Expression Analysis of Cyst Specific Protein (CSP21) and Cellulose Synthase II (CSII) Genes in Acanthamoeba castellanii Trophozoites Exposed to Silver Nanoparticles Conjugated with Elaeagnus umbellata

Abstract

Purpose: Acanthamoeba species are eucaryotic protozoa found predominantly in soil and water. They cause ulceration and vision loss in the cornea (Acanthamoeba keratitis) and central nervous system (CNS) infection involving the lungs (granulomatous amoebic encephalitis). Antiparasitic drugs currently used in the treatment of infections caused by Acanthamoeba species are not effective at the desired level in some anatomical regions such as the eye and CNS. The existence of an agent effective against both cysts and trophozoites has not yet been proven. Drugs used for treatment of Acanthamoeba infrections are still limited.

Method: The present study investigates amoebicidal activites of various concentrations of ethanolic fruit extract of E. umbellata (EU) (40, 20, 10, 5, 2.5, 1.25, 0.625 mM/mL), silver nanoparticles (AgNP) that are synthesized from EU and confirmed with characterization tests (20, 10, 5, 1, 0.5 mM/mL), and lauric acid (LA) in EU detected with gas chromatography-mass spectrometry (GC-MS) against A. castellanii trophozoites. In addition, DNA-preserving activities of EU, AgNP and LA were studied on pBR322 plasmid DNA, following damage induced with hydroxyl radical (-OH). Cytotoxicity of EU over HeLa cells was examined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Furthermore, the effects over the expression of SOD and CAT genes, which are coding oxidative stress enzymes in trophozoites, and expression of genes responsible for pseudocyst and cyst formation (CSII and CSP21, respectively) were investigated following methanol-induced stress, with reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR).

Results: At highest concentrations, EU, AgNP and LA showed lethal effects against majority of trophozites at 24 th h and against all trophozoites at 48th hour. EU at 5 mg/mL concentration and LA at 1, 0.8, 0.6, 0.4 mM/mL concentrations prevented DNA damage. A dose-dependent decrease in cell viability was observed, EU was found to be non-cytotoxic for 53.82% of HeLa cells at 72 nd h even at 40 mg/mL concentration. Greatest inhibitory effects were found with EU, AgNP and LA on CSII, EU on CAT, LA on CSP21, and hydrogen peroxide (H₂O₂) on SOD genes.

Conclusion: The findings of this study show that EU, LA and AgNPs can be used in a controlled manner to combat A. castellanii infections by reducing or blocking the activity of the parasite's antioxidant enzymes (SOD and CAT), without giving the parasite a chance to initiate the process of pseudocyst or proper cyst formation.

Keywords: Acanthamoeba castellanii; Elaeagnus umbellata; Catalase (CAT); Cellulose synthase II (CSII); Cyst specific protein (CSP21); Silver nanoparticle (AgNP); Superoxide dismutase (SOD).