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.

FIG. 1.

(A) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis band patterns of a recombinant fusion protein (lane 1) and an enterokinase-digested Nfa1 protein (lane 2) expressed from the nfa1 gene. (B) Results of Western blotting with an anti-Nfa1 polyclonal antibody (lane An) and normal mouse serum (lane N). Arrow, uncut 17-kDa fusion protein; arrowhead, cut 13.1-kDa Nfa1 protein; lane M, molecular size markers.

FIG. 2.

Antibody titers of anti-Nfa1 polyclonal sera (1:200 dilution) obtained from Nfa1-immunized mice. The recombinant Nfa1 protein (5 μg/ml) was used as the antigen. A₄₀₅ values for each serum sample (n = 5) were determined by an ELISA reader. OD, optical density; P, PBS control; S, anti-Nfa1 polyclonal sera; N, normal mouse sera (1:200 dilution).

FIG. 3.

Immunoreactivity of the Nfa1 protein with a peroxidase-labeled anti-Nfa1 antibody. Trophozoites of N. fowleri in cultivation were treated with an anti-Nfa1 antibody (A) and normal mouse serum (B). Trophozoites of N. fowleri were stained brown. Trophozoites of N. gruberi (C) did not react with an anti-Nfa1 antibody and did not stain brown. Magnification, ×200.

FIG. 4.

Mouse brain tissue infected with N. fowleri trophozoites was treated with an anti-Nfa1 antibody (A and C) and unimmunized control serum (B and D). Trophozoites (arrows) were stained brown. (A and B) The inflammatory region of the brain tissue; (C and D) the necrotic region of the brain tissue. Magnification, ×200.

FIG. 5.

Cellular localization of the Nfa1 protein in an N. fowleri trophozoite. N. fowleri trophozoites were immunostained with normal serum (A) and anti-Nfa1 antibody (B) by using peroxidase and diaminobenzidine. Positive findings (arrowheads) were observed on pseudopodia (P) and around food vacuoles (F) of a trophozoite. N, nucleus. Bar, 2.5 μm.

FIG. 6.

Microscopy findings for CHO cells (arrows) grown in cultures in EMEM (A) and grown in cocultures with N. fowleri trophozoites (arrowheads) (B) and with N. fowleri and an anti-Nfa1 polyclonal antibody (C) at 48 h. Magnification, ×200.

FIG. 7.

Visualization (using an LDH release assay) of the cytotoxicity of N. fowleri trophozoites against CHO cells. CHO cells were grown in cultures in EMEM medium alone (lane 1, rows A, B, and C), in cocultures with N. fowleri trophozoites (lane 1, rows D, E, and F), and in cocultures with N. fowleri trophozoites and an anti-Nfa1 antibody (lane 2, rows D, E, and F). Other wells represent control groups.