The Anti-Amoebic Activity of a Peptidomimetic against Acanthamoeba castellanii

Abstract

Acanthamoeba is a free-living protozoan known to cause keratitis most commonly, especially among contact lens wearers. Treatment of Acanthamoeba keratitis is challenging as Acanthamoeba can encyst from the active form, a trophozoite, into a hibernating cyst that is refractory to antibiotics and difficult to kill; therefore, there is a need for more effective anti-amoebic strategies. In this study, we have evaluated the anti-amoebic activity of the antimicrobial peptide mimic RK-758 against Acanthamoeba castellanii. RK-758 peptidomimetic was subjected to biological assays to investigate its amoebicidal, amoebistatic, anti-encystation, and anti-excystation effects on A. castellanii. The anti-amoebic activity of the peptide mimic RK-758 was compared with chlorhexidine against the Acanthamoeba castellanii ATCC30868 and Acanthamoeba castellanii 044 (a clinical strain) with the concentrations of both ranging from 125 µM down to 7.81 µM. All experiments were performed in duplicate with three independent replicates. The data were represented as mean ± SE and analysed using a two-sample t-test and two-tailed distributions. A p < 0.05 was considered statistically significant. The peptidomimetic RK-758 had anti-Acanthamoeba activity against both trophozoites and cysts in a dose-dependent manner. The RK-758 had amoebicidal and growth inhibitory activities of ≥50% at a concentration between 125 µM and 15.6 µM against the trophozoites of both Acanthamoeba strains. Inhibitory effects on the cyst formation and trophozoite re-emergence from cysts were noted at similar concentrations. Chlorhexidine had 50% activity at 7.81 µM and above against the trophozoites and cysts of both strains. In the haemolysis assay, the RK-758 lysed horse RBCs at concentrations greater than 50 µM whereas lysis occurred at concentrations greater than 125 µM for the chlorhexidine. The peptidomimetic RK-758, therefore, has activity against both the trophozoite and cyst forms of Acanthamoeba and has the potential to be further developed as an anti-microbial agent against Acanthamoeba. RK-758 may also have use as an anti-amoebic disinfectant in contact lens solutions.

Keywords: Acanthamoeba; anti-microbial peptides; free-living amoeba; peptidomimetics.

Figure 1

Amoebicidal activity of peptidomimetic RK-758 in comparison with chlorhexidine against Acanthamoeba castellanii ATCC30868 (A) and A. castellanii 044 (B). In brief, 5 × 10⁵ A. castellanii trophozoites were incubated with the peptidomimetics RK-758 and chlorhexidine at 30 °C for 24 h after which the viability was determined by staining with trypan blue using a Neubauer haemocytometer. The results show significant anti-Acanthamoeba activity when compared to the negative control (amoeba alone). * p < 0.05 using a two-sample t-test and two-tailed distribution.

Figure 2

Amoebistatic activity of the peptidomimetic RK-758 in comparison with chlorhexidine against Acanthamoeba castellanii ATCC30868 (A) and A. castellanii 044 (B). In brief, 2 × 10⁵ A. castellanii trophozoites were incubated with the peptidomimetics RK-758 and chlorhexidine at 30 °C for 48 h after which the viability was determined by staining with Trypan blue using a Neubauer haemocytometer. The results showed significant anti-Acanthamoeba activity when compared to the negative control (amoeba alone). ** p < 0.001; * p < 0.05 using a two-sample t-test and two-tailed distribution.

Figure 3

Inhibition of cysts formation by the peptidomimetic RK-758 in comparison with chlorhexidine against Acanthamoeba castellanii ATCC30868 (A) and A. castellanii 044 (B). In brief, 5 × 10⁵ A. castellanii trophozoites were incubated with the peptidomimetics RK-758 and chlorhexidine at 30 °C for 72 h after which cysts were determined by solubilising trophozoites adding 0.25% SDS. The number of cysts was counted using the Neubauer haemocytometer. The results showed significant anti-Acanthamoeba activity when compared to the negative control (amoeba alone). ** p < 0.001; * p < 0.05 using the two-sample t-test and two-tailed distribution.

Figure 4

Inhibition of trophozoites’ re-emergence from cysts by the peptidomimetic RK-758 in comparison with chlorhexidine, against Acanthamoeba castellanii ATCC30868 (A) and A. castellanii 044 (B). In brief, 5 × 10⁵ A. castellanii cysts were incubated with the peptidomimetics RK-758 and chlorhexidine at 30 °C for 72 h after which the trophozoites were determined by counting on a Neubauer haemocytometer. The results showed significant anti-Acanthamoeba activity when compared to the negative control (amoeba alone). ** p < 0.001; * p < 0.05 using a two-sample t-test and two-tailed distribution.

Figure 5

Inhibition of trophozoite emergence from cysts of Acanthamoeba castellanii ATCC30868 by the peptidomimetic RK-758 (A–E) in comparison with chlorhexidine (F–J), with concentrations of both ranging from 125 µM to 7.81 µM. In brief, 5 × 10⁵ A. castellanii trophozoites were incubated with the peptidomimetics RK-758 and chlorhexidine at 30 °C for 72 h and observed for the emergence of trophozoites. PYG alone was used as a control (K). PeptidomimeticsRK-758 inhibited the trophozoite emergence between concentrations of 125 µM and 31.25 µM (A–C) whereas the chlorhexidine inhibited their emergence between 125 µM and 7.81 µM (F–J). Arrowheads indicate trophozoites.

Figure 6

Inhibition of trophozoite emergence from cysts of Acanthamoeba castellanii 044 by the peptidomimetic RK-758 (A–E) in comparison with chlorhexidine (F–J), with concentrations of both ranging from 125 µM to 7.81 µM. In brief, 5 × 10⁵ A. castellanii trophozoites were incubated with the peptidomimetics RK-758 and chlorhexidine at 30 °C for 72 h and observed for the emergence of trophozoites. PYG alone was used as a control (K). Peptidomimetic RK-758 inhibited the trophozoite emergence between concentrations of 125 µM and 31.25 µM (A–C) whereas the chlorhexidine inhibited their emergence between 125 µM and 7.81 µM (F–J). Arrowheads indicate trophozoites.