Suk-Yul Jung

Molecular Cloning and Characterization of a Gene Encoding a 13.1 kDa Antigenic Protein of Naegleria fowleri

Abstract An antigen-related gene was cloned from a cDNA expression library of Naegleria fowleri by immunoscreening with sera obtained from mice that were either immunized with an amoebic lysate or infected with trophozoites. The coding nucleotide sequence of the cloned gene consisted of 357 bases that were translated into 119 amino acids. This gene was designated as nfa1. The predicted amino acid sequence of Nfa1 protein has two potential glycosylation and three potential phosphorylation sites, and its predicted secondary structure consists of four helices and three corners. The deduced amino acid sequence of Nfa1 protein shares 43% identity with the myohemerythrin (myoHr) protein from a marine annelid, Nereis diversicolor, including 100% identity in conserved regions and iron-binding residues. A phylogenetic tree constructed from amino acid sequences placed the N. fowleri Nfa1 protein outside of a cluster of myoHr proteins from eight invertebrates. A purified recombinant protein that migrated as a 13.1 kDa species in SDS-PAGE was produced. This recombinant protein exhibited a strong immunoreactivity with infected, immune, and anti-Nfa1 sera. In addition, an anti-Nfa1 serum reacted with an amoeba lysate in immunoblotting analysis. The present nfa1 gene encoding the myoHr-like protein is the first myoHr gene cloned from protozoa, and the Nfa1 antigen may be useful in diagnostic studies

Effect of Therapeutic Chemical Agents In Vitro and on Experimental Meningoencephalitis Due to Naegleria fowleri

ABSTRACT Naegleria fowleri is a ubiquitous, pathogenic free-living amoeba; it is the most virulent Naegleria species and causes primary amoebic meningoencephalitis (PAME) in laboratory animals and humans. Although amphotericin B is currently the only agent available for the treatment of PAME, it is a very toxic antibiotic and may cause many adverse effects on other organs. In order to find other potentially therapeutic agents for N. fowleri infection, the present study was undertaken to evaluate the in vitro and in vivo efficacies of miltefosine and chlorpromazine against pathogenic N. fowleri. The result showed that the growth of the amoeba was effectively inhibited by treatment with amphotericin B, miltefosine, and chlorpromazine. When N. fowleri trophozoites were treated with amphotericin B, miltefosine, and chlorpromazine, the MICs of the drug were 0.78, 25, and 12.5 μg/ml, respectively, on day 2. In experimental meningoencephalitis of mice that is caused by N. fowleri, the survival rates of mice treated with amphotericin B, miltefosine, and chlorpromazine were 40, 55, and 75%, respectively, during 1 month. The average mean time to death for the amphotericin B, miltefosine, and chlorpromazine treatments was 17.9 days. In this study, the effect of drugs was found to be optimal when 20 mg/kg was administered three times on days 3, 7, and 11. Finally, chlorpromazine had the best therapeutic activity against N. fowleri in vitro and in vivo. Therefore, it may be a more useful therapeutic agent for the treatment of PAME than amphotericin B.

Immunological Characterizations of a Cloned 13.1-Kilodalton Protein from Pathogenic Naegleria fowleri

ABSTRACT We previously cloned an antigenic gene (named nfa1) from a cDNA library of Naegleria fowleri by immunoscreening. The nfa1 gene had a coding nucleotide sequence consisting of 357 bases and produced a recombinant 13.1-kDa protein (Nfa1). In this study, to get more information regarding the recombinant Nfa1 protein (rNfa1), we produced an anti-Nfa1 polyclonal antibody from mice immunized with rNfa1 and used a peroxidase staining method to carry out immunocytochemistry experiments. In addition, we observed the effect of the presence of an anti-Nfa1 antibody on the in vitro cytotoxicity of N. fowleri against Chinese hamster ovary (CHO) cells. Trophozoites of N. fowleri in cultivation reacted strongly with a peroxidase-labeled anti-Nfa1 antibody. In inflammatory and necrotic regions of brain tissue infected with N. fowleri, labeled trophozoites that were stained brown were also observed. When examined using a transmission electron microscope, the Nfa1 protein showed pseudopodium-specific immunolocalization on a trophozoite of N. fowleri. When examined using a light microscope, CHO cells grown in cocultures with N. fowleri trophozoites (group I) for 48 h showed morphologically severe destruction but CHO cells grown in cocultures with N. fowleri trophozoites and an anti-Nfa1 polyclonal antibody (group II) showed less destruction. The results of a lactate dehydrogenase release assay showed that group I CHO cells exhibited 81% cytotoxicity and group II CHO cells exhibited 13.8% cytotoxicity.

Cytopathic Changes in Rat Microglial Cells Induced by Pathogenic Acanthamoeba culbertsoni: Morphology and Cytokine Release

ABSTRACT To determine whether pathogenic Acanthamoeba culbertsoni trophozoites and lysate can induce cytopathic changes in primary-culture microglial cells, morphological changes were observed by transmission electron microscopy (TEM). In addition, the secretion of two kinds of cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β), from microglial cells was observed. Trophozoites of pathogenic A. culbertsoni made contact with microglial cells and produced digipodia. TEM revealed that microglial cells cocultured with amoebic trophozoites underwent a necrotic process, accompanied by lysis of the cell membrane. TEM of microglial cells cocultured with amoebic lysate showed that the membranes of the small cytoplasmic vacuoles as well as the cell membrane were lysed. The amounts of TNF-α secreted from microglial cells cocultured with A. culbertsoni trophozoites or lysate increased at 6 h of incubation. The amounts of IL-1β secreted from microglial cells cocultured with A. culbertsonitrophozoites at 6 h of incubation was similar to those secreted from the control group, but the amounts decreased during cultivation with A. culbertsoni lysate. These results suggest that pathogenic A. culbertsoni induces the cytopathic effects in primary-culture rat microglial cells, with the effects characterized by necrosis of microglial cells and changes in levels of secretion of TNF-α and IL-1β from microglial cells.

Gene silencing of nfa1 affects the in vitro cytotoxicity of Naegleria fowleri in murine macrophages.

The gene nfa1 was isolated from the free-living pathogenic amoeba Naegleria fowleri. The protein Nfa1 is located in pseudopodia and specifically in food-cups. It is also involved in cytotoxicity. In this study, we used synthetic small interfering RNAs (siRNA) to examine the effects of nfa1 down-regulation. We observed the expression of nfa1 mRNA and Nfa1 protein using Northern and Western blots. We also examined the effects of nfa1 down-regulation on the in vitro cytotoxicity of N. fowleri. Four synthetic siRNAs were constructed, and of those, sinfa1-1 showed the highest down-regulation of an nfa1 mRNA and Nfa1 protein by 70 and 43%, respectively. In order to achieve long-lasting silencing of the transfected genes, we constructed two vectors which were pAct/SAGAH and pAct/asnfa1AGAH cloned with the sinfa1-1 and an antisense RNA to the nfa1 gene. In N. fowleri transfected with pAct/SAGAH, FACS revealed a 60 and 57% reduction in nfa1 mRNA and Nfa1 protein levels, respectively. To determine whether the Nfa1 proteins were related with in vitro cytotoxicity, LDH assays were used and showed that the cytotoxicity of these transfectants to macrophages was reduced by 26.4 and 36.2% at 17 and 24h, respectively. Moreover, after transfection with pAct/asnfa1AGAH, amoebic cytotoxicity decreased by 8.2 and 10% at 17 and at 24h, respectively. This is the first report to show the RNA interference in N. folweri trophozoites and also demonstrate the Nfa1 function in vitro for its cytotoxicity.