The Activity of Polyhomoarginine against Acanthamoeba castellanii

Simple Summary Acanthamoeba is free-living amoeba known to cause severe vision threatening eye infection. In most of the cases treating this infection is difficult due to the Acanthamoeba’s ability to switch between an active feeding stage (trophozoite) and inactive dormant stage (cyst). Arginine rich peptides are highly positively charged and can kill the microorganisms by acting on their negatively charged surface. This study assessed the anti-amoebic activity of polyhomoarginines of different lengths against Acanthamoeba castellanii trophozoites and cysts. Polyhomoarginine showed excellent anti-amoebic activity against both the stages of 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.


Introduction
Anti-microbial peptides (AMPs) are usually characterized as highly positively charged peptides and used as defense mechanisms by many organisms, including humans [1,2]. These peptides often contain a high abundance of arginine and lysine residues to give them their Biology 2022, 11, 1726 2 of 11 highly cationic nature and act by attaching to negatively charged microbial cell membranes thereby causing membrane damage [3,4]. Short, synthetic polyarginine and homoarginine compounds have been explored for activities such as mammalian cell-membrane penetration, disruption or lysis, and carriers for intracellular protein delivery [5][6][7][8]. Polymers of L or D-arginine with 6 or more amino acids tend to be taken up more effectively by cells [9] Additionally, these peptides can be easily surface-modified by copolymers to improve their cellular uptake and reduce cytotoxicity [9][10][11].
Polyarginine is active against systemic and mucocutaneous Candida albicans in a murine infection model which was mediated by charge interactions between the negatively charged cell membrane and positively charged polyarginine [12]. Poly-L-arginine is also active against Escherichia coli and Staphylococcus aureus, and its activity can be further enhanced by increasing the glycine content in the peptide [13]. Homoarginine is a cationic, non-proteinogenic amino acid derived from lysine and it differs structurally from arginine by having an extra methylene (CH 2 ) group on its side chain [7,14,15]. Poly-L-arginine and poly-L-homoarginine (PLHA) bind more strongly to negative surfaces in comparison to poly-L-lysine due to the guanidino chain of arginine binding 2.5 to 4 times higher to carboxylate anions [10,16]. L-homoarginine can inhibit the growth of Gram-positive bacteria [17]. Poly-(D, L)-homoarginine containing glutamic acid is active against highly fluconazole -resistant Candida albicans and, also had excellent in vivo biosafety [18].
Acanthamoeba keratitis (AK) is a rare but severe corneal infection caused by freeliving amoebae of the Acanthamoeba genus. Recent studies from the United Kingdom and The Netherlands have shown that the number of AK cases in 2010-2011 and 2015 had increased compared to historical numbers [19,20]. Acanthamoeba keratitis is difficult to diagnose and treat due to its transformation from the active trophozoites feeding stage to the resilient quiescent stage cysts, which are difficult to kill [21,22]. Polyhexamethylene biguanide or chlorhexidine are widely used to treat this infection as monotherapies or in combination [23]. However, treatment still requires a long duration and the infection can relapse upon ceasing the treatment [24]. Additionally, the age of the patient (>34 years), and the presence of secondary inflammatory complications with AK also can contribute to poor treatment outcomes [25,26]. Thus, there is a need to develop improved treatment options such as reducing relapse upon ceasing the treatment by reducing cyst formation in situ [27].
Protamine, an arginine-rich 33 amino acid-based peptide, has been shown to be active against Acanthamoeba trophozoites and cysts, bacteria, and fungi, specifically reducing the numbers of Acanthamoeba polyphaga and Acanthamoeba castellanii trophozoites by 2 log 10 and cysts by between 0.6 to 0.9 log 10 at 229 µM [28,29]. Melimine, a hybrid cationic peptide derived from protamine and melittin, has been shown to inhibit Acanthamoeba trophozoite adhesion to contact lenses by 1.2 log 10 [30,31]. To assess any new compound for its anti-amoebic activity, it is important to evaluate its activity against both trophozoites and cysts [21,22,24,32]. Protamine has been evaluated for the amoebicidal and encystment assay, but it is also important to assess its ability to stop trophozoite emergence from cysts. Similarly, melimine anti-amoebic activity was tested against the trophozoites only. However, these AMPs have not been evaluated for their ability to cause encystment or prevent excystment of Acanthamoeba. Based on the previously demonstrated antimicrobial activity of homoarginines, the current study assessed the activity of different lengths of homoarginines against Acanthamoeba castellanii.

Polyhomoarginines
Polyhomoarginine (PHA) peptides of different lengths (400 to 10 homoarginines) were purchased from Alamanda Polymers Inc. (Huntsville, AL, USA) and chlorhexidine (CLX) was obtained from Sigma Aldrich. All PHAs were used as received from the supplier and diluted from a stock solution in 1X PBS or PYG medium and freshly prepared before each experiment.

Amoebicidal Assay
This assay followed a previously published method [34]. Briefly, 5 × 10 5 trophozoites/mL were incubated with PHA 400, PHA 250, PHA 100, PHA 50, PHA 30 and PHA 10 in 24 well plates at concentrations ranging from 0.93 µM to 15 µM in PBS. Additionally, PHA 10 was tested at 30 µM and 60 µM. Chlorhexidine at 15 µM and 60 µM was used as a positive control [35]. PBS alone was used as a negative control. The trophozoites were incubated at 30 • C for 24 h. The number of viable trophozoites was determined by adding 0.1% trypan blue to each well. Dead trophozoites that stained blue and live trophozoites that remained unstained were counted using a Neubauer haemocytometer (Hirschmann, Germany) [36].

Amoebistatic Assay
This assay followed a previously published method [34]. Briefly, 2 × 10 5 trophozoites/mL were incubated with PHA 400, PHA 250, PHA 100, PHA 50, PHA 30 and PHA 10 in 24 well plates at concentrations ranging from 0.93 µM to 15 µM in PYG. Additionally, PHA 10 was tested at 30 µM and 60 µM. Chlorhexidine at 15 µM and 60 µM was used as a positive control. PYG medium alone was used as a negative control. After incubation at 30 • C for 48 h, the number of viable trophozoites was determined using a Neubauer haemocytometer after the addition of 0.1% of trypan blue to each well.

Encystation Assay
Based on the amoebicidal and amoebistatic activity, PHA 30 and PHA 10 were used for further experiments. To assess the ability of trophozoites to encyst in the presence or absence of PHA or chlorhexidine, an encystment medium (EM) was prepared by adding 50 mM MgCl 2 and 10% glucose to 1X PBS by filter sterilization [37]. Trophozoites (5 × 10 5 trophozoites/mL) were incubated with PHA 30 and PHA 10 in 24 well plates at concentrations ranging from 0.93 µM to 15 µM in this encystment media. Chlorohexidine at 15 µM was used as a positive control. The encystment medium alone was used as a negative control. The trophozoites were incubated at 30 • C for 72 h, and at the end of 72 h 0.25% (w/v) sodium-dodecyl sulphate was added to each well to burst trophozoites leaving cysts intact [37,38]. The number of cysts was determined by counting on a Neubauer haemocytometer.

Excystment Assay
For this assay [33,37], 5 × 10 5 cysts/mL were incubated with PHA 30, and PHA 10 in 24 well plates at concentrations ranging from 0.93 µM to 15 µM in PYG. Chlorohexidine at 15 µM was used as a positive control. PYG medium alone was used as a control. The plates were observed every day at 10× and 40× magnification to assess the emergence of trophozoites during incubation at 30 • C for 72 h. At the end of the incubation period, the number of trophozoites that re-emerged was counted using a Neubauer haemocytometer.

Lysis of Horse Red Blood Cells (HRBCs)
The haemolytic activities of the PHA 30 and 10 were determined using HRBCs (Oxid, Australia) as described previously [39,40]. Briefly, 10 mL of HRBCs were washed three times with PBS at 470× g for 5 min. PHA 30 and 10 peptide concentrations ranging from 0.93 µM to 60 µM were added to the washed HRBCs and incubated at 37 • C for 4 h. HRBCs in PBS was used as a negative control with the expectation of 0% lysis. Similarly, HRBCs in distilled water was used as positive controls to achieve 100% lysis. At the end of incubation, cells were pelleted at 1057× g for 5 min, and the supernatant was removed to assess the release of haemoglobin by measuring the optical density (OD) at 540 nm. The relative OD of HRBCs treated with compounds chlorohexidine and PHA were compared to that treated with distilled water were used to determine the relative percentage of haemolysis as follows:

Statistical Analyses
Statistical analyses were performed using GraphPad Prism 8.4.3 software (GraphPad Software, San Diego, CA, USA). All experiments are performed in duplicate with two independent replicates. Data were represented as mean ± SE and analysed using one-way ANOVA. p < 0.05 is considered statistically significant.

Amoebicidal Assay
PHA 400, PHA 250, PHA 100, PHA 50, PHA 30 and PHA 10 were used to study both their amoebicidal and amoebistatic activities against Acanthamoeba castellanii ATCC30868. The number of trophozoites in the negative control was 2.34 × 10 5 ± 2.2 × 10 4 at the end of the 24 h incubation period. Trophozoite killing activity and growth inhibition was observed in a dose-dependent manner with all the PHA peptides tested. All the PHA peptides showed statistically significant killing of the Acanthamoeba trophozoites at the lowest test concentration of 0.93 µM in comparison to the negative control (Amoebae in 1X PBS Figure 1). Except for PHA 10, all compounds showed 100% killing of trophozoites at 15 µM and 7.5 µM (p < 0.0001) whereas chlorohexidine gave 96% killing of trophozoites at 15 µM (p < 0.0001, Figure 1) in comparison to the negative control.

Figure 1.
Amoebicidal activity of polyhomoarginine (PHA) peptides against A. castellanii ATCC30868. In brief, 5 × 10 5 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 a two-sample t-test and two-tailed distribution).

Amoebistatic Assay
For the growth inhibition assay, the number of trophozoites enumerated in the PYG medium alone was 3.28 × 10 5 ± 4.1 × 10 4 trophozoites/mL at the end of 48 h incubation period. All the PHAs inhibited trophozoite proliferation by 100% at 15 µM (p < 0.0001) except for PHA 10 which gave 97% inhibition (7.5 × 10 3 ± 3.2 × 10 3 , p < 0.0001) when compared to the negative control (Amoebae in PYG, Figure 2). Chlorohexidine gave similar activity with 98% inhibition at 15 µM (5 × 10 3 ± 3.5 × 10 3 , p< 0.0001) compared to the negative control. PHA 400 gave the highest amount of growth inhibition of 100% between 15 µM to 0.93 µM (p < 0.0001, Figure 2). Similarly, PHA 250 inhibited growth by 100% at 15 µM (p < 0.0001) and 98% at 0.93 µM (5 × 10 3 ± 3.5 × 10 3 , p = 0.0002, Figure 2) when compared to the negative control. There is statistical significance seen between the activities of PHA 10 and the rest of all PHA compounds at 7.5 µM concentrations when comparing the activities among the PHA groups (p < 0.0001). Similarly, at 3.75 µM there is a statistically significant difference between the activities of PHA 400, PHA 250 and PHA 100 in comparison to PHA 30 and PHA 10 (p ≤ 0.006). Additionally, PHA 50 and PHA 10 showed statistical differences in inhibition activities between them (p = 0.01). PHA 400, PHA 250 and PHA 100 have showed a statistically significant difference in activities in comparison to PHA 50, PHA 30 and PHA 10 at 1.87 µM (p ≤ 0.01). Additionally, at 0.93 µM PHA 400 In brief, 5 × 10 5 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).

Amoebistatic Assay
For the growth inhibition assay, the number of trophozoites enumerated in the PYG medium alone was 3.28 × 10 5 ± 4.1 × 10 4 trophozoites/mL at the end of 48 h incubation period. All the PHAs inhibited trophozoite proliferation by 100% at 15 µM (p < 0.0001) except for PHA 10 which gave 97% inhibition (7.5 × 10 3 ± 3.2 × 10 3 , p < 0.0001) when compared to the negative control (Amoebae in PYG, Figure 2). Chlorohexidine gave similar activity with 98% inhibition at 15 µM (5 × 10 3 ± 3.5 × 10 3 , p< 0.0001) compared to the negative control. PHA 400 gave the highest amount of growth inhibition of 100% between 15 µM to 0.93 µM (p < 0.0001, Figure 2). Similarly, PHA 250 inhibited growth by 100% at 15 µM (p < 0.0001) and 98% at 0.93 µM (5 × 10 3 ± 3.5 × 10 3 , p = 0.0002, Figure 2) when compared to the negative control. There is statistical significance seen between the activities of PHA 10 and the rest of all PHA compounds at 7.5 µM concentrations when comparing the activities among the PHA groups (p < 0.0001). Similarly, at 3.75 µM there is a statistically significant difference between the activities of PHA 400, PHA 250 and PHA 100 in comparison to PHA 30 and PHA 10 (p ≤ 0.006). Additionally, PHA 50 and PHA 10 showed statistical differences in inhibition activities between them (p = 0.01). PHA 400, PHA 250 and PHA 100 have showed a statistically significant difference in activities in comparison to PHA 50, PHA 30 and PHA 10 at 1.87 µM (p ≤ 0.01). Additionally, at 0.93 µM PHA 400 and PHA 250 showed statistically significant differences in inhibition activities when compared to the rest of all PHA compounds (p < 0.001). 022, 11, x 6 of 12 and PHA 250 showed statistically significant differences in inhibition activities when compared to the rest of all PHA compounds (p < 0.001).

Figure 2.
Amoebistatic activity of polyhomoarginine (PHA) peptides against A. castellanii ATCC30868. In brief, 2 × 10 5 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 a two-sample t-test and two-tailed distribution). ## p<0.001, # p<0.05 Shows statistical significance among the PHA groups at each test concentration.
In the excystment assay, when incubated alone, 3.16 × 10 5 ± 1.5 × 10 4 trophozoites emerged. PHA 30 inhibited trophozoite re-emergence from cysts by 100% and PHA 10 by 99% at 15 µM and chlorohexidine inhibited re-emergence from cysts by 100% at the same concentrations (p < 0.0001, Figure 4) when compared to amoebae in PYG. Both PHA 30 and PHA 10 significantly inhibited the trophozoite emergence from cysts by ≥50% at 3.75 µM concentration (p = 0.0017, p = 0.01) when compared with PYG alone. There is no statistically significant difference between the activities of PHA 30 and PHA 10 at each test concentration (p > 0.05). In the excystment assay, when incubated alone, 3.16 × 10 5 ± 1.5 × 10 4 trophozoites emerged. PHA 30 inhibited trophozoite re-emergence from cysts by 100% and PHA 10 by 99% at 15 µM and chlorohexidine inhibited re-emergence from cysts by 100% at the same concentrations (p < 0.0001, Figure 4) when compared to amoebae in PYG. Both PHA 30 and PHA 10 significantly inhibited the trophozoite emergence from cysts by ≥50% at 3.75 µM concentration (p = 0.0017, p = 0.01) when compared with PYG alone. There is no statistically significant difference between the activities of PHA 30 and PHA 10 at each test concentration (p > 0.05).

Discussion
The current study demonstrated that the PHA peptides were highly effective against both the trophozoites and cysts of Acanthamoeba castellanii, with a dose-dependent and amino acid number dependence. To the best of our knowledge, this is the first study to examine the effects of homoarginine against Acanthamoeba.
The results are broadly similar to a previous study that found greater bacterial inhibition by homopeptides of arginine with an increasing number of residues and concentrations [17]. In that study, arginine homopeptides with 10 residues at 20 µM was able to inhibit the growth of Gram-positive bacteria Staphylococcus aureus by approximately (c.) 65%, Staphylococcus epidermidis by c. 95%, Micrococcus luteus by c. 95%, Listeria monocytogenes by c. 45%, Bacillus cereus by c. 100% but Bacillus subtilis was not affected. There was a moderate improvement in inhibition activity when evaluated at 30 µM [17]. A followup study using Gram-negative bacteria found that arginine homopeptides with 10 residues at 20 µM inhibited the growth of E.coli by 100%, Salmonella enterica typhimurium by c. 90%, S. enteritidis by c. 90%, Pseudomonas aeruginosa by c. 30%, Vibrio parahaemolyticus by c. 25% and Aeromonas hydrophilia by <10% [41].

Discussion
The current study demonstrated that the PHA peptides were highly effective against both the trophozoites and cysts of Acanthamoeba castellanii, with a dose-dependent and amino acid number dependence. To the best of our knowledge, this is the first study to examine the effects of homoarginine against Acanthamoeba.
The results are broadly similar to a previous study that found greater bacterial inhibition by homopeptides of arginine with an increasing number of residues and concentrations [17]. In that study, arginine homopeptides with 10 residues at 20 µM was able to inhibit the growth of Gram-positive bacteria Staphylococcus aureus by approximately (c.) 65%, Staphylococcus epidermidis by c. 95%, Micrococcus luteus by c. 95%, Listeria monocytogenes by c. 45%, Bacillus cereus by c. 100% but Bacillus subtilis was not affected. There was a moderate improvement in inhibition activity when evaluated at 30 µM [17]. A follow-up study using Gram-negative bacteria found that arginine homopeptides with 10 residues at 20 µM inhibited the growth of E.coli by 100%, Salmonella enterica typhimurium by c. 90%, S. enteritidis by c. 90%, Pseudomonas aeruginosa by c. 30%, Vibrio parahaemolyticus by c. 25% and Aeromonas hydrophilia by <10% [41].
Another study tested peptides containing four residues each of arginine and phenylalanine [3]. The peptide was poorly active, with minimum bactericidal concentrations (MBC, 99.9% inhibition) being >406 µM against S. aureus, S. epidermidis, P. aeruginosa and E. coli. However, they had slightly greater efficacy against Candida sp. giving MBCs of 206 µM against some strains [3]. In the current study, PHA 10 had excellent overall activity at 15 µM with 98% of trophozoite killing, 97% inhibition of trophozoite proliferation, 84% inhibition of cyst formation and, 99% inhibition of trophozoite re-emergence from cysts. This suggests that polyhomoarginine may have superior antimicrobial activity compared to peptides containing arginine. Indeed, protamine, an arginine-rich and cationic peptide, had amoebicidal activity at 57 µM, reducing growth by 90% for Acanthamoeba polyphaga and by 67% for A. castellanii [28].
Previous results have shown that polyhomoarginine (D, L-homoarginine) containing 28 residues had a reduction in the cell penetrating capability with increased cytotoxicity [18]. This may be due to the interaction of PHA with serum protein leading to aggregation, precipitation, and reduction in biological activity [18]. In the current study, PHA 10 and 30 had ≥70% killing of trophozoites at lowest test concentration (0.93 µM). The potential disadvantages of using PHA with greater numbers of residues and the excellent in vitro activity of PHA with smaller numbers of residues led to further evaluation of the activities of PHA 30 and 10 against Acanthamoeba cysts.
In the cytotoxicity assay, PHA 10 gave a low level of haemolysis (8%) at 15 µM and PHA 30 gave <40% haemolysis at 3.75 µM. Additionally, PHA 10 gave 23% of haemolysis at 60 µM. A linear correlation between toxicity and increasing mass percentage of arginine in membrane-permeable peptides has been previously shown [43]. Arginine homopeptides exhibit negligible cytotoxicity up to 11 residues at <100 µM concentrations [17]. This suggests the higher toxicity with PHA 30 may be due to the higher cationic charge with increasing mass percentage leading to the toxicity on the red blood cells. The results on cytotoxicity against HRBCs are encouraging and warrant further investigation on the evaluate the cytotoxicity in AK animal models in future experiments.

Conclusions
This is the first study to evaluate the in vitro activity of polyhomoarginine against Acanthamoeba castellanii ATCC30868. PHA10 had excellent anti-acanthamoeba activity against both trophozoites and cysts at 15 µM and low cytotoxicity against HRBcs. The results showing low cytotoxicity against HRBCs are encouraging. Based on these results it would be interesting to examine in more detail whether polyhomoarginine can be developed into a potential anti-acanthamoeba agent against Acanthamoeba by carrying out further pre-clinical and then in vivo experiments using AK animal model.