The recombinant Nf-actin-(His)6 fusion-protein expressed in E. coli containing the nf-actin gene showed a major protein band of about 50 kDa in SDS-PAGE (Fig. 4A). The purified rNf-actin fusion protein showed strong immunoreactivity to the anti-Nf-actin polyclonal antibody, but not to normal mouse sera (Fig. 4B).
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Fig. 4. SDS-PAGE and Western blot of the ITPG-induced recombinant Nf-actin protein. A; UN, uninduced E. coli extracts; IN, 1mM IPTG-induced E. coli extracts. P, recombinant Nf-actin protein (fused with His-tag) purified using Ni-NTA column. B;
Western blot of the rNf-actin reacted with normal mouse sera and mouse immunized with rNf-actin sera (IM). Normal mouse sera (N) did not show the reaction band. M indicated protein marker.
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D. Cellular localization of the Nf-actin by immunofluorescence assay (IFA)
Immunofluorescence assay using polyclonal anti-actin antibody showed that the Nf-actin protein was immunolocalized in cytosol, and compact signal was particularly shown in food-cup regions (Fig. 5).
Fig. 5. Cellular localization of the Nf-actin by IFA. Under a fluorescence microscope localization of the Nf-actin was strongly observed in food-cups (arrow). The light microscopic findings were shown in small box (x400).
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E. Localization of the Nf-actin in N. fowleri trophozoites co-cultured with target cells
To observe the role of the Nf-actin protein in N. fowleri co-cultured with target cells during cell contact, target cells were stained with CM-SNARF (red color) (Fig. 6). After N.
fowleri was co-cultured with CHO cells at 3 h, strong fluorescent signals were shown in pseudopodia of N. fowleri trophozoties and in food-cups structures (Fig. 7). In addition, the Nf-actin protein was often observed around area contacted with CHO cells (Fig. 7).
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Fig. 6. CHO cells stained with CM-SNARF. a; Red color from CHO cells observed by a fluorescent microscope, b; light microscopic finding (x200).
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Fig. 7. Localization of the Nf-actin protein in N. fowleri trophozoites co-cultured with CHO cells. Strong fluorescent signals were shown in pseudopodia and food-cups of N.
fowleri (arrow). a; CM-SNARF stained CHO cells (red), b; fluorescein isothiocyanate-labeled Nf-actin (green), c; DIC-merged images, d; merged images. Arrows indicate food-cup structures in N. fowleri trophozoites (x200).
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F. Function of the Nf-actin by treatment of actin inhibitor
1) Localization of the Nf-actin in N. fowleri treated with cytochalasin D
To observe the role of actin on the food-cup formation of N. fowleri, N. fowleri was treated with cytochalasin D, actin polymerization inhibitor. N. fowleri treated with Cytochalasin D showed shrinkage in food-cup structure. Morphologically, the N. fowleri trophozoites changed into cyst form at high concentration of cytochalasin D (Fig. 8). And then, On the results of inhibition of the Nf-actin in N. fowleri co-cultured with CHO cells, CHO cells stained with CM-SNARF showed red color (Fig. 9). When N. fowleri without cytochalasin D treatment was co-cultured with CHO cells at 3 h, N. fowleri trophozoites showed strong and compact Nf-actin signals around area contacted with CHO cells (Fig 9A).
In contrast, N. fowleri treated with cytochalasin D and co-cultured with CHO cells showed decreasing the numbers of food-cups and showed food-cup formation (Fig. 9B).
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Fig. 8. Localization of the Nf-actin in N. fowleri treated with cytochalasin D. N. fowleri were treated with 20 μM or 100 μM of Cytochalasin D for 1 h. N. fowleri treated with cytochalasin D showed reducing the numbers of food-cups structures and weak food-cup formation. a; fluorescein isothiocyanate-labeled Nf-actin (green), b; light microscopic finding (x200).
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Fig. 9. Inhibition of the Nf-actin with cytochalasin D in N. fowleri co-cultured with CHO cells. A; wild-type N. fowleri was co-cultured with CHO cells. B; N. fowleri with 20 uM cytochalasin D was co-cultured with CHO cells. a; CM-SNARF stained CHO cells (red), b; fluorescein isothiocyanate-labeled Nf-actin (green), c; DIC images, d; merged images.
Arrows indicate food-cup structures in N. fowleri trophozoites (x200).
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2) In vitro cytotoxicity against target cells in N. fowleri inhibited Nf-actin
To evaluate the role of Nf-actin on the N. fowleri cytotoxicity against CHO cells, LDH release assay was carried out. When CHO cells were co-cultured with N. fowleri trophozoites pretreated with cytochalasin D, the cytotoxicity of N. fowleri decreased 30 % at 6 h in comparison with untreated control (P < 0.001) (Fig. 10).
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Fig. 10. In vitro cytotoxicity of the Nf-actin-inhibited N. fowleri. Cytotoxicity was calculated at 3 and 6 h post-treated cytochalasin D and showed decreasing value in a time-dependent manner (P < 0.001). N. fowleri; untreated trophozoites, DMSO; dimethyl sulfoxide-treated trophozoites, Cytochalsin D; 20 μM cytochalasin D-treated trophozoites.
Values are means ± standard errors of three experiments in triplicate.
- 39 - G. Construction of eukaryotic expression vectors
Constuction of Ubi-pEGFP-C2/nf-actin and Ubi-pEGFP-C2/nfa1 vectors using modified pEGFP-C2 vector containing expression of the green fluorescence protein in mammalian cells. Instead of CMV promoter, ubiquitin promoter was replaced. In the Ubi-pEGFP-C2/nf-actin and Ubi-pEGFP-C2/nfa1 vectors, an nf-actin gene and nfa1 gene were inserted in downstream of the gene encoding EGFP which produced 7.7 kbp and 6.9 kbp, and nf-actin primer and nfa1 primer were used to amplify the 1.2 kb and 360 bp, respectively (Fig. 11). These results show that Ubi-pEGFP-C2/nf-actin and Ubi-pEGFP-C2/nfa1 vectors were correctly constructed.
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Fig. 11. Construction of eukaryotic expression vectors. In the Ubi-pEGFP-C2/nf-actin (A) or Ubi-pEGFP-C2/nfa1 (B) vectors, ubiquitin promoter was replaced with CMV, and nf-actin or nfa1 gene was inserted. Amplified PCR products of nf-nf-actin (A) or nfa1 (B) on Ubi-pEGFP-C2/nf-actin and Ubi-pEGFP-C2/nfa1 were shown. DNA size marker (M), Lane 1:
amplified PCR product of nf-actin gene of Ubi-pEGFP-C2/nf-actin, Lane 2: amplified PCR product of nfa1 gene of Ubi-pEGFP-C2/nfa1.
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H. Transfection of nf-actin and nfa1 gene and observation of Nf-actin and Nfa1 expresssion
On following 48 h post transfection and selection with 1 mg/ml of G418 for 7 days the expression of GFP in N. fowleri tranfected with C2/nf-actin or Ubi-pEGFP-C2/nf-actin vectors was examined. At 48 h post transfection, GFP expression was observed in the cytoplasm of N. fowleri transfected with C2/nf-actin or pEGFP-C2/nfa1 vector (Fig 12A). In N. fowleri transfected with pEGFP-C2/nf-actin or Ubi-pEGFP-C2/nfa1, GFP fusion proteins could be identified by Western blotting after 48 h of transfection (Fig. 12B).
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Fig. 12. Fluorescence microscope findings and Western blot analysis in nf-actin or nfa1 overexpressed N. fowleri. A; The fluorescence of GFP was observed at 48 h after transfection (x200). B; The lysates of wild-type N. fowleri (lane 1), N. fowleri transfected with Ubi-pEGFP-C2 (lane 2), Ubi-pEGFP-C2/nfa1 (lane 3) and Ubi-pEGFP-C2/nf-actin (lane 4) were reacted with GFP antibody.
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I. Knock-down of nf-actin or nfa1 gene using antisense oligomer
To observe the inhibited role of nf-actin or nfa1 gene in the food-cup formation of N.
fowleri, nf-actin or nfa1 gene were knock-downed using transfection system with antisense oligomer. The nf-actin mRNA levels were knock-downed about 42 %, and the Nf-actin protein expression was inhibited about 58.2 % (Fig. 13A). Under a fluorescent microscope, an nf-actin knock-downed N. fowleri showed decreasing numbers of food-cup and weak food-cup formation (Fig. 13B).
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Fig. 13. Knock-down of the Nf-actin expression in N. fowleri treated with antisense oligomer. A; RT-PCR and Western blotting analysis in untransfected or nf-actin knock-downed N. fowleri trophozoites. Total RNA profiles indicated that all loading sample quantity was equal. B; Immunofluorescence assay with polyclonal anti-Nf-actin antibody in untransfected or nf-actin knock-downed N. fowleri trophozoites (x200). N; wild-type N.
fowleri, T; nf-actin knock-downed N. fowleri.
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J. Expression of the Nf-actin and Nfa1 in transgenic N. fowleri
To confirm the effect of overexpression or knock-down of nf-actin or nfa1 on N.
fowleri trophozoites, RT-PCR and Western blot ananlysis were performed. In nf-actin overexpressed N. fowleri, the elevated mRNA level of nf-actin was induced compared to control groups (Fig. 14A). In nf-actin knock-downed N. fowleri, mRNA level and protein expression were reduced in comparison with control groups (Fig. 14A). In addition, nfa1 overexpressed N. fowleri showed increasing mRNA levels and protein expression in comparison with control groups (Fig. 14B). In nfa1 knock-downed N. fowleri, the reduced mRNA level and protein expression were showed in comparison with control groups (Fig.
14B).
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Fig. 14. Expression of the Nf-actin and Nfa1 in nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri. The expressions of the Nf-actin (A) and Nfa1 (B) in overexpressed or knock-downed N. fowleri were analyzed by RT-PCR and Western blotting.
Total RNA profiles indicated that all loading sample quantity was equal. Lane 1; wild type N.
fowleri, lane 2; N. fowleri transfected with empty vector, lane 3; nf-actin or nfa1 overexpressed N. fowleri, lane 4; nf-actin or nfa1 knock-downed N. fowleri
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K. Cytotoxicity against target cells in transgenic N. fowleri
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
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