The free-living amoeba, Naegleria fowleri, causes a fatal disease called primary amoebic meningoencephalitis (PAM) in humans and experimental animals. Of the pathogenic mechanism of N. fowleri concerning host tissue invasion, the adherence of amoeba to hose cells is the most important. We previously cloned the nfa1 gene from N. fowleri. The protein displayed immunolocalization in the pseudopodia, especially the food-cups structure, and was related to the contact-dependent mechanism of the amoebic pathogenicity in N. fowleri infection. The cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) have been used as potent mucosal adjuvants via the parenteral route of immunization in most cases. In this study, to examine the effect of protective immunity of the Nfa1 protein for N. fowleri infection with enhancement by CTB or LTB adjuvants, intranasally immunized BALB/c mice were infected with N. fowleri trophozoites for the development of PAM. The mean time to death of mice immunized with the Nfa1 protein using LTB or CTB adjuvant was prolonged by 5 or 8 days in comparison with that of the control mice. In particular, the survival rate of mice immunized with Nfa1 plus CTB was 100% during the experimental period. The serum IgG levels were significantly increased in mice immunized with Nfa1 protein plus CTB or LTB adjuvants. These results suggest that the Nfa1 protein, with CTB or LTB adjuvants, induces strong protective immunity in mice with PAM due to N. fowleri infection.

Naegleria fowleri, a free-living amoeba, has been found in diverse habitats throughout the world. It causes primary amoebic meningoencephalitis in children and young adults. The amoeba attaches to nasal mucosa, migrates along olfactory nerves and enters the brain. Astrocytes are involved in the defence against infection and produce inflammatory responses. In this study, we focus on the mechanism of immune responses in astrocytes. We showed, using RNase protection assay, RT-PCR and ELISA in an in vitro culture system, that N. fowleri lysates induce interleukin-1beta (IL-1β) and IL-6 expression of astrocytes. In addition, cytokine levels of astrocytes gradually decreased due to extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 inhibitors. To determine the transcription factor, we used transcription inhibitor (AP-1 inhibitor), which downregulated IL-1β and IL-6 expression. These results show that AP-1 is related to IL-1β and IL-6 production. N. fowleri-mediated IL-1β and IL-6 expression requires ERK, JNK and p38 mitogen-activated protein kinases (MAPKs) activation in astrocytes. These findings show that N. fowleri-stimulated astrocytes in an in vitro culture system lead to AP-1 activation and the subsequent expressions of IL-1β and IL-6, which are dependent on ERK, JNK and p38 MAPKs activation. These results may imply that proinflammatory cytokines have important roles in inflammatory responses to N. fowleri infection.

No abstract