파울러자유아메바의 actin 유전자 클로닝 및 특성분석
Characterization of an nf-actin gene in the pathogenic Naegleria fowleri
- 주제(키워드) Naegleria fowleri , actin gene , adhesion , contact-dependent mechanism , cytotoxicity , food-cups , pathogenicity , phagocytosis
- 발행기관 아주대학교
- 지도교수 신호준
- 발행년도 2014
- 학위수여년월 2014. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000015894
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Naegleria fowleri, a pathogenic free-living amoeba has been isolated from samples obtatined from soil,polluted water and chlorinated swimming pool waster and existed as a virulent pathogen which causes fatal primary amoebic meningoencephalitis (PAM) in experimental animal or humans. The cytotoxicity of N. fowleri on target cells requires the contact-dependent mechanism such as a phagocytosis and the contact-independent mechanism such as a secretion of proteases. The phagocytosis mechanisms involve a process of piecemeal ingestion of target cells by food-cups (amoebastomes). Phagocytosis is an actin dependent process that includes polymerization of monomeric G-actin into filamentous F-actin. Despite the numerous studies concerning with phagocytosis, the detailed role of actin in the N. fowleri food-cup formation has been poorly reported. In this study, we cloned and characterized an nf-actin gene for elucidation of the role of nf-actin in N. fowleri pathogenesis. The nf-actin gene is composed of 1,124 bp and the sequence identity was 82% with nonpathogenic N. gruberi. No sequence identity with other mammals or human actin gene. Immunofluorescence assay using anti-Nf-actin polyclonal antibody developed in BALB/c mice immunized with recombinant protein (rNf-actin fused with His-tag) revealed that the Nf-actin was localized in the cytoplasm and pseudopodia, especially, food-cup structure (amoebastome), of N. fowleri trophozoites. When N. fowleri were co-cultured with CHO cells, the Nf-actin was observed to localize around on phagocytic food-cups. When the Nf-actin protein was inhibited with cytochalasin D, the actin polymerization inhibitor, or nf-actin gene knock-downed by transfection with antisense oligomers, N. fowleri trophozoites showed reduced food-cup structures and in vitro cytotoxicity. It suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri. In addition, the nf-actin gene was inserted into Ubi-pEGFP-C2 vector for overexpression, and then Ubi-pEGFP-C2/nf-actin was transfected to N. fowleri trophozoites. The strong GFP fluorescence was observed in N. fowleri trophozoites transfected with Ubi-pEGFP-C2/nf actin, and the expression of EGFP-Nf-actin protein was detected by western blot. The nf-actin overexpressed transgenic N. fowleri showed significantly increasing adherence ability against extracellular matrix components such as fibronectin, collagen I and fibrinogen in comparison to wild type N. fowleri. Moreover, the nf actin overexpressed transgenic N. fowleri showed increasing phagocytotic ability and cytotoxicity in comparison with the wild type N. fowleri. Finally, these results suggest that the nf-actin gene plays an important role in cell adhesion, phagocytosis and cytotoxicity of N. fowleri.
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iii
LIST OF TABLE vi
LIST OF FIGURES vii
I. INTRODUCTION 1
A. Free-living amoeba, Naegleria fowleri 1
B. Primary amoebic meningoncephalitis 2
C. Mechanisms of pathogenicity 4
D. An antigenic nfa1 gene 5
E. Actin cytoskeleton 5
E. Purpose of this study 6
II. MATERIALS AND METHODS 8
A. Cultivation of N. fowleri and CHO cells 8
B. Gene cloning 8
C. Sequence analysis and homology alignment 10
D. Expression and purification of recombinant Nf-actin 10
E. Production of anti-Nf-actin polyclonal antibody 11
F. SDS-PAGE and immunoblotting 11
G. Immunofluorescence assay and confocal microscope practice 13
H. Inhibition assay of the Nf-actin 14
1) Effect of cytochalasin D treatment on Nf-actin expression 14
2) In vitro cytotoxicity 14
I. Construction of EGFP expression vector 15
J. Transfection and G418 selection 16
K. Observation of EGFP expression on N. fowleri 17
L. Knock-down system for inhibition of nf-actin gene 17
M. cDNA synthesis and RT-PCR 18
N. Adhesion assay 19
O. Phagocytosis assay 19
P. In vitro cytotoxicity analysis 20
Q. Statistical analysis 21
III. RESULTS 22
A. Cloning of an nf-actin gene 22
B. Homology analysis of the nf-actin gene 25
C. Characterization of recombinant protein (rNf-actin) and polyclonal
anti-Nf-actin antibody 28
D. Cellular localization of the Nf-actin by IFA 30
E. Localization of the Nf-actin in N. fowleri trophozoites co-cultured
with target cells 31
F. Function of the Nf-actin by treatment of actin inhibitor 34
G. Construction of eukaryotic expression vectors 39
H. Transfection of nf-actin and nfa1 gene and observation of Nf-actin and
Nfa1 expression 41
I. Knock-down of nf-actin or nfa1 gene using antisense oligomers 43
J. Expression of the Nf-actin and Nfa1in transgenic N. fowleri 45
K. Cytotoxicity against target cells in transgenic N. fowleri 47
L. Ability of adherence in transgenic N. fowleri 50
M. Phagocytic activity in transgenic N. fowleri 53
IV. DISCUSSION 56
V. CONCLUSION 62
REFERENCES 63
국문요약 72
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LIST OF TABLES
Table 1. Identity (%) of the nf-actin gene amino acid sequence with other species 27
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LIST OF FIGURES
Fig. 1. Complete nucleotide sequences of the nf-actin gene 23
Fig. 2. Amplified PCR products of the nf-actin gene from genomic DNA 24
Fig. 3. Multiple sequence alignments of the deduced amino acid sequence of nf-actin with those from other species 26
Fig. 4. SDS-PAGE and Western blot of the ITPG-induced recombinant Nf-actin 29
Fig. 5. Cellular localization of the Nf-actin by IFA 30
Fig. 6. CHO cells stained with CM-SNARF 32
Fig. 7. Localization of the Nf-actin protein in N. fowleri trophozoites co-cultured with CHO cells 33
Fig. 8 Lcalization of the Nf-actin in N. fowleri treated with cytochalasin D 35
Fig. 9. Inhibition of the Nf-actin with cytochalasin D in N. fowleri co-cultured with CHO cells 36
Fig. 10. In vitro cytotoxicity in Nf-actin inhibited N. fowleri 38
Fig. 11. Construction of eukaryotic expression vectors 40
Fig. 12. Fluorescence microscope finding and Western blot analsysis in nf-actin or nfa1 overexpressed N. fowleri 42
Fig. 13. Knock-down of the Nf-actin expression in N. fowleri treated with antisense nf-actin oligomer 44
Fig. 14. Expression of the Nf-actin and Nfa1 in nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri 46
Fig. 15. Cytotoxicity against CHO cells in nf-actin overexpressed or knock-downed N. fowleri 48
Fig. 16. Cytotoxicity against CHO cells in nfa1 overexpressed or knock-downed N. fowleri 49
Fig. 17. Adhesion activity to the ECM components in nf-actin overexpressed or knock-downed N. fowleri 51
Fig. 18. Adhesion activity to the ECM components in nfa1 overexpressed or knock-downed N. fowleri 52
Fig. 19. Phagocytosis assay in nf-actin overexpressed or knock-downed N. fowleri 54
Fig. 20. Phagocytosis assay in nfa1 overexpressed or knock-downed N. fowleri 55
