RNAi means post-transcriptional gene silencing and has been induced in most organisms by microinjection. In this present study, a lipid formulation was used for the transfection. Because there have not been reports showing the effect of transfection reagents for RNAi in free-living amoebae, TransMessenger or RNAiFect reagent was used for the transfection of single-stranded and dsRNA. As a northern blot analysis, it was observed that an nfa1 mRNA was successfully knockdowned in N. fowleri trophozoites transfected with dsnfa1 using RNAiFect reagent (Fig. 5A, 5B). Using TransMessenger reagent, no changes were observed in the level of the nfa1 gene mRNA. In the principle, TransMessenger reagent is recommended in the transfection of single-stranded RNA. Hybridizations of northern blotting with a probe specific for the nfa1 gene revealed that in the transfection of asnfa1 using TransMessenger reagent, no changes were observed in the level of the nfa1 gene mRNA and also, in ssnfa1 and only RNAiFect reagent, the level of the nfa1 gene mRNA was almost identical with it of untransfected N. fowleri. Therefore, it was suggested that dsnfa1 should be very effective in knockdowning the nfa1 gene mRNA. An nfa2 gene which used to normalize and show that RNAi was specific for
the nfa1 gene was not changed in the mRNA patterns (Fig. 5A). Therefore, it was suggested that, although long dsRNA but not siRNA had the problem of non-specificity, dsnfa1 could be specifically acted in N. fowleri. In Fig. 5B, it was shown that there were not any problems in nfa1 gene-specific probe due to the stringent experimental conditions. To more understand the level of knockdowned nfa1 gene mRNA, a quantitative analysis was performed (Fig. 5C). The transfection of dsnfa1 using RNAiFect reagent had the highest effect about 60% on knockdowning the mRNA gene. There were not the differences of dsnfa1 transfected using TransMessenger reagent with asnfa1 transfected using RNAiFect reagent. When asnfa1 was transfected using TransMessenger reagent, no change was also observed in the nfa1 gene mRNA (data not shown).
As a western blot analysis, the Nfa1 protein was not decreased highly as the nfa1 gene mRNA (Fig. 6). It was observed that the Nfa1 protein was higher knockdowned in N. fowleri trophozoites transfected with dsnfa1 than asRNA using RNAiFect or dsnfa1 using TransMessenger reagent (Fig. 6A). Similar amounts of the Nfa1 protein were detected in the controls and the patterns of the Nfa2 protein of 21 kDa were not changed (Fig. 6A). Quantitative estimation of the level of the Nfa1 protein showed higher decreasing effect about 30% in the N. fowleri transfected with dsnfa1 using RNAiFect reagent (Fig. 6B). On contrast, there were few changes in the dsnfa1 using TransMessenger or asnfa1 using RNAiFect reagent. These results indicate that dsnfa1 using RNAiFect reagent is the most efficient for knockdowning the nfa1 and Nfa1 protein
Fig. 5. Northern blotting and quantitative analysis of the nfa1 gene mRNA from N. fowleri trophozoites transfected with an asnfa1 or dsnfa1. (A) Northern
blotting of the nfa1 or nfa2 gene mRNA with a gene-specific probe. 25 µg of the mRNA samples were loaded. (B) Northern blotting of the nfa1 gene mRNA used as a loading control. C. Quantitative analysis of fig. 5A. F, RNAiFect reagent; T,
Fig. 6. Western blotting and quantitative analysis of the Nfa1 protein from N.
fowleri trophozoites transfected with an asnfa1 or dsnfa1. (A) Western blotting of
the Nfa1 or Nfa2 protein detected with a respective polyclonal antibody. 10 µg of the N. fowleri lysate was loaded. (B) Quantitative analysis of fig. 5A. F, RNAiFect
C. Knockdown of an nfa1 gene mRNA and Nfa1 protein by synthetic siRNAs
The transfection of siRNA into N. fowleri trophozoites was performed with RNAiFect reagent. It is not easy to find the most functional siRNA of any genes.
Although a lot of siRNAs are chosen, they don’t act to knockdown equally. In other words, in the present study, four siRNAs were randomly chosen as considerations of siRNA choices. Because siRNA has very little nucleotide and it is difficult to observe whether the transfection is done or not, we identified the transfection with the fluorescence of GFP-conjugated siRNA of Lamin A/C gene from human, irrespective of the nfa1 gene (Fig. 7). GFP fluorescence was observed 24 h after the transfection into N. fowleri trophozoites with the GFP-conjugated siRNA of Lamin A/C as compared with untransfected N. fowleri. GFP fluorescence was observed in the cytoplasm of transfected N. fowleri. This result showed that siRNA could be transfected using RNAiFect reagent. The fact of the decrease of the nfa1 gene and Nfa1 protein by a dsnfa1 transfection using RNAiFect reagent supported that the RNAi mechanism associated with siRNA not only exist in N. fowleri trophozoites but also siRNA act to knockdown the nfa1 gene. Thus, we transfected each siRNA (sinfa1-1, sinfa1-2, sinfa1-3 and sinfa1-4) using a RNAiFect reagent into N. fowleri trophozoites. All siRNAs were composed of each 21 nucleotides. The expression of the nfa1 gene mRNA in the siRNA transfectants was assessed by northern blot analysis with a specific probe for the nfa1 gene ORF (Fig. 8).
Fig. 7. GFP fluorescence in N. fowleri transfected with GFP-conjugated with siRNA of Lamin A/C gene. (A) and (C) under a light microscopy, and (B) and (D) under a fluorescence microscopy were observed 24 h after the transfection. (D) shows N. fowleri transfected with GFP-conjugated with siRNA of Lamin A/C compared with untransfected N. fowleri of fig. 7D. × 400.
B
D A
C
Although they didn’t show an identical effect, all siRNAs had an effect on knockdowning nfa1 gene mRNA. In particular, the sinfa1-1 showed the highest knockdown of the nfa1 gene mRNA by northern blotting (Fig. 8A). According to the quantitative analysis, even the sinfa1-2 or sinfa1-4 showed the effect from 55 ~ 57%
and the sinfa1-1 did the effect about 70% (Fig. 8B). No change of the nfa2 gene mRNA, which is not homologous with the nfa1 gene, was occurred by the transfection of siRNAs of the nfa1 gene (Fig. 8A). Western blots with anti-Nfa1 polyclonal antibody showed the highest decreasing effect of the Nfa1 protein levels by the sinfa1-1, in agreement with the northern blot data (Fig. 9). The Nfa1 protein transfected with each siRNA was not knockdowned as the nfa1 gene mRNA (Fig.
9A). According to the quantitative analysis of fig. 9A, the knockdowned Nfa1 protein was higher about 20% than the nfa1 protein (Fig. 9B). Even though it was done so, the sinfa1-1 showed the highest decreasing effect with about 43%. In the sinfa1-4, the nfa1 gene mRNA was knockdowned with about 45% but the Nfa1 protein with 7%, which was very different from the effect of other three siRNAs. The level of the Nfa2 protein was not affected by the transfection of siRNAs of the nfa1 gene (Fig.
9A). These results supported the functional analysis in vitro. However, when we observed the in vitro cytotoxicity of N. fowleri trophozoites knockdowned by the dsnfa1 or sinfa1-1 against murine macrophages, we didn’t observe the significant inhibition of the cytotoxicity of N. fowleri by the morphological analysis of murine macrophages and LDH release assay (data not shown).
Fig. 8. Northern blotting and quantitative analysis of the nfa1 gene mRNA from N. fowleri trophozoites transfected with the siRNAs of an nfa1 gene. (A)
Northern blotting of the nfa1 or nfa2 gene mRNA with a gene-specific probe. 25 µg of the mRNA samples were loaded. (B) Quantitative analysis of fig. 5A. N. fowleri
Fig. 9. Western blotting and quantitative analysis of the Nfa1 protein from N.
fowleri trophozoites transfected with the siRNAs of an nfa1 gene. (A) Western
blotting of the Nfa1 or Nfa2 protein detected with a respective polyclonal antibody.
10 µg of the N. fowleri lysate was loaded. (B) Quantitative analysis of fig. 5A. N.
fowleri trophzoites were transfected using RNAiFect reagent.
sinfa1-1
This result supported the use of a long-lasting system such as a vector. Therefore, we used the vector-based system with sinfa1-1 showing the highest decreasing effect of the nfa1 gene mRNA and Nfa1 protein.
D. RNAi function by a vector-based system
DNA-vector based technology has a lot of advantages comparing to chemically synthesized siRNA. It is more effective than synthetic siRNA for inhibition of gene expression and very stable and easy to handle (Yu et al., 2002). It also allows researcher to obtain a stable cell line, and observe long-term effects of RNAi. The vector with selectable markers and active promoters is required to transfect. A pRNAT−U6.1/Hygro vector was transfected into N. fowleri trophozoites using SuperFect reagent, but the GFP fluorescence (data not shown) as well as GFP transcript by reverse transcription-PCR were not observed. Also, when the nfa1 gene mRNA level was examined by northern blotting, it was not knockdowned in N.
fowleri trophzoites transfected with the pRNAT−U6.1/Hygro vector (data not shown).
It meant that U6 promoter in the vector did not act to transcribe a sinfa1-1. Therefore, all viral promoters and U6 promoter in the pRNAT−U6.1/Hygro vector were replaced with 5’ UTR of actin gene from nonpathogenic N. gruberi, and a sinfa1-1 showed the highest decreasing effect or asnfa1 was cloned to create a pAct/SAGAH or pAct/ asnfa1AGAH vector (Fig. 10). The size of the pAct/SAGAH and pAct/
asnfa1AGAH vector was 7040 and 7400 bp, respectively. Act means 5’ UTR of actin
Fig. 10. Vector construction for RNAi in N. fowleri. The pAct/SAGAH and pAct/asnfa1AGAH vector were derived from the pRNAT–U6.1/Hygro vector. All promoters in the pRNAT–U6.1/Hygro vector were replaced with 5’ UTR of actin gene. S, sinfa1-1; A, 5’ UTR of actin gene; G, GFP; H, hygromycin resistance gene.
pRNAT–U6.1/Hygro
gene. It has not been used in pathogenic N. fowleri yet. When 5’ UTR cloned into the pAct/SAGAH vector was applied to this RNAi study, it efficiently transcribed the GFP and hygromycin resistance gene, which were observed by reverse transcription-PCR (Fig. 11). On the other hand, GFP gene was not transcribed in N. fowleri transfected with a pAct/SAGAH vector using a lipid formulated-Lipofectamine 2000 (Invitrogen) compared (Fig. 11A). In other pAct/AGAH, pAct/SAGAH, and pAct/
asnfa1AGAH vector, the hygromycin resistance gene was transcribed by reverse transcription-PCR (Fig. 11B). However, no fragments were amplified in controls of untransfected and mock-transfected N. fowleri. These result showed the possibility of 5’ UTR of actin gene as a promoter, and that SuperFect reagent could be used to transfect a mammalian vector into N. fowleri trophozoites.
The knockdown of an nfa1 gene and Nfa1 protein was observed by northern and western blotting. When the pAct/SAGAH vector with sinfa1-1 was transfected into N.
fowleri, the nfa1 gene mRNA was significantly knockdowned as compared with pAct/AGAH vector without sinfa1-1 by northern blot analysis (Fig. 12A).
Surprisingly, in the transfectants of the pAct/asnfa1AGAH vector with asnfa1, the nfa1 gene mRNA was a little knockdowned. In the study of asnfa1 which was not cloned into a vector, when asnfa1 was transfected into N. fowleri trophozoites, no changes were occurred in the level of the nfa1 gene mRNA and Nfa1 protein. It was suggested that the vector-based system cloned with asnfa1 should be more stable to knockdown the nfa1 gene mRNA than asnfa1 alone. This suggestion was supported by quantitative analysis (Fig. 12B). About 30% of the nfa1 gene mRNA was
Fig. 11. Feasibility of transfection reagents for transfection into N. fowleri and gene transcription by reverse transcription-PCR. (A) Reverse transcription-PCR data of GFP gene from N. fowleri transfected with each vector using Lipofectamine 2000 or SuperFect reagent. pDNA was used as a positive control of plasmid DNA from E. coli. (B) Reverse transcription-PCR data of hygromycin resistance gene from N. fowleri transfected with a vector with 5’ UTR of actin gene using SuperFect reagent. pDNA was used as a positive control of plasmid DNA from E. coli.
M
Fig. 12. Northern blotting and quantitative analysis of the nfa1 gene mRNA
knockdowned in N. fowleri transfected with the pAct/asnfa1AGAH vector. In the transfection of the pAct/SAGAH vector, the nfa1 gene mRNA was higher knockdowned with about 60% than in the pAct/asnfa1AGAH vector. By western blot analysis, identical patterns of the Nfa1 protein were shown (Fig. 13). The Nfa1 protein from N. fowleri transfected with a pAct/SAGAH vector was knockdowned with about 28% compared with N. fowleri transfected with a pAct/asnfa1AGAH vector with about 17% (Fig. 13B). However, the level of the Nfa1 protein was more increased about 29% in the pAct/SAGAH vector and 12% in the pAct/asnfa1AGAH vector than that of the nfa1 gene mRNA. This increased result was similar with the effect of dsnfa1 and asnfa1 without cloning into a vector. There were no changes in the level of the Nfa1 protein from the controls of a pAct/AGAH vector and SuperFect alone and Nfa2 protein used to normalize or prove Nfa1 protein-specific (Fig. 13A). The results supported that the RNAi vectors should be transfected into N.
fowleri trophzoites using SuperFect reagent and used to knockdown the specific genes.
E. Expression of an Nfa1 protein in N. fowleri transfected with a RNAi vector
Immunocytochemistry was used to observe the knockdown of a Nfa1 protein in N. fowleri transfected with a pAct/SAGAH or pAct/asnfa1AGAH vector.
Fig. 13. Western blotting and quantitative analysis of the Nfa1 protein from N.
fowleri trophozoites transfected with the RNAi vector. (A) Western blotting of the
Nfa1 or Nfa2 protein detected with a respective polyclonal antibody. 10 µg of the N.
fowleri lysate was loaded. (B) Quantitative analysis of fig. 5A. N. fowleri trophzoites were transfected using SuperFect reagent.
Prior to IFA test, the GFP expression of N. fowleri transfected with a RNAi vector mentioned above was observed under immunofluorescence microscope (data not shown). It was observed very weakly in N. fowleri transfected with the vector. When the transfection efficiency was analyzed with fluorescence activated cell sorting (FACS), 0.10 ~ 0.24% N. fowleri showing weak or strong GFP fluorescence was observed (data not shown). Although the transfection efficiency was very low, fluorescent N. fowleri trophozoites were sorted using FACS followed by immunocytochemistry. After N. fowleri trophozoites were fixed by 10% formalin and permeabilized, they were treated with the anti-Nfa1 antibody, followed by rhodamine-conjugated anti-IgG antibody. The fluorescence of red colored-rhodamine of the Nfa1 protein was observed under a fluorescence microscopy (Fig. 14). N.
fowleri trophozoites transfected with the pAct/SAGAH vector showed the weakest fluorescence among other samples. The fluorescence of N. fowleri transfected with the pAct/asnfa1AGAH vector was a little stronger than that transfected with pAct/SAGAH vector. Other samples as controls showed the strongest fluorescence.
These results were similar patterns with northern or western blotting by RNAi vectors.
Fig. 14. The fluorescence of the Nfa1 protein by immunocytochemistry. N.
fowleri trophozoites were treated with the anti-Nfa1 polyclonal antibody, followed by rhodamine-conjugated anti-IgG. (A) Untransfected N. fowleri. (B) N. fowleri transfected with a pAct/AGAH vector. (C) N. fowleri transfected with a pAct/SAGAH vector. (D) N. fowleri transfected with a pAct/asnfa1AGAH vector.
(E) mock-transfected N. fowleri. × 400.
A B C
D E
None pAct/AGAH pAct/SAGAH
pAct/asnfa1AGAH SuperFect
F. In vitro cytotoxicity of N. fowleri transfected with a RNAi vector
To test whether an nfa1 gene-knockdowned N. fowleri trophzoites could destroy murine macrophages, the in vitro cytotoxicity was performed by LDH release assay, where a red color represents the extent of in vitro cytotoxicity. LDH is released from destroyed mammalian cells. All transfected or untransfected N. fowleri trophozoites were maintained with the Nelson medium containing hygromycin (100 µg /ml). As experimental time increased, murine macrophages were severely damaged by untransfected N. fowleri, but murine macrophages cocultured with N. fowleri transfected with a pAct/SAGAH or pAct/asnfa1AGAH vector were less damaged for 17 h and 24 h than murine macrophages cocultured with N. fowleri transfected with a control, pAct/AGAH vector (Table 1). The calculated cytotoxicity of untransfected N.
fowleri was highest, with a mean of 67% for 17 h and 89% for 24 h. The cytotoxicity of N. fowleri transfected with the pAct/SAGAH vector was about 40% for 17 h and 52% for 24 h, lower than that of untransfected N. fowleri (t test; P < 0.01). The inhibition of the cytotoxicity of N. fowleri transfected with the pAct/AGAH vector was not occurred. The in vitro cytotoxicity of N. fowleri with added hygromycin antibiotics showed no differences from that of normal N. gruberi. Therefore, an increase in the cytotoxicity of N. fowleri transfected with the pAct/SAGAH vector was not the result of hygromycin antibiotics selection. These results show that N.
fowleri trophozoites knockdowned with the nfa1 gene be inhibited in the cytotoxicity against murine macrophages.
Table 1. In vitro cytotoxicity of N. fowleri against murine macrophages. Macrophage + N. fowleri transfected with a pAct/SAGAH vector
---79.1 ± 1.1 Macrophage + N. fowleri transfected with a pAct/asnfa1AGAH vector
----86.4 ± 0.2 Macrophage + N. fowleri transfected with only SuperFect reagent
---85.1 ± 2.6 Macrophage + N. fowleri transfected with a pAct/AGAH vector
---85.7 ± 3.1 Macrophage + N. fowleri + Hygromycin antibioticsc
---Cytotoxicity (%) Macrophage + N. fowleri transfected with a pAct/SAGAH vector
---79.1 ± 1.1 Macrophage + N. fowleri transfected with a pAct/asnfa1AGAH vector
----86.4 ± 0.2 Macrophage + N. fowleri transfected with only SuperFect reagent
---85.1 ± 2.6 Macrophage + N. fowleri transfected with a pAct/AGAH vector
---85.7 ± 3.1 Macrophage + N. fowleri + Hygromycin antibioticsc
---Cytotoxicity (%)
a5 × 104cells.
b5 × 104trophozoites.
c100 μg/ml of hygromycin antibiotics.
Group
IV. 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
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