Novel Antiamoebic Tyrocidine-Derived Peptide against Brain-Eating Amoebae

Abstract

Acanthamoeba castellanii (A. castellanii) can cause Acanthamoeba keratitis, a sight-threatening infection, as well as a fatal brain infection termed granulomatous amoebic encephalitis, mostly in immunocompromised individuals. In contrast, Naegleria fowleri (N. fowleri) causes a deadly infection involving the central nervous system, recognized as primary amoebic encephalitis, mainly in individuals partaking in recreational water activities or those with nasal exposure to contaminated water. Worryingly, mortality rates due to these infections are more than 90%, suggesting the need to find alternative therapies. In this study, antiamoebic activity of a peptide based on the structure of the antibiotic tyrocidine was evaluated against A. castellanii and N. fowleri. The tyrocidine-derived peptide displayed significant amoebicidal efficacy against A. castellanii and N. fowleri. At 250 μg/mL, the peptide drastically reduced amoebae viability up to 13% and 21% after 2 h of incubation against N. fowleri and A. castellanii., whereas, after 24 h of incubation, the peptide showed 86% and 94% amoebicidal activity against A. castellanii and N. fowleri. Furthermore, amoebae pretreated with 100 μg/mL peptide inhibited 35% and 53% A. castellanii and N. fowleri, while, at 250 μg/mL, 84% and 94% A. castellanii and N. fowleri failed to adhere to human cells. Amoeba-mediated cell cytopathogenicity assays revealed 31% and 42% inhibition at 100 μg/mL, while at 250 μg/mL 75% and 86% A. castellanii and N. fowleri were inhibited. Assays revealed inhibition of encystation in both A. castellanii (58% and 93%) and N. fowleri (73% and 97%) at concentrations of 100 and 250 μg/mL respectively. Importantly, tyrocidine-derived peptide depicted minimal cytotoxicity to human cells and, thus, may be a potential candidate in the rational development of a treatment regimen against free-living amoebae infections. Future studies are necessary to elucidate the in vivo effects of tyrocidine-derived peptide against these and other pathogenic amoebae of importance.

Figure 1

Design and validation of the tyrocidine-derived peptide. (a) The FRET-peptide library was screened with A. castellanii culture supernatant. Key: black = lack of activity (F/min, <5); dark green = low activity (F/min, 5–25), and light green = moderate activity (F/min, 25–125). (b) Design of the tyrocidine-derived linear peptides based on the cyclic structure of the antibiotic compound of tyrocidine A. The arrows depict where the cyclic structure was opened. The names and sequences of the derived peptides are denoted within the box. L-amino acids are denoted as upper-case letters, d-amino acids as lower-case letters. (c) Proteolytic interaction between the tyrocidine-peptides and A. castellanii. Culture supernatant was treated with 16 μM FRET-peptide substrate. Fluorescence was measured for 1 h at 37 °C. Data are depicted as the mean ± standard error of three independent experiments.

Figure 2

Tyrocidine-derived peptide exhibiting significant amoebicidal activities against A. castellanii and N. fowleri. (a) Amoebae were treated with the peptide for 2 h and (b) challenged with peptide for 24 h. The data are representative of three independent experiments and presented as the mean ± standard error. AmpB and chlorhexidine were used as positive control, while for negative control amoebae were cultured in RMPI alone. P-values were established utilizing the two-sample t test, two-tailed distribution; (*) is P ≤ 0.05, (**) is P ≤ 0.01, and (***) is P ≤ 0.001.

Figure 3

Tyrocidine-derived peptide inhibiting binding of A. castellanii and N. fowleri to human cells. In brief, A. castellanii and N. fowleri (5 × 10⁵ amoebae) were pretreated with peptide for 2 h at 30 °C followed by a binding assay to HeLa cells. The data are representative of three independent experiments and presented as the mean ± standard error. P-values were established via the two-sample t test, two-tailed distribution; (*) is P ≤ 0.05, (**) is P ≤ 0.01, and (***) is P ≤ 0.001.

Figure 4

Effect of the tyrocidine-derived peptide on amoebae trophozoites encystation. The results revealed that peptide significantly inhibited the encystation process as compared to the negative control. The data are representative of three independent experiments and presented as the mean ± standard error. P-values were ascertained via two-sample t test, two-tailed distribution; (*) is P ≤ 0.05 and (***) is P ≤ 0.001.

Figure 5

Tyrocidine-derived peptide showing minimal cytotoxicity. A confluent monolayer of human cells (HeLa) were challenged with varying amounts of tyrocidine-derived peptide (25, 50, 100, and 250 μg/mL). At all tested concentrations, the peptide showed limited cytotoxicity toward human cell lines. Data are depicted as mean ± standard error. Experiments were accomplished in duplicates.

Figure 6

Tyrocidine-derived peptide inhibiting amoebae-mediated cytotoxicity against human cells. Briefly, 5 × 10⁵ amoebae were treated with 100 and 250 μg/mL peptide at 30 °C for 2 h. Pretreated amoebae were then transferred to HeLa cells and kept overnight at 5% CO₂ at 37 °C. The results revealed inhibition of amoebae-mediated host cytotoxicity when compared to amoeba alone (untreated). The data are representative of three independent experiments and presented as the mean ± standard error. P-values were ascertained with the sample t test, two-tailed distribution; (*) is P ≤ 0.05 and (**) is P ≤ 0.01.