laminin with Entamoeba histolytica cysteine proteinases and its effect on amebic pathogenesis. Infect Immun 63:4150–4153, 1995
46. Nizet V, Kim KS, Stins M, Jonas M, Chi EY, Nguyen D, Rubens CE: Invasion of brain microvascular endothelial cells by group B streptococci. Infect Immun 65:5074–
5081, 1997
47. Loschiavo F, Ventura-Spagnolo T, Sessa E, Bramanti P: Acute primary meningoencephalitis from entamoeba Naegleria fowleri. Acta Neurol 15:333–340, 1993
48. Ma P, Visvesvara GS, Martinez AJ, Theodore FH, Gaggett PM, Sawyer TK:
Naegleria and Acanthamoeba infections: review. Rev Infect Dis 12:490–513, 1990 49. Marciano-Cabral FM, Patterson M, John DT, Bradley SG: Cytopathogenicity of N.
fowleri and N. gruberi for established mammalian cell cultures. J Parasitol 68:1110–
1116, 1982
50. Marciano-Cabral F, John DT: Cytopathogenicity of Naegleria fowleri to rat neuroblastoma cell cultures; scanning electron microscopy study. Infect Immun 40:1214–1217, 1983
51. Marciano-Cabral F: Biology of Naegleria spp. Microbiol Rev 52:114–133, 1988 52. Marciano-Cabral F, Cabral GA: The immune response to Naegleria fowleri amebae
and pathogenesis of infection. FEMS Immunol Med Microbiol 51:243–259., 2007.
53. Niederkorn JY, Alizadeh H, Leher H, McCulley JP: The pathogenesis of
- 69 -
Acanthamoeba keratitis. Microbes Infect 1(6):437–44, 1999
54. Oh YH, Jeong SR, Kim JH, Song KJ, Kim K, Park S, Sohn S, Shin HJ: Cytophatic changes and pro-inflammatory cytokines induced by Naegleria fowleri trophozoites in rat microglial cells and protective effects of an anti-Nfa1 antibody. Parasite Immunol 27:453–459, 2005
55. Okada M, Huston CD, Mann BJ, Petri Jr WA, Kita Kb, Nozaki T: Proteomic analysis of phagocytosis in the enteric protozoan parasite Entamoeba histolytica. Eukaryot Cell 4(4):827-31, 2005
56. Ondarza R: Drug targets from human pathogenic amoebas: Entamoeba histolytica, Acanthamoeba polyphaga and Naegleria fowleri. Infect Disord Drug Targets 7:266–
280, 2007
57. Pollard TD, Satterwhite L, Cisek L, Corden J, Sato M, Maupin P: Actin and myosin biochemistry in relation to cytokinesis. Ann N Y Acad Sci 582:120-30, 1999
58. Reisler E: Actin molecular structure and function. Curr Opin Cell Biol 5(1):41-7, 1993
59. Rocha-Azevedo B, Jamerson M, Cabral GA, Silva-Filho FC, Marciano-Cabral F: The interaction between the amoeba Balamuthia mandrillaris and extracellular matrix glycoproteins in vitro. Parasitology 134:51–58, 2007
60. Rocha-Azevedo BD, Jamerson M, Cabral GA, Silva-Filho FC, Marciano-Cabral F:
Acanthamoeba interaction with extracellular matrix glycoproteins; biological and biochemical characterization and role in cytotoxicity and invasiveness. J Eukaryot Microbiol 56, 270–278, 2009
- 70 -
61. Robertson AS, Smythe E, Ayscough KR: Functions of actin in endocytosis. Cell Mol Life Sci 66(13):2049-65, 2009
62. Seidel JS, Harmatz P, Visvesvara GS, Cohen A, Edwards J, Turner J: Successful treatment of primary amebic meningoencephalitis. N Engl J Med 11:346–348, 1982 63. Shibayama M, Serrano-Luna JJ, Rojas-Herna´ndez S, Campos-Rodrı´guez R,
Tsutsumi V: Interaction of secretory immunoglobulin A antibodies with Naegleria fowleri trophozoites and collagen type I. Can J Microbiol 49:164–170, 2003
64. Shin HJ, Cho MS, Jung SY, Kim HI, Park S, Kim HJ, Im KI: Molecular cloning and characterization of a gene encoding a 13.1 kDa antigenic protein of Naegleria fowleri.
J Eukaryot Microbiol 48:713–717, 2001
65. Shin HJ, Im KI: Pathogenic free-living amoebae in Korea. Korean J Parasitol 42:93–
119, 2004
66. Song, KJ, Song KH, Na BK, Kim JH, Kwon D, Park S, Pak JH, Im KI, Shin HJ:
Molecular cloning and characterization of a cytosolic heat shock protein 70 from Naegleria fowleri. Parasitol Res 100(5)1083-1089, 2007
67. Song KJ, Song KH, Kim JH, Sohn HJ, Lee YJ, Park CE, Shin HJ: Heat shock protein 70 of Naegleria fowleri is important factor for proliferation and in vitro cytotoxicity.
Parasitol Res 103(2):313-317, 2008
68. Swanson JA, Baer SC: Phagocytosis by zippers and triggers. Trends Cell Biol 5:89–93, 1995
69. Taylor WM, Pidherney MS, Alizadeh H, Niederkorn JY: In vitro characterization of Acanthamoeba castellanii cytopathic effect. J Parasitol 81(4):603–609, 1995
- 71 -
70. Tuppeny M: Primary amoebic meningoencephalitis with subsequent organ procurement; a case study. J Neurosci Nurs 43:274-9, 2011
71. Visvesvara GS: Free-living amebae as opportunistic agents of human disease. J Neuroparasitol 1, 2010
72. Visvesvara GS, De Jonckheere JF, Sriram R, Daft B: Isolation and molecular typing of Naegleria fowleri from the brain of a cow that died of primary amebic meningoencephalitis. J Clin Microbiol 43:4203-4, 2005
73. Wulff BS, O’Hare MM, Boel E, Theill LE, Schwartz TW: Partial processing of the neuropeptides Υ precursor in transfected CHO cells. FEBS Lett 261:101-5, 1990 74. Yoder JS, Eddy BA, Visvesvara GS, Capewell L, Beach MJ: The epidemiology of
primary amoebic meningoencephalitis in the USA, 1962-2008. Epidemiol Infect 138(7):968-75, 2010
- 72 -
- 73 - 클로닝(Ubi-pEGFP-C2/nf-actin)하였다. 클로닝된 Ubi-pEGFP-C2/nf-actin을 PEI를 통해 transfection 한 후, G418을 처리하여 stable transgenic 아메바를 만들었으며,
- 74 -
결과, nf-actin이 과발현된 transgenic 아메바의 식세포작용이 대조군에 비해 증가됨을 보였다. 또한, actin 유전자의 기능을 억제시키기 위해 antisense actin oligomer를 파울러자유아메바에 transfection하여 억제 실험을 확인한 결과, nf-actin 유전자의 발현이 억제 되었을 때 표적세포에 대한 세포독성과 ECM에 대한 부착성 그리고 식세포 작용이 저해됨을 관찰하였다. 따라서 본실험을 통해 파울러자유아메바의 접촉성기전과 관련된 nf-actin 유전자는 아메바의 부착성과 세포독성에 중요한 기능을 담당하며 아메바의 병원성 작용기전에 크게 관여하는 것으로 생각된다. .
Key words: 파울러자유아메바; phagocytosis; food-cup; trogocytosis; actin