Expression analysis of cyst-specific protein (CSP21) and cellulose synthase II (CSII) genes i̇n acanthamoeba castellanii trophozoites exposed to silver nanoparticles conjugated with Elaeagnus umbellata

Acanthamoeba species, which are free-living parasites, cause corneal ulceration and vision loss (Acanthamoeba keratitis) and Central Nervous System (CNS) infection (Granulomatous amoebic encephalitis) involving the lungs. Antiparasitic drugs currently used in the treatment of infections caused by Acanthamoeba species are not as effective as desired in some anatomical regions such as the eye and CNS. The existence of an effective agent against both cysts and trophozoites has not yet been proven. In this study, ethanolic fruit extract (EU) of Elaeagnus umbellata Thunb., silver nanoparticles (AgNPs) synthesized by green method using this extract and confirmed by characterization tests (UV-Vis, EDX, SEM, FTIR), and Lauric Acid (LA), which was detected in E. umbellata extract by Gas Chromatography-Mass Spectrometry (GS MS), were investigated for their percentage (%) viability effects on A. castellanii trophozoites. Under different conditions, the expression of Superoxide dismutase (SOD) and Catalase (CAT) genes encoding oxidative stress enzymes of A. castellanii and Cellulose synthase (CSII) and Cyst specific protein (CSP21) genes responsible for pseudocyst and cyst formation of the parasite, respectively, were analyzed by Reverse-Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR). DNA protective activity of EU, LA and AgNPs was tested on pBR322 plasmid DNA. In addition, the MTT Cytotoxicity assay of EU was performed on HeLa cell line. E. umbellata extract had a lethal effect on A. castellanii trophozoites at a concentration of 40mg/ml at the end of 24 h. at the same concentration, no viable trophozoites were found after 48 h. The approximate IC50 values of EU at 72, 48 and 24 hours were 0.9, 1.09 and 1.15mg/ml, respectively. The highest viability was measured at 0.625mg/ml and the lowest viability was measured at 40mg/ml. The highest viability was measured at the end of 24th hour and the lowest at the end of 72nd hour. The % viability results of all extract concentrations on A. castellanii trophozoites were tested by Principal Component Analysis (PCA). According to PCA analysis, there was a negative correlation between the doses at 40, 20, 10, 5 and 2.5mg/ml and the control, 1.25 and 0.625mg/ml doses. After the synthesized AgNPs were confirmed by characterization tests, their amoebicidal activity on A. castellanii trophozoites was investigated. AgNPs at a concentration of 20mM/ml showed a lethal effect on most of the trophozoites at the end of 24 hours, no viable trophozoites were found after 48 hours at the same concentration and the approximate IC50 values on A. castellanii trophozoites at 72, 48 and 24 hours were found to be 0.65, 0.8 and 1mM/ml, respectively. The highest viability was measured at 0.5mM/ml and the lowest at 20mM/ml. According to PCA results of all AgNP concentrations, doses at 20, 10, 5, 1mM/ml, control and 0.5mM/ml were negatively correlated. In this study, the amoebicidal activity of LA on A. castellanii trophozoites was investigated. It was determined that LA at a concentration of 1mM/ml had a lethal effect on most of the trophozoites at the end of 24 hours and no viable trophozoites were found after 48 hours at the same concentration. The approximate IC50 values on A. castellanii trophozoites at 72, 48 and 24 hours were 0.19, 0.25 and 0.34mM/ml, respectively. According to the PCA results of all LA concentrations, it was determined that there was a negative correlation between the doses at 1, 0.8, 0.6 and 0.4mM/ml and the control and 0.2mM/ml doses. In the present study, the activity of EU, LA and AgNPs in inhibiting Hydroxyl radical (-OH) induced DNA damage was examined with pBR322 plasmid DNA. It was found that 5mg/ml concentration of EU and all concentrations of LA (1, 0.8, 0.6, 0.4mM/ml) had effects on the inhibition of DNA damage induced by -OH. All concentrations of AgNPs (20, 10, 5, 1, 0.5mM/ml) were found to have no effect on the inhibition of DNA damage. In the study, the amoebicidal activity of LA on A. castellanii trophozoites was examined. It was determined that LA had a lethal effect on the majority of trophozoites at the end of the 24th hour at a concentration of 1mM/ml, and no viable trophozoites were found after the 48th hour at the same concentration. Approximate IC50 values on A. castellanii trophozoites at the 72nd, 48th and 24th hours were found to be 0.19, 0.25 and 0.34mM/ml, respectively. According to the PCA results of all LA concentrations, it was determined that there was a negative correlation between the doses at 1, 0.8, 0.6 and 0.4mM/ml and the control and 0.2mM/ml doses. In the study, the cytotoxic effect of EU, ethanolic fruit extract at different concentrations (40, 20, 10, 5, 2.5, 1.25, 0.625, 0.312mg/ml) on mammalian cells was evaluated by MTT analysis using the HeLa cell line. It has been determined that EU reduces cell viability depending on the dose. However, at the end of the 72nd hour, it was determined that it did not have a cytotoxic effect on more than half of the cells (53.82%) even at the highest concentration (40mg/ml) and it is thought that the IC50 value may be found at doses higher than 40mg/ml. In the study, A. castellanii trophozoites exposed to stress with methanol and EU, AgNPs and LA were treated separately and investigated in comparative studies with the positive control (PK) exposed to stress only with methanol and the control (K) not exposed to any substance. Accordingly, EU showed the most inhibitory effect on the expression of the CSII gene with 1.88±0.94, while the maximum inhibition was measured as 1.65±0.87 at the 30th minute. According to the results of PCA analysis, K, EU, AgNPs show a negative correlation between LA and PK. When the expression data were evaluated in all conditions and applied periods, the expression at the 30th minute was accepted as the threshold value. While LA had the most inhibitory effect on the expression of the CSP21 gene with 2.64±0.73, the maximum inhibition was measured as 2.56±1.05 at the 24th hour. According to the results of PCA analysis; EU shows a negative correlation between AgNP's, LA and PK. When expression data were evaluated under all conditions and applied periods, the expression at the 24th hour was accepted as the threshold value. When examined in terms of expression of CAT, the gene responsible for antioxidant activity in A. castellanii trophozoites, when compared with 3-AT, which is the specific inhibitor of PK, K and CAT, EU showed the highest inhibitory effect among the groups with 2.25±0.83, the highest inhibition was measured as 2.33±0.90 in the 1st hour. According to the results of PCA analysis, a negative correlation was detected between K, EU, LA, 3-AT and PK, AgNPs. In all conditions and applied times, a negative correlation was observed between the expression values of the CAT gene at the 30th minute, 1st and 12th hour, and the expression values at the 2nd, 24th and 48th hour. When examined in terms of expression of the other gene responsible for antioxidant activity, SOD, when compared with H2O2, which is the specific inhibitor of PK, K and SOD, H2O2 showed the most inhibitory effect among the groups with 1.86±0.63, while the highest inhibition was measured as 1.68±0.76 at the 30th minute. A significant negative correlation was observed on the expression of the SOD gene in all conditions and applied periods in terms of 30th minute, 1st and 48th hour expression values and 2nd, 12th and 24th hour expression values. The existence of an effective treatment against both cysts and trophozoites of Acanthamoeba species has not yet been proven. It was concluded that EU, AgNPs and LA can be used as potential candidates for alternative or combined treatment methods in the treatment of diseases caused by Acanthamoeba.