DISCUSSION

PAME caused by N. fowleri is an acute, fulminant, and rapidly progressing fatal illness that usually affects children and young adults. The olfactory neuroepithelium is the route of invasion in PAME due to N. fowleri. Invasions of the olfactory mucosa and the olfactory bulbs, with hemorrhagic necrosis of both cerebral gray and white matters and an acute inflammatory infiltrate, are the histopathologic characteristics (Maritra et al., 1976). Naegleria has an intranuclear mitosis, called promitosis, following the classical pattern of chromosome separation, but the chromosomes are too small to be counted by conventional histological techniques (Fulton, 1970).

However, it has been possible to enumerate the chromosomes with the use of pulsed field gel electrophoresis. The number of chromosomes and their size differ between species and even between strains of the same species. Two stains of N. gruberi sensu lato have 23 chromosomes, but the size of some chromosomes differs (Clark et al., 1990). The ploidy of the Naegleria genome is still not known. The sum of the chromosome sizes (approximately 19 Mb) does not equal the expected genome size (approximately 104 Mb), which indicates that Naegleria might be polyploidy (Clark et al., 1990). Isoenzyme studies of Naegleria spp. usually imply diploidy (Cariou and Pernin, 1987). These organisms have been long recognized as attractive models for a variety of studies in basic cellular and molecular biology. They have a relatively large size, rapid growth in axenic culture, active motility and phagocytosis, and they

exhibit unicellular differentiation. Despite the attractiveness of Naegleria, it has been underutilized as a model system. So far there has been no evidence for sexual reproduction. Therefore, classical mapping and genetic analysis is limited. The earliest stage post-inoculation of N. fowleri was 24 h (Jarolim et al., 2000). As the recent study (Rojas-Hernández et al., 2004), the events occurring during the first 8 h post-inoculation are as follows: N. fowleri trophozoites make contact with the surface of the mucous layer of the olfactory epithelium; some of them move across the mucous layer and reach the apical pole of the epithelial cells, apparently without disruptionand/or depletion of the mucosa. Several trophozoites are eliminated by being embedded in the mucous layer that sometimes forms a lump containing inflammatory cells. The binding of N. fowleri trophozoites to the mucous layer can be mediated by specific cell surface lectins that recognize carbohydrates in mucin glycoproteins, as has been described during intestinal colonization by Entamoeba histolytica (Chadee et al., 1987, 1988). The binding of N. fowleri trophozoites to mucins may facilitate the adhesion and invasion of the parasite. After 96 h, N. fowleri trophozoites in the olfactory bulb were abundant, suggesting that they have proliferated. Furthermore, abundant inflammatory cells and severe tissue damage were found. This damage could be provoked by both N. fowleri and neutrophils.

Trophozoites may invade and enhance tissue damage by releasing cysteinproteases and other enzymes which degrade components of the extracellular space and have a cytopathic effect on mammalian cells (Aldape et al., 1994). In Acanthameoba of same free-living amoeba, the mechanism by which Acanthamoeba produces

granulomatous amoebic encephalitis and amoebic keratitis (AK) has not been fully elucidated. However, it is generally accepted that the two major predisposingfactors in the pathogenesis of AK are minor corneal trauma causedby contact lens wear or other noxious agents and exposure to contaminated solutions including lens care products and tapwater (Kilvington et al., 2004; Larkin et al., 1990). The adhesion of parasites to the host cells isclearly a critical first step in the pathogenesis of infection (Moore et al., 1991; Panjwani et al., 1997; van Klink et al., 1992). Subsequent to adhesion, the parasites produce a potentcytopathic effect leading to target cell death (Cao et al., 1998; De Jonckeere, 1980; Larkin et al., 1991; van Klink et al., 1992).

That the Acanthamoeba may adhere to host cells via a carbohydrate-bindingprotein has been suggested by studies demonstrating that: (i)the adhesion of Acanthamoeba to corneal epithelial cells inculture as well as to the surface of the corneal buttons canbe inhibited by free methyl-α-mannopyranoside (α-Man) but notby a number of other sugars (Cao et al., 1998; Morton et al., 1991; Panjwani et al., 1997; Yang et al., 1997), (ii) Acanthamoebaebind to a neoglycoprotein, mannosylated-bovine serum albuminbut not to galactose-bovine serum albumin (Cao et al., 1998), (iii) mannose-relatedsaccharides that inhibit amoeba binding to corneal epithelialcells are also potent inhibitors of the amoeba-induced cytopathic effect (Cao et al., 1998). In addition, preliminary studies have shown that Acanthamoebae express a putative mannose-binding protein (MBP) of 136 kDa (Yang et al., 1997). These findings suggest that the adhesion ofAcanthamoeba to the corneal surface is mediated by interactions between a mannose-specific lectin on the surface of the amoebaand

mannose residues of glycoproteins of corneal epithelium, and that the mannose-mediated cross-talk between amoeba andcorneal epithelial cells is a key component of the Acanthamoeba-inducedcytopathic effect. On the mechanism of pathogenicity of N. fowleri, the adherence of the amoeba to host cells is most important, and the specific pseudopodial projection, so called as amoebastome, is formed (Derr-Harf and De Joncheere, 1984). In addition, it was reported that killing of host cells is mediated by a pore-forming peptide known as amoebapore (Herbst et al., 2002) and proteolysis of the host’s extracellular matrix is mediated by cysteine proteinases (Aldape et al., 1994). We previously reported that the Nfa1 protein expressed from nfa1 gene was located in pseudopodia and around vacuoles (Shin et al., 2001). When CHO target cells were cocultured with N. fowleri trophozoites, the Nfa1 protein was specifically localized at amoebastomes of phagocytic evidence (Kang et al., 2005). It powerfully supported that the Nfa1 protein might be related with the cytotoxicity of N. fowleri. Moreover, the treatment of anti-Nfa1 antibody decreased the cytotoxicity of N. fowleri against CHO cells (Jeong et al., 2004). Recently, to elucidate an Nfa1 protein with cytotoxicity of transgenic N. gruberi to CHO cells, transfection study was performed (Jeong et al., 2005). N. gruberi trophozoites were transfected with a pEGFP–C2/nfa1UTR vector, and the trophozoites induced in vitro cytotoxicity to CHO cells. And also, the treatment of anti-Nfa1 antibody to transgenic N. gruberi decreased the cytotoxicity to CHO cells.

In Naegleria, because of the presence of multiple copies of the genome, it is difficult to study gene function in N. fowleri by classical genetical methods or to

isolate mutants. Mammalian gene function has been determined traditionally by methods such as disruption of murine genes, the introduction of transgenes, the molecular characterization of human hereditary diseases, and targeting of genes by antisense or ribozyme techniques. In addition, microinjection of specific antibodies into cultured cells or binding of antibodies to cell surface-exposed receptors may provide information on the function of the targeted protein. A new alternative to these reverse genetic approaches has now become available with the discovery of small interfering RNAs, which are able to trigger RNA interference in mammalian somatic cells (Caplen et al., 2001; Elbashir et al., 2001). RNAi is a sequence-specific posttranscriptional gene silencing mechanism, which is triggered by dsRNA and causes degradation of mRNAs homologous in sequence to the dsRNA (Fire et al., 1988; Montgomery et al., 1998). Our strategy involves the use of antisense RNA or dsRNAi, which have been used effectively in other protozoa, E. histolytica (Kaur and Lohia, 2004), Leishmania donovani (Zhang and Matlashewski, 2000), Trypanosoma brucei (Tschudi et al., 2003), Plasmodium falciparum (Malhotra et al., 2002), and other organisms.

In the present study, to observe the association of an Nfa1 protein with cytotoxicity of N. fowleri infection, we have applied antisense RNA or dsRNA interference strategy to the nfa1 gene, which is post-transcriptional gene silencing mechanism, to knockdown an nfa1 gene and the Nfa1 protein. When antisense RNA or dsRNA of the nfa1 gene was transfected into N. fowleri trophozoites, the nfa1 gene and the Nfa1 protein were efficiently knockdowned. However, dsRNA

transfection was more effective than antisense RNA transfection. In other words, the effect of dsRNA using RNAiFect transfection reagent was higher about 45% in an nfa1 gene and 29% in an Nfa1 protein than antisense RNA. It was presumed that exogenously transfected antisense RNA should be unstable. The fact that dsRNA induced to knockdown the nfa1 gene supported that RNAi mechanism should be in N.

fowleri trophozoites. Initially, dsRNA have to be excised by enzyme, e.g., RNAaseIII-like endonuclease to form siRNAs to specifically knockdown a gene. It was supported that endonuclease identically functioning with RNAaseIII-like endonuclease should be in N. fowleri trophozoites. There was no change in the level of an nfa2 mRNA and Nfa2 protein used to normalize and show specific function.

The nfa2 gene was cloned by immunoscreening with immune and infected sera identically used for the nfa1 gene in our previous study (Jeong et al., 2004). It was homologous to calcineurin B gene related with signal transduction. The reason why the nfa2 gene was used to normalize and show specific function was that any house-keeping gene like glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in mammalian cells has not yet been cloned in pathogenic N. fowleri trophozoites. The quantity of the nfa2 mRNA and Nfa2 protein has no changes, although dsRNA treatment has the possibility of nonspecific function, which supported that dsRNA and antisense RNA of the nfa1 gene was specifically functioned in N. fowleri trophozoites. Using siRNAs, although dsRNA of nfa1 gene ORF was processed to siRNAs, all siRNAs did not function identically. However, a randomly chosen sinfa1-1 of nfa1 gene was more effective with about 13.8 ~ 24.6% than sinfa1-2,

sinfa1-3, and sinfa1-4. The sinfa1-1 was corresponded to nucleotides 340–360 closed by ending codon of the nfa1 gene. There were no changes in the level of the nfa2 gene and Nfa2 protein in this siRNA transfection study. In vitro cytotoxicity experiment was performed with the nfa1 gene knockdowned-N. fowleri trophozoites against murine macrophages. When N. fowleri transfected with dsRNA or sinfa1-1 of the nfa1 gene was applied for the in vitro cytotoxicity, we could not observe the obscure inhibition of cytotoxicity of the amoeba. Thus, we applied a vector-based system of plasmid to transfect into N. fowleri trophozoites. The vector-based system supplies more stable system and marker select transfected N. fowleri. A pRNAT–

U6.1/Hygro vector was used, and it carries GFP and hygromycin selectable marker transcriptable by viral promoters. In particular, U6.1 is a promoter from human and can efficiently transcript short RNAs. When a pRNAT–U6.1/Hygro vector was transfected into N. fowleri, we could observe neither GFP fluorescence (data not shown) nor any fragment transcripted by a viral promoter using reverse transcription-PCR. Therefore, viral promoters and U6.1 promoter were replaced with 5’ UTR of an actin gene in nonpathogenic N. gruberi by cohesive ligation to make a pAct/SAGAH vector. When the vector was transfected into N. fowleri trophzoites, the nfa1 gene and Nfa1 protein efficiently were knockdowned. Moreover, the GFP gene and hygromycin resistance gene transcript were observed by reverse transcription-PCR.

Also, we observed the weak GFP fluorescence in the transfected N. fowleri. Even though it was done so, it was supported that 5’ UTR of an actin gene could be used as a promoter to transcript siRNA of sinfa1-1 in a pAct/SAGAH vector. In addition, a

pAct/asnfa1AGAH vector transcriptable to antisense RNA of the nfa1 gene was cloned to compare with a pAct/SAGAH vector. There was no GFP fluorescence in transfected N. fowleri until five days of transfection with a pAct/SAGAH or pAct/asnfa1AGAH vector. We did not observe any amplified fragment by reverse transcription-PCR using even 1 µg of cDNA. The result was different from general transfection data in mammalian cells or other protozoa. In our transfection system, GFP fluorescence, and GFP and hygromycin resistance gene transcript were detected since six days of the transfection. In particular, the nfa1 gene mRNA and Nfa1 protein were efficiently knockdowned in N. fowleri transfected with a pAct/SAGAH vector. In the case of a pAct/asnfa1AGAH vector, the knockdown effect was less than a pAct/SAGAH vector. The nfa1 gene mRNA and Nfa1 protein in N. fowleri transfected with the pAct/SAGAH vector were knockdowned with about 60% and 30%, respectively. On the other hand, by the pAct/asnfa1AGAH vector, the nfa1 gene mRNA and Nfa1 protein were knockdowned with about 30% and 18%, respectively. There were no changes in the nfa2 gene and Nfa2 protein. Following the observation of the nfa1 gene and Nfa1 protein knockdown, the transfected N.

fowleri trophozoites were selected with hygromycin antibiotics. After N. fowleri trophzoites were selected with hygromycin antibiotics two times, they were used to experiment in vitro cytotoxicity against macrophages, which have similar characteristics with murine microglial cells of target cells by the infection of N.

fowleri. In our previous study, in vitro cytotoxicity of other free-living amoeba, A.

culbertsoni, was performed with primary cultured rat microglial cells (Shin et al.,

2001). Recently, in vitro cytotoxicity of N. fowleri was performed with primary cultured rat microglial cells (unpublished). Because it was difficult in obtain sufficient numbers of primary cultured rat microglial cells, and CHO target cells often used for in vitro cytotoxicity did not have characteristics similar to microglial cells, immortalized murine macrophages were used in this study. Almost all (89% at 24 h) macrophages cocultured with N. fowleri at a ratio of 1 to 1 were destroyed, whereas N. fowleri transfected with a pAct/SAGAH and pAct/asnfa1AGAH vector destroyed 52.9% and 79.1% of macrophages at 24 h, respectively. However, the cytotoxicity of N. fowleri transfected with a pAct/AGAH vector as a control vector, was about 66.5% at 17 h and 85.7% at 24 h, which was little difference with the cytotoxicity of normal N. fowleri. Moreover, the in vitro cytotoxicity of N. fowleri with added hygromycin antibiotics showed no difference from that of normal N.

fowleri. Therefore, the decrease in the cytotoxicity of N. fowleri transfected with a pAct/AGAH or pAct/asnfa1AGAH vector was not the result of G418 selection. The lower cytotoxic effect of transgenic N. fowleri transfected with a pAct/AGAH or pAct/asnfa1AGAH vector suggests that Nfa1 protein contributes to in vitro cytotoxicity against macrophages.