Repurposing drugs: Ca²⁺ ion dependency that can be exploited to treat keratitis caused by Acanthamoeba castellanii

Acanthamoeba castellanii is a free-living amoeba (FLA) and a protist pathogen that has persisted in the environment since the emergence of early unicellular eukaryotic life forms on this planet. Unlike other FLA, Acanthamoeba spp. are the causative agent of Acanthamoeba keratitis (AK), which is a vision-threatening corneal infection difficult to manage with present-day drug therapy. AK in humans has emerged as a challenging ocular infection to treat due to the prolonged treatment regimen and if left untreated, the disease causes blindness. Even in the patients who complete the treatment for over a year, residual corneal damage impacting the vision is seen. Though considered to be rare, epidemics have been recently reported in south-east England with an increased emergence of cases with AK [1]. As Acanthamoeba spp. survive in water and soil as FLA, use of contaminated water to clean contact lens has continued to be a source of acquiring AK. Factors like antimicrobial drug resistance and lack of specific drugs directed against discrete molecular targets in Acanthamoeba are the possible reasons that contribute toward treatment failures in AK.

Investigating the cardinal needs that are vital for the survival and growth of Acanthamoeba spp. is expected to reveal nutrients and minerals that can be depleted to target this protist pathogen. Acanthamoeba spp. have not been studied in greater details for its calcium influx regulating mechanisms and its dependency on calcium–calmodulin interaction. Mitochondrial calcium accumulation has been studied in Acanthamoeba, but a channel system that allows Ca²⁺ to get through the outer mitochondrial membrane in A. castellanii has not been explored previously [2] and the same is the case with our knowledge regarding the movement of this ion across the cell membrane. With the rationale that Ca²⁺ ions play a vital role in Acanthamoeba spp., and the recently reported expression of human-like Ca²⁺ ion-channels and the effects of Ca²⁺-channel blockers [3] in trophozoite forms of keratitis isolate of Acanthamoeba castellanii, we tested chelators of Ca²⁺ ions in trophozoite forms of this protist pathogen. Our intention was to elucidate the role of extracellular Ca²⁺ in the biology of Acanthamoeba spp. The eukaryotic cells have intracellular storage of Ca²⁺ ions but these storage sites get depleted if Ca²⁺ ions are not replenished from extracellular resources. Recent studies have reported that the depletion of cytosolic Ca²⁺ by blocking human-like voltage-gated Ca²⁺ channels and calmodulin in Acanthamoeba castellanii with the drugs loperamide [3], amlodipine and gabapentin [4] exert amoebistatic and amoebicidal effect in low and high doses respectively. However, the effects of Ca²⁺ chelation in extracellular space in A. castellanii have not been tested yet.

Herein, we show the effects of known Ca²⁺ chelating agents, Ethylenediaminetetraacetic acid (EDTA) and pirenoxine eye drop, on growth and viability of the trophozoite forms of Acanthamoeba obtained from a patient with AK. Fura-2 AM staining was done to establish the effects of the Ca²⁺ chelating agents on intracellular levels of Ca²⁺ in trophozoites forms of A. castellanii. We show that both, EDTA and pirenoxine, exert significant antiproliferative effects as compared with their solvent controls PYG (Fig. 1) with the induction of morphological changes within 20 min of exposure to the drugs. This is reflective of an acute cellular Ca²⁺ depletion stress (data not shown).

Fig. 1

Effects of Ethylenediaminetetraacetic acid (EDTA) (A1) and pirenoxine (A2) on the growth and proliferation of A. castellanii trophozoites. EDTA and Pirenoxine in a dose of 10 and 25 µg/ml showed amoebicidal (Trypan blue positive) effects on the trophozoites. A muscarinic receptor agonist, pilocarpine, which after binding on mAChR1 and mAChR3 induces Ca²⁺ influx in eukaryotic cells, failed to rescue the trophozoites exposed to EDTA and pirenoxine

Fura-2 AM staining of EDTA exposed trophozoites showed reduced intracellular staining as compared with that of the controls, reflecting the effect of EDTA on the cytosolic Ca²⁺ (Fig. 2 first-row). Similar results were seen with pirenoxine dissolved in its solvents (Fig. 2 middle-row). Muscarinic receptor agonist pilocarpine, that causes calcium influx upon binding these receptors (Fig. 2 last-row), was unable to revert the effects of EDTA (Fig. 2 last-row) that chelates Ca²⁺ ions in extracellular space.

Fig. 2

×40 images of Fura-2AM stained trophozoites of A. castellanii. Control without EDTA shows Fura-2AM staining which is similar to EDTA 5µg/ml (EDTA-1). Note that, EDTA 10 µg/ml (EDTA-2) shows reduced trophozoite staining. Note the trophozoite turning spherical with EDTA-2. Pirenoxine dissolved in its solvent in a dose of 20 µg/ml (Pirenoxine-1) and 15 µg/ml (Pirenoxine-2) did not affect the intracellular Fura-2 AM staining significantly as compared with when combined with EDTA (EDTA + Perinoxine). Pilocarpine increased the Fura-2AM staining in a dose of 75 µg/ml alone (Pilocarpine), reflecting its Ca²⁺ influx inducing effects. Note, pilocarpine 75 µg/ml was not effective in inducing Ca-influx when given with 5 and 10 µg/ml of EDTA (EDTA-1 + Pilocarpine 75 and EDTA-2 + Pilocarpine 75: last row), respectively

Exploiting the voltage-gated Ca²⁺ channels and calmodulin in unicellular microbes like Acanthamoeba spp. has proven to be effective in killing this microbe [3, 4]. Ca²⁺ ion chelation with EDTA and structural analogues can repurpose them for the treatment of AK and possibly granulomatous amoebic encephalitis. In-depth understanding of the dependencies of this protist pathogen on cardinal nutrients and vital minerals is expected to uncover mechanisms and pathways which can be targeted. In addition, the recently reported use of EDTA in the eye for breaking Ca²⁺ ion mediated adhesions in keratopathy and the use of pirenoxine in prevention of the progression of presbyopia [5, 6] reflect the safety of their use in the human eye.

In Acanthamoeba spp., Ca²⁺ influx, its passage through voltage-gated channels [3], and cardinal cellular events that occur subsequent to the binding of this divalent cation to calmodulin [4] are novel molecular targets that can be exploited to gain therapeutic benefits in AK. By inhibition of the influx of Ca²⁺ in the trophozoites of this protist pathogen, we show the vulnerability of Acanthamoeba spp. to this divalent cation, which could be implemented in human clinical trials to treat AK. To the best of our knowledge, this is the first report of drug-induced chelation of Ca²⁺ ion in trophozoites of A. castellanii that has shown antiproliferative and amoebicidal effects. We advocate that these two agents could be exploited in the management of AK. Firstly, adding to contact lens cleaning liquids may minimise the risk of acquiring AK infection in frequent contact lens users; and secondly, through drug development and translational clinical trials, repurposing of agents like EDTA and pirenoxine as an adjunctive topical therapy for patients with AK may aid in the management of this disease. These measures could have substantial impact on the prevention of long-term corneal damage and sight loss in AK.