Abstract
Free-living amoeba, Naegleria fowleri, destroys target cells through contact-dependent mechanisms, such as phagocytosis and/or trogocytosis. A previous experiment showed that the nf-actin gene consisted of 1.2 kbp, produced a 50.1kDa recombinant protein (Nf-actin), and was localized on the cytoskeleton, pseudopodia and amoebastome. In this study, cellular characterization of the nf-actin gene concerned with contact-dependent mechanisms in Nfowleri was performed. The nf-actin gene was amplified from a gene-cloned vector, pEXQP5-T7/NT TOPO. The nf-actin gene was introduced into the Ubi-pEGFP-C2 vector, and Ubi-pEGFP-C2/nf-actin was transfected into Nfowleri trophozoites. Strong GFP fluorescence was detected in Nfowleri trophozoites transfected with Ubi-pEGFP-C2/nf-actin. Expression of EGFP-Nf-actin protein was detected by Western blot analysis. The nf-actin-overexpressing Nfowleri showed significantly increased adhesion activity against extracellular matrix components, fibronectin, collagen I and fibrinogen, compared with wild-type Nfowleri. Moreover, nf-actin-overexpressing Nfowleri showed increased phagocytic activity and cytotoxicity in comparison with wild-type Nfowleri. In summary, the overexpressed nf-actin gene has an important function in ability to increase cell adhesion, cytotoxicity and phagocytosis by Nfowleri.
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