The Nf-actin gene is an important factor for food-cup formation and cytotoxicity of pathogenic Naegleria fowleri

Abstract

Naegleria fowleri destroys target cells by trogocytosis, a phagocytosis mechanism, and a process of piecemeal ingestion of target cells by food-cups. Phagocytosis is an actin-dependent process that involves polymerization of monomeric G-actin into filamentous F-actin. However, despite the numerous studies concerning phagocytosis, its role in the N. fowleri food-cup formation related with trogocytosis has been poorly reported. In this study, we cloned and characterized an Nf-actin gene to elucidate the role of Nf-actin gene in N. fowleri pathogenesis. The Nf-actin gene is composed of 1,128-bp and produced a 54.1-kDa recombinant protein (Nf-actin). The sequence identity was 82% with nonpathogenic Naegleria gruberi but has no sequence identity with other mammals or human actin gene. Anti-Nf-actin polyclonal antibody was produced in BALB/c mice immunized with recombinant Nf-actin. The Nf-actin was localized on the cytoplasm, pseudopodia, and especially, food-cup structure (amoebastome) in N. fowleri trophozoites using immunofluorescence assay. When N. fowleri co-cultured with Chinese hamster ovary cells, Nf-actin was observed to localize around on phagocytic food-cups. We also observed that N. fowleri treated with cytochalasin D as actin polymerization inhibitor or transfected with antisense oligomer of Nf-actin gene had shown the reduced ability of food-cup formation and in vitro cytotoxicity. Finally, it suggests that Nf-actin plays an important role in phagocytic activity of pathogenic N. fowleri.

Diversity of Heterolobosea

“…The heterolobosean amoebae do not possess any cytoskeleton-underlain cytostomes. However, the amoeba of Naegleria fowleri forms socalled amoebastomes, sucker-like surface structures that aid in phagocytosis (Sohn et al, 2010). The amoeboid stage is unknown (and possibly completely lost) in genera Lyromonas, Pharyngomonas, Pleurostomum, Percolomonas, and Stephanopogon.…”

NLRP3 Inflammasome Activation in THP-1 Target Cells Triggered by Pathogenic Naegleria fowleri

“…We previously reported that the pathogenicity of N. fowleri is a complex process which involves contact-independent pathways as well as contact-dependent pathways (29). In the contact-dependent mechanisms, the Nfa1 protein and actin protein were characterized and identified in N. fowleri infections (11,30). In addition, excretory-secretory proteins (ESPs) released from N. fowleri have also been identified, and these may play an important role in its pathogenicity (31).…”

Development of a high- versus low-pathogenicity model of the free-living amoeba Naegleria fowleri

“…In order to assess transcriptional differences between N. fowleri trophozoites grown in the three different media, a quantitative real-time RT-PCR using primers against potential pathogenicity factors (Aldape et al, 1994;Herbst et al, 2002Herbst et al, , 2004Hu et al, 1991Hu et al, , 1992Jung et al, 2009;Lee et al, 2007;Sohn et al, 2010) was performed. The primers used for the PCR are listed in Table 1.…”

“…After biochemical and functional characterization, naegleriapores have been found to be cytotoxic to human cells (Herbst et al, 2004(Herbst et al, , 2002. Another protein involved in N. fowleri-induced cell destruction is Nfactin, which is specifically located around phagocytic foodcups and therefore plays an important role in phagocytic activity (Sohn et al, 2010). Studies using mAbs against Nfactin ( Lee et al, 2007) and gene silencing by small interfering RNAs targeting the Nf-actin gene (Jung et al, 2009) also suggested that Nf-actin is involved in in vitro cytotoxicity.…”