Cloning of nf-profilin and intercellular interaction with nf-actin in Naegleria fowleri cysts

Abstract

Naegleria fowleri is a free-living amoeba found in lakes, soil, hot springs, and poorly chlorinated swimming pools. It is pathogenic to humans, causing a rare and fatal brain infection known as primary amoebic meningoencephalitis (PAM). A previous study utilized RNA-seq analysis to examine genes expressed in N. fowleri cysts and trophozoites, focusing on the nf-profilin gene, which showed high expression in cysts. Profilin is a small actin-binding protein that regulates nf-actin polymerization and cell movement. Sequence analysis revealed 83% similarity with non-pathogenic N. gruberi and 38% similarity with Acanthamoeba castellanii. Nf-profilin was found to be associated with N. fowleri lysates but not with lysates from other amoebae, as shown by Western blot analysis. Immunofluorescence assays demonstrated that nf-profilin primarily localized to the cell membrane in N. fowleri cysts, while nf-actin localized to the cytoplasm, pseudopodia, and food-cup structures. Real-time RT-PCR indicated higher expression of the nf-profilin gene in cysts compared to trophozoites. In co-culture experiments with target cells, Nf-profilin was initially expressed in the cytoplasm of N. fowleri cysts and the morphology of cyst gradually transitioned to the trophozoite form. Concurrently, the expression of Nf-profilin protein decreased, while Nf-actin protein began to appear in the pseudopodia and food-cups of trophozoites. In conclusion, the nf-profilin and nf-actin genes exhibited complementary expression patterns based on the life stage of N. fowleri, indicating their critical roles in the survival and proliferation. This study emphasizes the significance of actin-binding proteins in understanding the infection and pathogenic mechanisms of N. fowleri.

Keywords: Naegleri fowleri; Actin-binding protein; Pathogenicity mechanisms; Profilin.

Fig. 1

Live image of N. folweri (A) trohpozoites and (B) encystic forms (× 200).

Fig. 2

Nf-profilin and nf-actin gene expression level of N. foweleri trophozoites and cysts. T, N. fowleri trophozoites; C, N. fowleri cysts; nfa1, pseudopodia-specific gene of N. fowleri; Nae3 and Nf-GAPDH were used for the control genes. M; DNA size marker.

Fig. 3

(A) Construction of expression vector. The pET-30a vector containing His-tag. Cloning of nf-profilin gene into the pET-30a vector. (B) Amplified PCR product of nf-profilin gene of pET-30a/nf-profilin. M, DNA size marker; lane A, amplified PCR product of nf-profilin gene of pET-30a/nf-profilin.

Fig. 4

(A) Band patterns of SDS-PAGE and Western blot of the IPTG-induced recombinant protein (Nf-profilin). M, molecular weight marker; UN, uninduced E. coli lysate, IN, IPTG induced E. coli lysate; PU, purified recombinant Nf-profilin protein using Ni-NTA resin; N, normal mouse sera (1:1000); Nf-P, anti-Nf-profilin antibody (1:1000). (B) Cross reactivity of anti-Nf-profilin antibody. lane 1, A. castellanii; lane 2, A. polyphaga; lane 3, N. fowleri.

Fig. 5

Cellular expression of the Nf-profilin in trophozoites and cysts by immunofluorescence assay. The light microscopic findings were shown in the inner box (× 400). Upper panels, N. fowleri trophozoites; Lower panels, N. fowleri cysts. Scale bar, 10 μm.

Fig. 6

Localization of (A) Nf-profilin and (B) Nf-actin in trophozoites and cysts by confocal microscope. Green fluorescence (FITC) indicates Nf-profilin or Nf-actin, and blue fluorescence (DAPI) indicates cell nuclei. The bright fields were shown in the inner box (× 800). Upper panels, N. fowleri trophozoites; Lower panels, N. fowleri cysts. Scale bar, 10 μm.

Fig. 7

Microscopic observation of N. fowleri co-cultured with CHO cells. Control G I, CHO cells in DMEM; Control G II, CHO cells in DMEM mixed with Nelson’s medium; Exp G I, CHO cells with N. fowleri trophozoites; Exp G II, CHO cells with N. fowleri cysts (× 400). Arrow, N. fowleri, Scale bar, 20 μm.

Fig. 8

Nf-profilin gene expression levels in N. fowleri co-cultured with CHO cells using RT-PCR. lane 1, CHO cells in DMEM (Control G I); lane 2, CHO cells in mixture medium with DMEM and Nelson’s medium (Control G II); lane 3, CHO cells with N. fowleri trophozoites (Exp G I); lane 4, CHO cells with N. fowleri cysts (Exp G II). The nf-actin and nfa1 genes were used for the trophozoite-dominant gene, CHO GAPDH, Nf-GAPDH and Nae3 genes were used for the controls. M, DNA size marker.

Fig. 9

Nf-profilin gene expression level of N. fowleri co-cultured with CHO cells using quantitative RT-PCR analysis. Control G I, CHO cells in DMEM; Control G II, CHO cells in DMEM mixed with Nelson’s medium; Exp G I, CHO cells with N. fowleri trophozoites; Exp G II, CHO cells with N. fowleri cysts.

Fig. 10

Localization of Nf-profilin protein in N. fowleri cysts co-cultured with CHO cells by immunofluorescence assay (× 400). Arrow, N. fowleri cysts and trophozoites; Arrow head, CHO cells. Scale bar, 20 μm.

Fig. 11

Localization of Nf-profilin protein in N. fowleri trophozoites co-cultured with CHO cells by immunofluorescence assay (× 400). Arrows, N. fowleri cysts and trophozoites; Arrow head, CHO cells. Scale bar, 20 μm.

Fig. 12

Localization of Nf-actin protein in N. fowleri cysts co-cultured with CHO cells by immunofluorescence assay (× 400). Arrows, N. fowleri cysts and trophozoites; Arrow head, CHO cells. Scale bar, 20 μm.

Fig. 13

Localization of Nf-actin protein in N. fowleri trophozoites co-cultured with CHO cells by immunofluorescence assay (× 400). Arrows, N. fowleri cysts and trophozoites; Arrow head, CHO cells. Scale bar, 20 μm.