Immunological characterizations of a cloned 13.1-kilodalton protein from pathogenic Naegleria fowleri.
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.
Cho
Myoung-Soo
MS
Department of Microbiology, School of Medicine, Ajou University, Suwon 442-749, Korea.
Jung
Suk-Yul
SY
Park
Sun
S
Kim
Kyongmin Hwang
KH
Kim
Hyung-Il
HI
Sohn
Seonghyang
S
Kim
Han-Jip
HJ
Im
Kyung-Il
KI
Shin
Ho-Joon
HJ
D000562:Amebiasis / Q000276:immunology; D000818:Animals; D000913:Antibodies, Protozoan / Q000276:immunology*; D000953:Antigens, Protozoan / Q000235:genetics / Q000276:immunology*; D015153:Blotting, Western; D001921:Brain / Q000382:microbiology; D016466:CHO Cells; D003001:Cloning, Molecular; D018920:Coculture Techniques; D006224:Cricetinae; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D007150:Immunohistochemistry; D051379:Mice; D008854:Microscopy, Electron; D016848:Naegleria fowleri / Q000276:immunology* / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology
D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; C460560:Nfa1 protein, Naegleria fowleri; D015800:Protozoan Proteins; D011994:Recombinant Proteins