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Infection and Immunity, July 2005, p. 4098-4105, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4098-4105.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Expression of the nfa1 Gene Cloned from Pathogenic Naegleria fowleri in Nonpathogenic N. gruberi Enhances Cytotoxicity against CHO Target Cells In Vitro

Seok-Ryoul Jeong,1 Sang-Chul Lee,1 Kyoung-Ju Song,1 Sun Park,1 Kyongmin Kim,1 Myung-Hee Kwon,1 Kyung-il Im,2 and Ho-Joon Shin1*

Department of Microbiology, Ajou University School of Medicine, Suwon 442-749,1 Department of Parasitology, Yonsei University College of Medicine, Seoul 121-752, Korea2

Received 6 August 2004/ Returned for modification 10 September 2004/ Accepted 25 February 2005

The pathogenic amoeba Naegleria fowleri has a 360-bp nfa1 gene that encodes the Nfa1 protein (13.1 kDa), which is located in the pseudopodia of the amoeba, and an anti-Nfa1 antibody reduces N. fowleri-induced mammalian-cell cytotoxicity in vitro. In contrast, an anti-Nfa1 antibody cannot detect Nfa1 protein expression in the nonpathogenic amoeba Naegleria gruberi, which also possesses the nfa1 gene. In the present study, the nfa1 gene cloned from pathogenic N. fowleri was transfected into nonpathogenic N. gruberi to determine whether it was related to pathogenicity. The nfa1 gene was initially inserted into a eukaryotic transfection vector, pEGFP-C2, containing a cytomegalovirus promoter and the green fluorescent protein (GFP) gene, and was designed as pEGFP-C2/nfa1UTR (nfa1UTR contains 5' upstream regions, the nfa1 open reading frame, and 3' downstream regions). After transfection, the green fluorescence was observed in the cytoplasm of N. gruberi trophozoites. These transfectants were preserved for more than 9 months after selection. The transfected nfa1 gene was observed by PCR using nfa1- and vector-specific primers in the genomic DNA of N. gruberi transfected with the pEGFP-C2/nfa1UTR vector. In addition, the nfa1 and GFP genes were identified by reverse transcription-PCR in transgenic N. gruberi. The Nfa1 protein expressed in transgenic N. gruberi was identified as a 13.1-kDa band by Western blotting using an anti-Nfa1 antibody. Finally, N. gruberi transfected with the pEGFP-C2/nfa1UTR vector was found to have enhanced cytotoxicity against CHO cells compared with naïve N. gruberi.

* Corresponding author. Mailing address: Department of Microbiology, Ajou University School of Medicine, Suwon 442-749, Korea. Phone: 82 31 219 5076. Fax: 82 31 219 5079. E-mail: hjshin{at}ajou.ac.kr.

Editor: J. F. Urban, Jr.

Infection and Immunity, July 2005, p. 4098-4105, Vol. 73, No. 7
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.7.4098-4105.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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