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PAM is a rare disease and rapidly fatal disease which occurs generally in previously healthy children and young adults with a history of swimming in freshwater lakes or ponds.

Presumably, infection results from introduction of water containing amoeba into the nasal cavity and subsequent passage of the organisms to the CNS via the olfactory apparatus (Carter, 1968; 1970; 1972). The incubation period of this disease produced by N. fowleri may vary from 2 to 3 days to as long as 7 to 15 days. It has an acute onset with severe headache, nausea, fever ataxia, sign of meningeal irritation, encephalitis (Jonh and John,

- 3 - 1989; Jonckheere, 2011).

As of 2012 year, approximately 128 cases of amoebic meningoencephalitis had been reported throughout the U.S and PAM lead to death in most cases (Yoder et al., 2012). The mortality of patients with PAM is greater than 98 % because of the rapid progression of the disease, delayed diagnosis, and lack of effective healing agents. For the diagnosis and treatment, the antigen related gene producing the antigenic molecule has been largely unsuccessful. Most human infections with N. fowleri have been associated with swimming in warm water but other poorly reports of infects include tap water (Yoder et al., 2012) and host baths.

The final diagnosis of PAM is based on the isolation and culture of free-living amoeba from CSF (cerebrospinal fluid) or the demonstration of amoebic trophozoites in biopsied brain tissue. Antibodies may be detected in serum; however, serologic tests usually are of no value in the diagnosis of infection with free-living amoeba (Visvesvara, 2005; Tuppeny, 2011). Amphotericin B is the only agent with clinical efficacy in PAM and has been reported in some patients treated with amphotericin B alone or in combination with other drugs (Apley et al., 1970; Seidel et al., 1982; Loschiavo et al., 1993). Unfortunately, to date, there is no completely effectively therapeutic drug for PAM.

- 4 - C. Mechanisms of Pathogenicity

The elucidation of pathogenicity of N. fowleri is very important in the mechanisms of parasite-host interaction. The factors that determine the pathogenicity of N. fowleri have not clearly established. In similar free living amoeba, Acanthamoeba, cytopathogenic effects of amoeba on host cells reported in the adhesion of amoeba to host cell, phagocytosis, and amoebic proteolytic enzymes include serine proteases (Kim et al., 2009), contact-dependent metalloproteases (Ondarza, 2007), elastases, cysteine proteases (Hysmith and Franson, 1982;

Barbour and Marciano-Cabral, 2001), and cytotoxic proteinases (Aldape et al., 1994;

Serrano-Luna et al., 2007) induced by mannose-mediated adhesion.

The adhesion is one of the crucial steps for the pathogenicity of amoeba. In addition, Khan et al (2000) reported that pathogenicity would involves both contact-dependent and contact-independent pathways in order to kill host cells quickly and to reduce the degree to which defense can be induced.

In contact-dependent mechanism, Naegleria trophozoites exhibit food-cups or amoebastomes, which are cytoplasmic extension of the surface. The food-cups, which vary in size and number depending on the species and strain if N. fowleri is used to ingest bacteria, yeast cells, and cellular debris concluded that N. fowleri injured target cells by nibbling, a process termed “trogocytosis” (Cursons and Brown, 1978; Marciano-Cabral and John, 1984;

John et al., 1984). N. fowleri destroys target cells by trogocytosis, a process of piecemeal ingestion of target cells by food-cups. This may suggest that the cytoplasmic extensions termed food-cups may play a role in attachment of amoebae to substrates in addition to being

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used for ingestion of particulate material (Marciano-Cabral et al., 1982; 1988).

D. An antigenic nfa1 gene

The pseudopodia-specific nfa1 gene was reported that it participates in the cytopathic activity of N. fowleri on rat microglial cells in a contact-dependent pathogenic mechanism (Oh et al., 2005; Lee et al., 2007). An nfa1 cloned from a cDNA library of N. fowleri by immunoscreening had a coding nucleotide sequence consisting of 360 bases and produced a recombinant protein of 13 kDa (Shin et al., 2001). The Nfa1 protein was mainly located on tips of the pseudopodia and especially food-cups in N. fowleri trophozoties co-cultured with target cells. And the treatment of an anti-Nfa1 antibody is able to induced the decreasing effect on the cytotoxicity of N. fowleri trophozoties (Jeong et al., 2004; Kang et al., 2005).

Therefore, the nfa1 gene is the key molecule concerned with cytotoxicity against host cells in regard to contact-dependent mechanisms of the N. fowleri pathogenesis.

E. Actin cytoskeleton

The actin cytoskeleton is abundant protein in most eukaryotic cells. The fibrous actin (F-actin) consists of a helical polymer of globular polypeptide chain with G-actin (Pollard et al., 1990). Actin plays a important role in a variety of cellular process like motility (Lazarides, 1974), cytokinesis, cell-to-cell and cell-substrate interactions, intracellular transport, endocytosis (Robertson, 2009), exocytosis and phagocytosis. All these processes

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are made possible through remodeling of diverse F-actin structures arising by interactions among actin filaments and regulatory components. Actin-binding proteins contribute to the structural organization of the actin cytoskeleton by filament cross-linking (e.g. filamin, a-actinin) or bundling (e.g. villin, fimbrin), some anchor cytoskeletal structures to the plasma membrane, nuclear envelope or cell-to-cell adhesion sites (e.g. spectrin, interaptin, plectin), others, such as the myosins, mediate movement of cargoes along F-actin tracks (Rivero and Cvrčková, 2006).

A number of infectious diseases regulate the production of actin or of it associated protein. Also the protection of actin is important to the process of pathogenic microorganism’s infection and related to the pathogenicity of intracellular pathogen such as Salmoella, Shigella, Listeria, Toxoplasma gondii, have evolved their own actin cytoskeletal systems (Reisler, 1993; Goode and Eck, 2007).

The expression of pathogenic activity requires a dynamic cytoskeleton that allows rapid movement, tissue penetration, and changes in parasite morphology. There are many studies reported that actin cytoskeleton plays a structural and dynamic role during phagocytosis in other parasites, Entamoeba histolytica, Acanthamoeba castellanii, Acanthamoeba healyi (Hostos, 1993; 1999; Eleonor et al., 2005; Okada et al., 2005).

F. Purpose of this study

N. fowleri destroys target cells through the contact-dependent mechanism such as a phagocytosis and the contact-independent mechanism such as a secretion of proteases. The

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actin cytoskeleton is playing a key of role in the pseudopodia formation and related with the phagocytosis in various protozoa. In D. discoideum, coronin has been found to play important roles in actin activity such as cell motility, cytokinesis, endocytosis and phagocytosis (Hostos, 1993; 1999; Eleonor et al., 2005; Okada et al., 2005). Actin and myosin IB was shown that localizes to the phagocytic cup and phagosomes during ingestion of target cells in E. histolytica (Okada et al., 2005). However despite the numerous studies concerning with pahgocytosis (food-cup formation or amoebastomes), the role of cytoskeleton protein of N. fowleri has been poorly reported.

In this study, I cloned and characterized an actin gene of N. fowleri to understand the role of actin gene in the food-cup formation and cytotoxicity against target cells of N. fowleri.

And the nf-actin gene have transfected into N. fowleri in transfection system which show synergic effect of pathogenicity in N. fowleri. For the transfection of the nf-actin gene into N.

fowleri, pEGFP-C2 and pEGFP-C2/nf-actin containing a ubiquitin promoter were constructed, and knock-down of nf-actin using antisense oligonucleotides was carried out.

After transfection, expressed GFP was observed under a fluorescent microscope and nf-actin gene product was detected by PCR. GFP/Nf-actin fusion protein was detected by western blotting using GFP antibody. And then the influence of Nf-actin on phagocytosis, cytotoxicity and adhesion activity response were observed by overexpression or knock-down system.

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II. MATERIALS AND METHODS

A. Cultivation of N. fowleri and CHO cells

N. fowleri trophozoites (Carter NF69; ATCC No. 30215) were axenically cultured at 37 °C in Nelson’s medium containing 10 % fetal bovine serum (Willaert, 1971). Chinese hamster ovary (CHO) cells were cultured with Dulbecco's modified Eagle's minimal essential medium (DMEM; Gibco BRL, Gaithersburg, MD, USA) containing 10 % fetal bovine serum at 37 °C in 5 % CO2 incubator.

B. Gene cloning

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

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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

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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.

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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

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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

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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

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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.

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After incubation for 5 h post-transfection, 1 ml of complete Nelson's media was added to each well.

After incubation for 5 h post-transfection, 1 ml of complete Nelson's media was added to each well.