On the observation by a fluorescent microscopic, CHO cells co-cultured with N. fowleri trophozoites for 24 h showed severe destruction (data not shown). On the contrary, CHO cells co-cultured with nf-actin or nfa1 knock-downed N. fowleri showed less destruction than wild type or overexpressed N. fowleri (data not shown). In nf-actin overexpressed N.
fowleri, the level of cytotoxicity was the highest about 74 % among the all experimental groups. However, in case of N. fowleri transfected with empty vector, the level of cytotoxicity was 31 % (Fig. 15). In case of nfa1 gene, the nfa1 overexpressed N. fowleri showed increasing cytotoxicity levels from 45 % to 72 % in a time-dependent manner (Fig.
16). In addition, the nfa1 knock-downed N. fowleri showed weekly increasing cytotoxicity levels from 6 % to 22 %, respectively (Fig. 16).
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Fig. 15. Cytotoxicity against CHO cells in nf-actin overexpressed or knock-downed N.
fowleri by LDH release assay. CHO cells co-cultured with nf-actin overexpressed N.
fowleri showed a high cytotoxicity in a time dependent manner, but the cytotoxicity was decreased in nf-actin knock-downed N. fowleri. Experiments were performed in triple and the data was shown with mean ± SD (P < 0.05).
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Fig. 16. Cytotoxicity against CHO cells in nfa1 overexpressed or knock-downed N.
fowleri by LDH assay. CHO cells co-cultured with nfa1 overexpressed N. fowleri showed a
high cytotoxicity in a time dependent manner, but the cytotoxicity was decreased in nfa1 knock-downed N. fowleri. Experiments were performed in triple and the data was shown with mean ± SD (P < 0.05).
- 50 - L. Ability of adherence in transgenic N. fowleri
To examine the ability of adherence in nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri, adhesion assay to extracellular matrix (ECM) was performed. The nf-actin overexpressed N. fowleri had increased ability to attach ECM components in comparison with bovine serum albumin (BSA; negative control). Particularly, nf-actin overexpressed N. fowleri have a significantly higher adhesion in fibronectin and fibrinogen (Fig. 17). Similarly, in case of nfa1 overexpressed N. fowleri, the ability of attachment to ECM components such as fibronectin was increased, and nfa1 knock-downed N. fowleri showed less adherence than control groups (Fig. 18).
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Fig. 17. Adhesion activity to the ECM components in nf-actin overexpressed or knock-downed N. fowleri. The nf-actin overexpressed N. fowleri showed a significantly higher adhesion to fibronectin, collagen I, collagen IV, laminin l and fibrinogen, and nf-actin knock-downed N. fowleri showed less adherence than control groups. Experiments were performed in triple and the data was shown with mean ± SD (P < 0.05).
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Fig. 18. Adhesion activity to the ECM components in nfa1 overexpressed or knock-downed N. fowleri. The nfa1 overexpressed N. fowleri increased the ability of attachment to fibronectin, collagen I, collagen IV and laminin I. Experiments were performed in triple and the data was shown with mean ± SD (P < 0.05).
- 53 - M. Phagocytic activity in transgenic N. fowleri
To examine the phagocytic activity of nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri, phagocytosis assay using zymosan particles was performed. As shown in Fig. 19 and 20, nf-actin or nfa1 overexpressed N. fowleri led to increase the phagocytic activity in comparison with control groups (wild type N. fowleri and N. fowleri transfected with empty vector). On the other hand, nf-actin or nfa1 knock-downed N. fowleri was decreasing phagocytic activity in comparison with control groups (Fig. 19, 20).
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Fig. 19. Phagocytosis assay in nf-actin overexpressed or knock-downed N. fowleri. To examine the phagocytic activity of N. fowleri, phagocytosis assay was performed in triple, and the data was shown with mean ± SD (P < 0.05). Lane N; Negative control, Lane 1; wild type N. fowleri, Lane 2; N. fowleri transfected with empty vector, Lane 3; nf-actin overexpressed N. fowleri, Lane 4; nf-actin knock- downed N. fowleri.
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Fig. 20. Phagocytosis assay in nfa1 overexpressed or knock-downed N. fowleri. To examine the phagocytic activity of N. fowleri, phagocytosis assay was performed in triple, and the data was shown with mean ± SD (P < 0.05). Lane N; Negative control, Lane 1; wild type N. fowleri, Lane 2; N. fowleri transfected with empty vector, Lane 3; nfa1 overexpressed N. fowleri, Lane 4; nfa1 knock-downed N. fowleri.
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IV. DISCUSSION
N. fowleri causes an acute lethal CNS disease called PAM. N. fowleri trophozoites enter the nasal cavity, then attach and invade the nasal mucosa and the olfactory nerve (Carter, 1968; Carter, 1972; Ma et al., 1990). PAM is typically leads to death with 1 to 2 weeks from the onset of symptoms (Apley et al. 1970; Shin and Im 2004).
Kim et al. (2008) reported that the contact-independent killing mechanisms of N.
fowleri induced by extracellular secreted proteins were related with amoebic pathgenicity, secreted protein and could play a major role in immune response. Moreover, pathogenic mechanism of N. fowleri is destruction of host cells through a ‘food-cups’ structure on the amoeba surface (Cline et al., 1986 and Marciano-Cabral et al., 1983). Concerned with host-invasion, the adhesion to target cells is most important step in an contact-dependent mechanism of pathogenicity. N. fowleri trophozoties are able to enter the nervous system through the olfactory nerve and digest neuronal tissue by cytolysis and phagocytosis (Carter 1968; Anderson and Jamieson 1972). In addition, subsequent to adhesion the parasite produces a potent cytopathic effect leading to target host cell death in Acanthamoeba (De Jonckheere 1980). Although the food-cups or amoebastomes in N. fowleri were mentioned and described in previous studies, there are no studies related to the attachment and trogocytosis of trophozoites (Marciano-Cabral and John 1983).
In the previous study, an nfa1 gene was characterized, and this gene was located in pseudopodia and food-cup structure (Kang et al. 2005). So, the food-cup formation of N.
fowleri and what molecules are involved in the formation of food-cup structure were my
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interest. To resolve this question, first, I screened with cytoskeleton proteins by using immunofluorescence assay with commercial usable antibodies. As a result, among the three common cytoskeletal proteins, myosin and tubulin were shown in dispersed cellular location of N. fowelri (data not shown), but actin was shown in cytosolic, pseudopodia, and food-cup structure with concentrated and compacted signals. There are many studies that reported actin cytoskeleton protein involved in diverse functions of cell such as adhesion (Gumbiner 1996), motility (Bretscher 1991), and phagocytosis (Swanson and Baer 1995). Therefore, I cloned and characterized an nf-actin gene to evaluate the role of nf-actin gene in pathogenic N. fowleri. The nf-actin gene had the coding sequence of 1.2 kbp, producing a 50 kDa recombinant fusion protein (Nf-actin). The sequence identity was 82 % with nonpathogenic N. gruberi, but has no sequence identity with other mammals and human actin gene (Table 1). In immunofluorescence assay, the Nf-actin was located on the cytoplasm, pseudopodia, and especially, food-cup structure in N. fowleri trophozoites. When N. fowleri co-cultured with CHO target cells, the Nf-actin was strongly expressed on food-cup structure concerning trogocytosis. Although the presence of food-cups does not correlate with pathogenicity (Marciano-Cabral 1988), N. fowleri treated with cytochalasin D, actin polymerization inhibitor, reduced ability of food-cup formation (Fig. 9, 10) and in vitro cytotoxicity in this study. It suggests that food-cup formation was reduced by actin inhibition, and then, cytotoxicity of N. fowleri was reduced because of decreased attachment ability. These results suggest that Nf-actin play an important role in phagocytic activity and pathgoenicity of N.
fowleri.
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In addition, eukaryotic transfection vectors, C2/nf-actin and Ubi-pEGFP-C2/nfa1 vector were constructed, and then, the effect of cell adhesion, cytotoxicity and phagocytosis in N. fowleri were observed. After transfection, GFP fluorescence using a microscope was observed in N. fowleri transfected with pEGFP-C2/nf-actin and Ubi-pEGFP-C2/nfa1 vectors. Ubiquitin promoter was used in this study, because ubiquitin is a small regulatory protein in almost all eukaryotic cells (Jentsch S, 1992). It is involved in several basic cellular functions; making protein for rapid degradation, mediation of gene transcription, DNA repair, cell cycle progression, stress response and the modulation of the immune response. The ubiquitin gene was shown to be regulated during Acanthamoeba development from the actively growing stage toward the dormant cyst stage (Ahn and Henny 1994; Wulff et al., 1990). In Ubi-pEGFP-C2/nf-actin and Ubi-pEGFP-C2/nfa1 vectors, a CMV promoter was replaced with an ubiquitin promoter in order to transcribe the nf-actin or nfa1 gene in transgenic N. fowleri, because the CMV promoter could poorly transcribe the nf-actin or nfa1 gene. Western blotting and RT-PCR was performed to indentify an expressed Nf-actin or Nfa1 from nf-actin or nfa1 overexpressed N. fowleri. Also N. fowleri transfected with antisense oligonucleotides of nf-actin or nfa1 gene was examined by knock-down system. Antisense nf-actin or nfa1 oligomers were designed to anneal on the nf-actin or nfa1 ATG start codon. In this study, nf-actin or nfa1 knock-downed N. fowleri showed decreasing, number of food-cup and reducing of nf-actin or nfa1 mRNA and protein levels.
These results show that the Nf-actin and Nfa1 proteins are the key molecules to contact and kill the target cells.
In the mechanisms of pathogenicity, the adherence of Naegleria trophozoites to the host
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cell is the most important step in the establishment and invasiveness of this infectious disease. Previously, it has been reported that protozoa are recognized by components of the ECM (Gordon et al., 1993; Han et al., 2004; Rocha-Azevedo et al., 2007, 2009; Shibayama et al., 2003). E. histolytica bind to ECM compontets and this interaction may play an important role in its penetration of intestinal mucosa (de Lourdes Mun˜oz et al., 2001; Li et al., 1995). In addition, it has been reported that Acanthamoeba binds to laminin-1, collagen IV and fibronectin (Gordon et al., 1993). In case of N. fowleri, amoeba binds to fibronectin in a concentration-dependent manner through the mediaction of a 60 kDa fibronectin-binding protein (Han et al., 2004). In the present study, nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri was selected for assessment in attachment to ECM components. An nf-actin overexpressed N. fowleri have a significantly higher adhesion in fibronectin and fibrinogen in comparison with wild-type N. fowleri. Similarly, in case of nfa1 overexpressed N. fowleri, the ability of attachment to ECM components such as fibronection was increased.
These results suggest that the Nf-actin and Nfa1 are recognized as an important process for the adhesion to target cells in pathogenicity of N. fowleri.
Phagocytosis plays an important role in the pathogenesis of protozoa (Niederkorn et al.
1999; Taylor et al., 1995). Previous studies have identified that actin polymerization plays an important role in bacterial entry into the host cell as shown in Legionella pneumophila entry into human monocytes (Coxon et al. 1998), E. coli K1 invasion to CNS (Khan et al.2002), group B Streptococcus (Nizet et al. 1997) and Citrobacter (Badger et al. 1999) invasion of human brain microvascular endothelial cells and Salmonella typhimurium invasion of epithelial cells (Finlay and Ruschkowski 1991). These studies indicate that actin-mediated
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cytoskeletal rearrangements play an important role in bacterial entry into the eukaryotic cells.
In this study, the phagocytic activity using pre-labelled zymosan particles was observed in nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri. These results show that the phagocytic activity of nf-actin or nfa1 overexpressed N. fowleri was strongly increased in comparison with control groups (wild-type N. fowleri and nf-actin or nfa1 knock-downed N.
fowleri). These results indicated that Nf-actin and Nfa1 play important roles in phagocytic activity of N. fowelri.
In addition, when N. fowleri trophozoites co-cultured with microglial cells, the high cytotoxicity of amoeba on microglial cells was increased in a time dependent manner (Jeong et al., 2004; Oh et al., 2005), but after anti-Nf-actin antibody was added co-cultivation system, the cytotoxicity was decreased than groups without antibody. In addition, the proliferation and in vitro the cytotoxicity of N. fowleri against CHO target cells were observed by adding an anti-Nfa1 antibody in the co-culture system to elucidate the antibody effect. As a result, the cytotoxicity of anti-Nfa1 antibody treated N. fowleri was decreased, when anti-Nfa1 antibody was added in a dose dependent manner (Jeong et al., 2004, 2005;
Kang et al., 2005). In this study, in vitro cytotoxicity of N. fowleri against target CHO cells was measured in nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri. N. fowleri co-cultured with CHO cells at 3 h were shown 28% of cytotoxicity. On the other hand, cytotoxicity of nf-actin overexpressed or knock-downed N. fowleri were 45 % and 19 %, respectively. And nfa1 overexpressed or knock-downed N. fowleri showed similar levels of cytotoxicitiy as a result of nf-actin. These results indicate that nf-actin or nfa1 overexpressed N. fowleri has more cytotoxic effect than control groups, and consequently Nf-actin and
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Nfa1 can induce the cytotoxic effect against target cells in the mechanism of pathogenesis.
Finally, these results suggest that Nf-actin and Nfa1 play important roles in phagocytic activity and pathogenicity of pathogenic N. fowleri. Furthermore, it is necessary to continuously research for the direct correlation between nf-actin and nfa1 gene on pathogenesis of N. fowleri.
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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.
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