The anti-amoebic activity of Pinus densiflora leaf extract against the brain-eating amoeba Naegleria fowleri

Abstract

Naegleria fowleri invades the brain and causes a fatal primary amoebic meningoencephalitis (PAM). Despite its high mortality rate of approximately 97%, an effective therapeutic drug for PAM has not been developed. Approaches with miltefosine, amphotericin B, and other antimicrobials have been clinically attempted to treat PAM, but their therapeutic efficacy remains unclear. The development of an effective and safe therapeutic drug for PAM is urgently needed. In this study, we investigated the anti-amoebic activity of Pinus densiflora leaf extract (PLE) against N. fowleri. PLE induced significant morphological changes in N. fowleri trophozoites, resulting in the death of the amoeba. The IC50 of PLE on N. fowleri was 62.3±0.95 μg/ml. Alternatively, PLE did not significantly affect the viability of the rat glial cell line C6. Transcriptome analysis revealed differentially expressed genes (DEGs) between PLE-treated and non-treated amoebae. A total of 5,846 DEGs were identified, of which 2,189 were upregulated, and 3,657 were downregulated in the PLE-treated amoebae. The DEGs were categorized into biological process (1,742 genes), cellular component (1,237 genes), and molecular function (846 genes) based on the gene ontology analysis, indicating that PLE may have dramatically altered the biological and cellular functions of the amoeba and contributed to their death. These results suggest that PLE has anti-N. fowleri activity and may be considered as a potential candidate for the development of therapeutic drugs for PAM. It may also be used as a supplement compound to enhance the therapeutic efficacy of drugs currently used to treat PAM.

Keywords: Naegleria fowleri; Pinus densiflora; RNA sequencing; anti-amoebic activity; therapeutic drug candidate.

Fig. 1

Anti-amoebic activity of PLE on N. fowleri trophozoites. (A) Morphological change. Treatment with PLE (500 or 1,000 μg/ml) for 48 h induced significant morphological changes in N. fowleri trophozoites. (B) Viability. Different concentrations of PLE (0 to 1,000 μg/ml) were added to N. fowleri trophozoites or C6 cells and incubated for 48 h. PLE caused a remarkable reduction in the viability of N. fowleri trophozoites with an IC₅₀ value of 62.33±0.95 μg/ml. However, it did not significantly affect the viability of the C6 glial cells. The viability of the amoebae and C6 cells is presented as a percentage relative to the negative controls (not treated with PLE). The results are presented as the mean and standard deviation (error bar) from 3 independent assays. *P<0.01, **P<0.001, ***P<0.0001.

Fig. 2

Transmission electron microscopy (TEM) analysis of N. fowleri trophozoites treated with PLE. Naeglaria fowleri trophozoites treated with PLE (500 μg/ml) for 24 h and 48 h showed remarkable ultrastructural morphological changes. Red circles indicate putative apoptotic bodies. Blebs (blue arrowheads). Autophagy-like vacuoles (yellow arrowheads). Magnification, 6,000× or 10,000×. CV, contractile vacuoles; M, mitochondria; N, nucleus.

Fig. 3

DEGs in the PLE-treated and non-treated N. fowleri. (A) MA plot for the DEGs. The red crosses represent significantly upregulated or downregulated genes, and the black spots indicate the non-DEGs. Pink spots represent DEGs, but they were not statistically significant. (B) DEG expression pattern. RNA-seq analysis revealed 5,846 DEGs (2,189 upregulated and 3,657 downregulated) in the PLE-treated N. fowleri. (C) Gene ontology (GO) analysis. The top 11 significantly enriched GO terms of the target genes were related to biological processes, cellular components, and molecular function.

Fig. 4

Semi-quantitative RT-PCR. The expression levels of genes encoding N. fowleri Kelch repeat protein (NF0105540), attractin-like protein 1 (ATRNL1; NF0044360), NADH azoreductase (NF0044710), and pyridoxine 5-phosphate oxidase (NF0073400) were comparatively analyzed in the PLE-treated and non-treated N. fowleri. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as an internal control.