Cellular characterization of actin gene concerned with contact‐dependent mechanisms in Naegleria fowleri

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 50.1 kDa 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 N fowleri 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 N fowleri trophozoites. Strong GFP fluorescence was detected in N fowleri trophozoites transfected with Ubi‐pEGFP‐C2/nf‐actin. Expression of EGFP‐Nf‐actin protein was detected by Western blot analysis. The nf‐actin ‐overexpressing N fowleri showed significantly increased adhesion activity against extracellular matrix components, fibronectin, collagen I and fibrinogen, compared with wild‐type N fowleri . Moreover, nf‐actin ‐overexpressing N fowleri showed increased phagocytic activity and cytotoxicity in comparison with wild‐type N fowleri . In summary, the overexpressed nf‐actin gene has an important function in ability to increase cell adhesion, cytotoxicity and phagocytosis by N fowleri .