Gene silencing of nfa1 affects the in vitro cytotoxicity of Naegleria fowleri in murine macrophages.
Jung, SY; Kim, JH; Song, KJ; Lee, YJ; Kwon, MH; Kim, K; Park, S; Im, KI; Shin, HJ
Molecular and biochemical parasitology, 165(1):87-93, 2009
Molecular and biochemical parasitology
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.
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.
Total Visit :2,390,659
Total Download :1,019,054
Today View :41
Ajou University Medical Information & Media Center 164 Worldcup-ro Yeongtong-gu Suwon 16499 Korea / TEL : 031-219-5312 / FAX : 031-219-5314 Copyright (c) Ajou University Medical Information & Media Center All Rights Reserved. AJOU Open Repository는 국립중앙도서관 OAK 보급사업으로 구축되었습니다.