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.