Acanthamoeba castellanii is a widespread unicellular eukaryote found in diverse environments, including tap water, soil, and swimming pools. It is responsible for severe infections, such as Acanthamoeba keratitis and granulomatous amebic encephalitis, particularly in individuals with immunocompromisation. The ability of protozoans to form dormant and persistent cysts complicates treatment, as current therapies are ineffective against cyst stages and suffer from poor specificity and side effects. Nitroxoline, a quinoline derivative with well- established antibacterial, antifungal, and antiviral properties, is a promising therapeutic candidate. This study aimed to elucidate cellular signalling events that counteract the effects of nitroxoline. In this study, nitroxoline significantly reduced the viability of A . castellanii trophozoites in a dose- and time-dependent manner, inducing morphological changes and apoptosis. Transcriptomic analysis revealed substantial alterations in gene expression, including enrichment of metabolic pathways, DNA damage responses, and iron ion binding. Nitroxoline treatment upregulated genes involved in DNA repair and oxidative stress response while regulating genes in the methionine and cysteine cycles. It also decreased the mitochondrial membrane potential, HAS production, and total iron amount in A . castellanii. Bioinformatic analyses and molecular docking studies suggest direct interactions between nitroxoline and several A . castellanii proteins. Our research provides a comprehensive molecular map of the response of A . castellanii to nitroxoline, revealing significant changes in gene expression related to the stress response and metabolic pathways. These findings underscore the potential of nitroxoline as a potent anti- Acanthamoeba agent, offering new insights into its mechanism of action and paving the way for effective combinational therapeutic strategies.

PurposeAcanthamoeba 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.MethodThe 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).ResultsAt 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.ConclusionThe 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.