The Ubi-pEGFP-C2/nf-actin or Ubi-pEGFP-C2/nfa1 were transfected to N. fowleri on a 12 well plate using transfection reagent (PEI; Polyscience, Warington, PA, USA), according to the manufacturer’s instructions. Briefly, 40 μl of transfection reagent, polyethylenimine (PEI), was added to 200 μl of serum-reduced medium (Opti-MEM; Life Technologies, Rockville, MD, USA). After 200 μl of Opti-MEM containing 10 μg DNA (Ubi-pEGFP-C2/nf-actin or Ubi-pEGFP-C2/nfa1) was added, the total sample was mixed by gently swirling the plate, and after incubation for 10 min at room temperature to allow formation of DNA-polyethylenimine complexes in final volume of 500 μl of Opti-M, samples were added to each well of 6 well plate (Costar, Cambridge, MA, BK) containing 5 × 105 trophozoites.
Two days after transfection, N. fowleri were added a lethal dose the antibiotic G418 (Geneticin; Gibco BRL) (1 mg/ml) was used to select against untransfected N. fowleri.
Transfected N. fowleri trophozoites were subcultured every week until 70 % confluent.
- 17 - K. Observation of EGFP expression on N. fowleri
The expression of EGFP in transfected N. fowleri was observed by fluorescent microscopy (Olympus) using standard fluorescein isothiocyanate excitation/emission filters (488 nm/500 nm). The strong EGFP fluorescence was observed in N. fowleri transfected with Ubi-pEGFP-C2/nf-actin or Ubi-pEGFP-C2/nfa1.
L. Knock-down system for inhibition of nf-actin or nfa1 gene
Expression of nf-actin was knocked down transiently by transfection with nf-actin-specific antisense oligonucleotides. Phosphorothioate antisense oligonucleotides were designed to anneal on the nf-actin ATG start codon (5′-TGC TTG AAC GTC GTA ACA CAT) or the nfa1 ATG start codon (5′-TGG TGA TGG AAT TGT GCC CAT).
Oligonucleotides were introduced into N. fowleri trophozoites by using the methods of transient transfection. Briefly, 10 μl of transfection reagent, polyethylenimine (PEI), was added to 50 μl of serum-reduced medium (Opti-MEM; Life Technologies, Rockville, MD, USA). After 50 μl of Opti-MEM containing 5 μg DNA (the antisense effect was observed to be dependent on oligonucleotide; amount was 5 μg of DNA determined by repeated concentration optimization experiments) was added, the total sample was mixed by gently swirling the plate, and after incubation for 10 min at room temperature to allow formation of DNA-polyethylenimine complexes in final volume of 500 μl of Opti-M, samples were added to each well of 6 well plate (Costar, Cambridge, MA, BK) containing 5 × 105 trophozoites.
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After incubation for 5 h post-transfection, 1 ml of complete Nelson's media was added to each well.
M. cDNA synthesis and Reverse Transcription Polymerase Chain Reaction (RT-PCR)
RNA of N. fowleri and nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri were isolated by RNeasy®Mini kit (QIAGEN, Valencia, CA, USA) according to instructions of manufacturer. The isolated RNA was quantitated by spectrophotometry and equalized and the purity was checked on 1 % agarose gel.
The cDNA was synthesized from 5 μg of total RNA in reaction mixture containing oligo (dT) primer. The mixtures was incubated at 42 °C for 1 h and then subsequently for 5 min at 94 °C and at 4 °C, respectively according to the instructions of the manufacturer. RT-PCR was performed using primer specific for nf-actin gene. RT-PCR condition was 95 °C for 5 min, 30 cycles at 95 °C for 45 sec, 50 °C for 45 sec, 72 °C for 45 sec and then final extension for 10 min at 72 °C.
The amplified products were separated by electrophoresis on EtBr-stained 1 % agarose gel and detected under UV light.
- 19 - N. Adhesion assay
For adhesion assay, the CytoSelect™ Cell Adhesion Assay Kit (Cell Biolabs, San Diego, CA, USA) utilizing an ECM protein-coated plate was carried out according to the manufacturer’s protocol. The ECM-plate coated with fibronectin, collagen type I, collagen type IV, laminin type I, fibrinogen and BSA as a negative control. N. fowleri and nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri were seed 1 x 106 cells/ml incomplete Nelson’s media to the each well (BSA-coated wells are provided as a negative control) at 37 °C for 3 h and the removal of unattached N. fowleri by PBS washes. And 200 μl of cell stain solution at RT for 10 min and removed the cell strain solution and DW washes and let the wells air dry. And 200 μl of Extraction solution per well and then incubate 10 min on an orbital shaker. Finally the extracted samples were transferred to a 96 well plate and measured the OD 560 nm in a microplate reader.
O. Phagocytosis assay
For phagocytosis assay, the CytoSelect™ 96-well Phagocytosis Assay (Cell Biolabs, San Diego, CA, USA) using prelabeled Zymosan particles as a phagocytosis pathogen was carried out. N. fowleri and nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri were cultured a monolayer using 96 well cell culture plate (Nunc A/S, Roskilde, Denmark) in Nelson's media and added 10 μl of zymosan to the each well at 37 °C for 1 h. Amoeba were washed with complete Nelson’s media to remove media and added of 100 μl of
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Fixation solution at RT for 5 min. Amoeba was removed fixation solution and added of 1X Blocking solution at RT for 1 h on orbital shaker. Also amoeba was removed solution and added 100 μl of 1X Permeabilization solution at RT for 5 min and then removed solution and added 100 μl of 1X Detection solution at RT for 1 h on orbital shaker. Finally, amoeba was gently removed solution followed by the addition of 50 μl of Detection buffer at RT for 10 min on orbital shaker. And 100 μl of substrate buffer at 37 °C for 20 min incubation and then stop solution was added. The reactants were measure the OD 405 nm in a plate reader.
P. In vitro cytotoxicity analysis
CHO cells are useful in observing in vitro cytotoxicity of the transfected N. fowleri (Jeong et al., 2004; Oh et al 2005). CHO cells were cultured as monolayer using 24 well cell culture plate (Nunc A/S, Roskilde, Denmark) in DMEM (Gibco BRL, Gaithersburg, MD) at 37 °C.
In control group, 3 x 104 CHO cells were cultured only in 500 μl of DMEM and 3 x 104 CHO cells cultured with trophozoites of N. fowleri with the vector, nf-actin (or nfa1) overexpressed (or knock-downed) N. fowleri was experimental groups for 24 h at 37 °C.
Lactate dehydrogenase (LDH) release assay was practiced to measure in vitro cytotoxicity because the LDH could be released from lysed cells. For LDH assay, 50 µl of reacted supernatant in each well was transferred on 96 well assay plates (Nunc A/S, Roskilde, Denmark). After 50 µl of the reconstituted substrate mix buffer in CytoTox96® Non-radioactive Cytotoxicity Assay Kit (Promega, Madison, WI, USA) for LDH release assay
- 21 -
was added, the plate was incubated 30 min at RT and then 50 µl of stop solution was added.
The reactants were read at 490 nm with ELISA reader. The formula of in vitro cytotoxicity was as follows:
Cytotoxicity (%) =
experimental release - spontaneous release
X 100 maximum release - spontaneous release
Q. Statistical analysis
All experiments were repeated at least three times for each determination. Statistical differences between groups or samples were determined with Student′s t-test. The difference was considered significant when P was < 0.05.
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III. RESULTS
A. Cloning of an nf-actin gene
A 1.2 kb DNA fragment from N. fowleri, tentatively identified as nf-actin, was amplified by degenerated oligonucleotide primer which was designed upon nf-actin of N.
fowleri lee strain (GenBank accession number: U37719). The open reading frame (ORF) of nf-actin was subcloned into pEXP5-NT-TOPO cloning expression vector to produce Nf-actin-(His)6 fusion protein. The full sequence of cDNA was contained an ORF of 1.2 kb encoding a 375 amino acid protein with a deduced molecular weight of 41.3 kDa (50 kDa, fused with His tag). The nucleotide sequence of Nf-actin has been deposited in the GenBank database with the accession number EU890397 (Fig. 1). There are no introns in the nf-actin gene in genomic DNA of N. fowleri (Fig. 2).
- 23 -
Fig. 1. Complete nucleotide sequences of the nf-actin gene. The coding region was shown
together with deduced amino acid translation.
- 24 -
Fig. 2. Amplified PCR products of the nf-actin gene from genomic DNA. DNA size marker (M), Lane 1: amplified PCR product of nf-actin gene from genomic DNA. Lane 2:
amplified PCR product of nf-actin gene from cDNA. The nf-actin gene was presented 1.2 kb.
- 25 - B. Homology analysis of the nf-actin gene
Homology search demonstrated that the amino acid sequence of the nf-actin gene is closely related to the nf-actin gene from amoeba to Yeast. The whole deduced amino acids sequences of the nf-actin gene had 82 % identity with actin gene of N. gruberi. The extent of homology of deduced amino acid sequence of the nf-actin gene ranged from 81 % with actin gene of Saccharomyces cerevisiae to 79 % with actin gene of Entamoeba histolytica (Fig. 3, Table 1).
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Fig. 3. Multiple sequence aligments of the deduced amino acid sequence of nf-actin with those from other species. Gaps were introduced to maximize alignment. The aligned sequences were actin gene from N. fowleri (Lee; U37719), N. gruberi (AAF37002), Yeast (Saccharomyces cerevisiae; AAA34391), and Entamoeba histolytica (HM-1:IMSS;
XP001913786).
- 27 -
Table 1. Identity (%) of the nf-actin gene amino acid sequence with other species
Other species Identity (%)
Naegleria fowleri (Lee) actin
mRNA, complete cds 99 Reference strain
Naegleria gruberi (act1) gene,
complete cds 82 Non-pathogenic Amoeba
Yeast gene for actin 81 Fungi
Entamoeba histolytica;
HM-1:IMSS actin, putative, mRNA 79 Protozoa
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C. Characterization of recombinant protein (rNf-actin) and polyclonal anti-Nf-actin antibody
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.
- 30 -
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).
- 32 -
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).
- 34 -
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).
- 37 -
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).
- 38 -
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.
- 40 -
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.
- 41 -
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).
- 42 -
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.
- 43 -
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).
- 44 -
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.
- 45 -
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).
- 46 -
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
- 47 -
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).
- 48 -
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).
- 49 -
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).
- 51 -
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).
- 52 -
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
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