Total RNA was prepared from trophozoites of N. fowleri using an isolation kit RNeasy®Mini kit (QIAGEN, Valencia, CA. USA). Briefly presented, after 600 μl of RLT buffer was mixed with pellet of 1 x 106 trophozoites by pipetting. It was centrifuged at 13,000 rpm for 3 min at room temperature (RT). The supernatant was transferred to a new eppendorf tube and reacted with 600 μl of 70 % ethanol. The reagent was transferred to a new spin column and centrifuged at 13,000 rpm for 1 min at RT. Add 700 μl of RW1 buffer to the spin column and centrifuged at 13,000 rpm for 1 min at RT. Add 500 μl of RPE buffer to the column and centrifuged at 13,000 rpm for 1 min. RNA yield and quality were checked by using spectrophotometrically and by analytical agarose gel electrophoresis. For reverse transcription polymerase chain reaction (RT-PCR), a superscript first strand synthesis
- 9 -
System kit (Invitrogen, Carlsbad, CA, USA) was used to generate cDNA from 5 μg of total RNA.
For amplification of cDNA by PCR, degenerated oligonucleotide primers were designed on conserved region of actin gene in N. fowleri Lee strain, and Oligo dT primer was used for 3’ end.
The genomic DNA was prepared from trophozoites of N. fowleri using DNeasy Tissue kit following the manufacturer’s instruction (QIAGEN, Valencia, CA. USA). For the amplification of genomic DNA by PCR, primers were designed on actin gene of N. fowleri (Nf-actin forward primer: ATG TGT GAC GAC GTT CAA GCA CTC, Nf-actin reverse primer: AAG ATC TTT CTG TGG ACA ATA CCT GGA). The amplification PCR product was purified from the gel and subcloned for sequence analysis into pCR2.1 TOPO TA vector (Invitrogen). The nucleotide sequence of cloned cDNA fragments was determined using an ABI Perkin Elmer 373A automated DNA sequencer (Applied Biosystems, Forster City, CA, USA). The open reading frame of Nf-actin was subcloned into the pCR-T7/NT-TOPO cloning /expression vector (Invitrogen) to produce Nf-actin-(His)6 fusion protein.
The recombinant expression plasmid pCR-T7/NT-TOPO: nf-actin was sequenced to ensure that the inserts were in the correct reading frame. Briefly presented, 4 μl of fresh amplified nf-actin ORF by PCR was mixed to 1 μl of PCR-T7/NT-TOPO expression vector (Fig. 2) and incubated for 5 min at room temperature. One μl of the 6 x TOPO cloning stop solution was added and mix for 10 sec. The reaction tube was placed on ice. PCR subsequently, the E. coli strain BL21 (DE3) pLysS was transformed with pCR-T7/NT-TOPO:
nf-actin.
- 10 - C. Sequence analysis and homology alignment
Deduced amino acid sequences were analyzed with the EditSeq.V 1.0.3 program and Clustal of the MegAlign program, a multiple-alignment program of the DNASTAR package (DNASTAR, Madison, WI, USA).
D. Expression and purification of recombinant Nf-actin
A fresh overnight culture form the E. coli cells containing pCR-T7/NT-TOPO: nf-actin was 1:100 in Luria-Bertani medium supplemented with 100 μg/ml ampicillin and 34 μg chloramphenicol, and grown at 37 °C until the OD600 reached 0.5. Expression was initiated with 1 mM isopropyl β-D-thiogalactopyranoside. The cells were grown for additional 4 h at 37 °C before being harvested by centrifugation at 8,000 rpm for 5 min at 4 °C. Expressed recombinant antigen was purified using the probondTM Purification System (Invitrogen).
Harvested cells were resuspened in native binding buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8) and sonicated with two or three 10 second bursts at a medium intensity setting while holding the suspension on ice. Sonicated cells were freezed immediately in liquid nitrogen and quickly thawed at 37 °C. Final concentration of 5 μg/ml RNase and 5 μg/ml DNase was treated on ice for 15 min. Insoluble debris was removed by centrifugation at 12,000 rpm for 20 min. The recombinant gene product was eluted using ProbondTM resin column (Invitrogen) by increasing imidazol concentration. The eluted protein was dialyzed and concentrated by freeze-dry. Recombinant Nf-actin was purified
- 11 -
from supernatant by chelating chromatography on Ni-NTA agarose resin (Invitrogen) by increasing imidazol concentration. The eluted proteins concentrated with Amicon Ultra-15 (Milipore, Bedford, MA, USA).
E. Production of anti-Nf-actin polyclonal antibody
For the production of polyclonal antibody, the Nf-actin protein (50 μg) was mixed an equal volume of complete Freund’s adjuvant (Sigma), and injected intraperitioneally into 7-week-old female BALB/c mice (Korea). The mouse was boosted biweekly for another 4 weeks with the Nf-actin protein (25 μg) containing an equal volume of incomplete Freund’s adjuvant (Sigma). After the third boosting the Nf-actin protein (5 μg) was injected intravenously without the adjuvant. Four day later, a polyclonal anti-Nf-actin antibody was collected from the mouse blood by centrifuging at 2,500 x g for 30 min at 4 °C.
F. SDS-PAGE and immunoblotting
12 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition was performed for the fraction of amoeba lysates. The lysates samples were suspended in reducing sample buffer (62.2 mM Tris, pH 6.8; 10 % glycerol; 10 % 2-mercaptoethanol; 3 % SDS and 0.1 % bromophenol blue), boiled for 5 min prior to loading onto the gel and then separated by electrophoresis. For Western blotting, proteins were transferred onto Polyvinylidene difluoride (PVDF) sheet and reacted with polyclonal
anti-- 12 anti--
Nf-actin antibody (1:1,000 dilutions) and normal sera (1:1,000 dilutions) at 4 °C for overnight. After washing with 0.05 % PBST three times, peroxidase-conjugated goat anti-mouse whole IgG (1:2,000 dilutions) (Sigma Chemical Co., St. Louis, USA) was added on each sheet. After washing with 0.05 % PBST three times, they were soaked in enhanced chemiliminescence (ECL) solution (Intron, Dajon, Korea) and exposed to an X-ray film (Konica, Tokyo, Japan).
Transgenic amoeba was extracted by cell lysis buffer (Intron, Dajon Korea) and quantified by centrifuged to remove cell pellets. The supernatants were quantified the Brafored method (Bradford, 1976). The sample was analyzed by 12 % SDS-PAGE using reducing sample buffer (62.2 mM Tris, pH 6.8; 10 % 2-mercaptoethanol; 3 % SDS; and 0.1 % bromophenol blue). For Western blotting, proteins were transfected onto PVDF membrane for 2 h at 250 mA. The membrane was blocked with 5 % skim milk for 2 h at RT and reacted with anti-Nf-actin antibody (1:1,000 dilutions with 3 % BSA), anti-Nfa1 antibody (1:1,000 dilutions with 3 % BSA) and anti-GFP antibody (1:1,000 dilutions with 3 % BSA, abcam) for overnight at 4 °C. The membrane was washed with PBS containing Tween-20 (PBST) 3 times for 15 min and reacted with secondary antibody of a goat anti-mouse IgG conjugated with horseradish peroxidase (1:2,000 dilutions with 3 % BSA) for 2 h at room temperature.
After washing with PBST 3 times, they were soaked in enhanced chemical luminescence’s solution and exposed to X-ray film.
- 13 -
G. Immunofluoroscence assay and confocal microscope practice
To observe the cellular localization of Nf-actin protein in N. fowleri, immunofluroscence assay was performed according to the method in a precious paper.
Amoeba trophozoites were fixed with 10 % formalin and treated with 1 % NH4OH ammonium hydroxide), and attached to a glass slide. Polyclonal anti-Nf-actin antibody (1:100 dilutions) was added to a slide glass. After incubating the glass slide at 4 °C for 2 h, a goat anti-mouse whole immunoglobulin (1:200 dilutions) conjugated with a fluorescein isothiocyanate (FITC, Sigma. USA) was added. Fluorescent localization in intact trophozoites was observed under a fluorescence microscope.
For immunofluorescene studies, cultivating trophozoites of N. fowleri or CHO cells used as target cells were fixed in 10 % formaladehyde for 10 min at room temperature, permeabilized in 1 % NH4OH, washed in Tween 20 for 5 min and extensively washed with 0.82 % saline. N. fowleri was incubated overnight at 4 °C with an anti-Nf-actin polyclonal antibody diluted serially with 3 % BSA. This was followed by incubation with the appropriate FITC-conjugated anti-mouse antibody (Sigma) a dilution of 1:100 at 4 °C for 2 h.
As the controls, N. fowleri trophozoites and CHO cells stained with the FITC-conjugated anti-mouse antibodies. For labeling the living target cells, CHO cells were incubated 30 min at 37 °C. DMSO stock solutions (100 μl in 5-(and-6)-chlormethyl seminaphthorhodafluor-1 acetate (CM-SNARF) stock) are typically diluted 1:1,000 into loading buffer should be serum-free because often contains esterase activity. Total fluorescence of cells was visualized in optical section produced by confocal microscope (Olympus FV-500). The
- 14 -
collected images were processed using Adobe Photoshop 7.0 software.
H. Inhibition assay of the Nf-actin
Cytochalasin D (Sigma) is a cell permeable and potent inhibitor of actin polymerization.
It disrupts actin microfilaments and activates the p53-dependent pathways causing arrest of the cell cycle at the G1-S transition. It is believed to bind to G actin and prevent polymerization of actin monomers.
1) Effect of cytochalasin D treatment on Nf-actin expression
To determine viability of N. fowleri by cytochalasin D treatment, trophozoites of N.
fowleri was removed from a 75 T flask. After twice washing with PBS, the trophozoites were treated at 20 μM, 100 μM concentration of cytochalasin D in 6 well plates, and incubated at 37 °C for 30 min, 1 h. Cytochalasin D was dissolved in dimethyl sulfoxide (DMSO). The same concentration of DMSO was used as a negative control.
2) In vitro cytotoxicity
As CHO cells are useful in observing in vitro cytotoxicity of Nf-actin inhibited N.
fowleri trophozoites (Shin et al., 2001). CHO cells were cultured as monolayer using 24 well cell culture plate (Nunc A/S, Roskilde, Denmark) in DMEM at 37 °C. When 3 x 104 CHO
- 15 -
cells were co-cultured with N. fowleri trophozoties pretreated with cytochalasin D. 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 was added, the plate was incubated 30 min at room temperature 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.
The eukaryotic expression vectors transfected into N. fowleri were the pEGFP-C2 vector (Clontech). The pEGFP-C2 vector containing the cytomegalovirus (CMV) promoter encodes a red-shifted variant of wild-type enhanced GFP (EGFP), which has been optimized to maximize fluorescence and expression in mammalian cells and was used as a control construct. We cloned to modify the ubiquitin promoter was inserted into the CMV promoter was deleted. The ubiquitin promoter might be able to act on nf-actin and express it more
- 16 -
efficiently than CMV promoter. The amplified ubiquitin promoter was cloned into CMV promoter-deleted pEGFP-C2 vector.
The nf-actin gene were inserted downstream of the gene encoding EGFP in the pEGFP vector. We constructed of Ubi-pEGFP-C2/nf-actin, which produced 7.7 kbp fragments.
In previously study, the nfa1 gene (GenBank Accession No. AF230370) was cloned from nfa1 gene-cloned vector, PCR-T7/NT TOPO (Shin et al., 2001). The nfa1 gene was inserted into Ubi-pEGFP-C2 vector, which produced 6.8 kbp fragments.
J. Transfection and G418 selection
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.
- 18 -
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
- 20 -
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.
- 22 -
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).
- 26 -
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
- 28 -
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).
- 29 -
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).
- 31 -
E. Localization of the Nf-actin in N. fowleri trophozoites co-cultured with target cells
E. Localization of the Nf-actin in N. fowleri trophozoites co-cultured with target cells