Effect of synthetic antimicrobial peptides on Naegleria fowleri trophozoites.

We evaluated the effect of tritrpticin, lactoferrin, killer decapeptide and scrambled peptide in vitro against Naegleria fowleri trophozoites compared with amphotericin B. Tritrpticin (100 microg/ml) caused apoptosis of N. fowleri trophozoites (2x10(5) cells/ml), while lactoferrin, killer decapeptide and scrambled peptide did not. On Gormori trichrome staining, tritrpticin affected the elasticity of the surface membrane and reduced the size of the nuclei of N. fowleri trophozoites. The ultrastructure surface membrane and food cup formation of the trophozoites were 100% inhibited. These results are consistent with inhibition of the nfa1, Mp2CL5 of the treated trophozoite, which plays a role in food cup formation. Tritrpticin 100 microg/ml was not toxic against SK-N-MC cells. Our findings suggest tritrpticin has activity against the surface membrane and nfa1 and Mp2CL5 of N. fowleri trophozoites and could be developed as a potential therapeutic agent.

Cytopathogenesis of Naegleria fowleri Thai strains for cultured human neuroblastoma cells.

The aim of this study is to evaluate cellular interaction between free-living amoebae Naegleria fowleri strains and mammalian target cells in vitro. Two Thai strains of N. fowleri; Khon Kaen strain from the environment and Siriraj strain from the patient's cerebrospinal fluid and the Center of Disease Control VO 3081 strain from Atlanta (US) were studied. Human neuroblastoma (SK-N-MC) and African Green monkey Kidney (Vero) cells were used as target cells. Each cell line was inoculated with each strain of N. fowleri at a ratio of 1:1 and observed for 7 days. The uninoculated target cells and each strain of N. fowleri were used as control. The numbers of the challenged and unchallenged cells as well as the free-living amoebae were counted three times by trypan blue exclusion method. The inoculation began when the amoebae attached to the cell membrane and ingested the target cells. In this study, extensive cytopathogenesis with many floating inoculated cells and abundant number of amoebae were observed. The destruction pattern of both inoculated SK-N-MC and Vero target cells were similar. Interestingly, SK-N-MC was more susceptible to N. fowleri strains than the Vero cell. In addition, N. fowleri Siriraj strain showed the highest destruction pattern for each target cell. Our findings suggest that the SK-N-MC should be used as a base model for studying the neuropathogenesis in primary amoebic meningoencephalitis patients.

Scanning electron microscopic study of human neuroblastoma cells affected with Naegleria fowleri Thai strains.

In order to understand the pathogenesis of Naegleria fowleri in primary amoebic meningoencephalitis, the human neuroblastoma (SK-N-MC) and African green monkey kidney (Vero) cells were studied in vitro. Amoeba suspension in cell-culture medium was added to the confluent monolayer of SK-N-MC and Vero cells. The cytopathic activity of N. fowleri trophozoites in co-culture system was elucidated by scanning electron microscope at 3, 6, 9, 12, and 24 h. Two strains of N. fowleri displayed well-organized vigorous pseudopods in Nelson's medium at 37 degrees C. In co-culture, the target monolayer cells were damaged by two mechanisms, phagocytosis by vigorous pseudopods and engulfment by sucker-like apparatus. N. fowleri trophozoites produced amoebostomes only in co-culture with SK-N-MC cells. In contrast, we could not find such apparatus in the co-culture with Vero cells. The complete destruction time (100%) at 1:1 amoeba/cells ratio of SK-N-MC cells (1 day) was shorter than the Vero cells (12 days). In conclusion, SK-N-MC cells were confirmed to be a target model for studying neuropathogenesis of primary amoebic meningoencephalitis.