The Activity of Polyhomoarginine against Acanthamoeba castellanii

Abstract

Arginine-rich peptides can have broad-spectrum anti-bacterial and anti-fungal activities. Polyhomoarginine consists of highly cationic residues which can act on the negatively charged microbial cell membranes. Acanthamoeba is a free-living protozoan known to cause a rare corneal infection which is difficult to diagnose and treat. This study evaluated the activity of the polyhomoarginines against Acanthamoeba castellanii. Acanthamoeba amoebicidal, amoebistatic, encystation and excystment assays were performed using protocols described in the literature. The activity of polyhomoarginines (PHAs) of different lengths (10 to 400 residues) was measured against the trophozoites and cysts of Acanthamoeba castellanii ATCC30868 in concentrations ranging from 0.93 μM to 15 μM. Data were represented as mean ± SE and analysed using one-way ANOVA. Overall, PHAs demonstrated good anti-acanthamoeba activity against both trophozoites and cysts. PHA 30 reduced the number of viable trophozoites by 99%, inhibited the formation of cysts by 96% and the emergence of trophozoites from cysts by 67% at 3.75 μM. PHA 10 was similarly active, but at a slightly higher concentration of 15 μM, reducing the numbers of viable trophozoites by 98%, inhibiting cyst formation by 84% and preventing the emergence of trophozoites from cysts by 99%. At their greatest anti-amoeba concentrations, PHA 10 gave only 8% haemolysis at 15 μM while PHA 30 gave <40 % haemolysis at 3.75 μM. Polyhomoarginine 10 showed excellent anti-amoebic activity against both forms of Acanthamoeba castellanii and was non-toxic at its most active concentrations. This implies that polyhomoarginines can be developed into a potential therapeutic agent for Acanthamoeba keratitis. However, there is a need to carry out further pre-clinical and then in vivo experiments in the AK animal model.

Keywords: Acanthamoeba; antimicrobial peptides; arginine peptides; free-living amoeba; polyhomoarginine.

Figure 1

Amoebicidal activity of polyhomoarginine (PHA) peptides against A. castellanii ATCC30868. In brief, 5 × 10⁵ A. castellanii trophozoites were incubated with PHA peptides and chlorhexidine at 30 °C for 24 h after which viability was determined by staining with Trypan blue using a Neubauer haemocytometer. The results show significant anti-acanthamoeba activity when compared to amoeba+ PBS only (** p < 0.001 using one way ANOVA).

Figure 2

Amoebistatic activity of polyhomoarginine (PHA) peptides against A. castellanii ATCC30868. In brief, 2 × 10⁵ A. castellanii trophozoites were incubated with PHA peptides and chlorhexidine at 30 °C for 48 h after which no trophozoites were enumerated by staining with Trypan blue using a Neubauer haemocytometer. The results show significant anti-acanthamoeba activity when compared to the amoeba + PYG medium (** p < 0.001, * p < 0.05 using one way ANOVA). ^## p < 0.001, ^# p < 0.05 Shows statistical significance among the PHA groups at each test concentration.

Figure 3

Inhibition of cysts formation by polyhomoarginine (PHA) peptides against A. castellanii ATCC30868. In brief, 5 × 10⁵ A. castellanii trophozoites were incubated with PHA peptides and chlorhexidine at 30 °C for 72 h after which cysts were determined by solubilizing trophozoites adding 0.25% SDS. The Number of cysts was counted using the Neubauer haemocytometer. The results show significant anti-acanthamoeba activity when compared to the amoeba + PBS with encystment medium (EM). (** p < 0.001, * p < 0.05 using one way ANOVA).

Figure 4

Inhibition of trophozoites re-emergence from cysts by polyhomoarginine (PHA) against A. castellanii ATCC30868. In brief, 5 × 10⁵ A. castellanii cysts were incubated with PHA peptides and chlorhexidine at 30 °C for 72 h after which trophozoites were determined by counting on a Neubauer haemocytometer. The results show significant anti-acanthamoeba activity when compared to the amoeba + PYG medium (** p < 0.001, * p < 0.05 using one way ANOVA).