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Acanthamoeba is a free-living protist pathogen, capable of causing a blinding keratitis and fatal granulomatous encephalitis. The factors that contribute to Acanthamoeba infections include parasite biology, genetic diversity, environmental spread and host susceptibility, and are highlighted together with potential therapeutic and preventative measures. The use of Acanthamoeba in the study of cellular differentiation mechanisms, motility and phagocytosis, bacterial pathogenesis and evolutionary processes makes it an attractive model organism. There is a significant emphasis on Acanthamoeba as a Trojan horse of other microbes including viral, bacterial, protists and yeast pathogens.

Abstract Acanthamoeba is an opportunistic protozoan that is widely distributed in the environment and is well recognized to produce serious human infections, including a blinding keratitis and a fatal encephalitis. This review presents our current understanding of the burden of Acanthamoeba infections on human health, their pathogenesis and pathophysiology, and molecular mechanisms associated with the disease, as well as virulence traits of Acanthamoeba that may be targets for therapeutic interventions and/or the development of preventative measures.

Free-living amoeba (FLA) is widely distributed in the natural environment. Since these amoebae are widely found in various waters, they pose an important public health problem. The aim of this study was to detect the presence of Acanthamoeba , B . mandrillaris , and N . fowleri in various water resources by qPCR in Izmir, Turkey. A total of (n = 27) 18.24% Acanthamoeba and (n = 4) 2.7% N . fowleri positives were detected in six different water sources using qPCR with ITS regions (ITS1) specific primers. The resulting concentrations varied in various water samples for Acanthamoeba in the range of 3.2x10 5 -1.4x10 2 plasmid copies/l and for N . fowleri in the range of 8x10 3 -11x10 2 plasmid copies/l. The highest concentration of Acanthamoeba and N . fowleri was found in seawater and damp samples respectively. All 27 Acanthamoeba isolates were identified in genotype level based on the 18S rRNA gene as T4 (51.85%), T5 (22.22%), T2 (14.81%) and T15 (11.11%). The four positive N . fowleri isolate was confirmed by sequencing the ITS1, ITS2 and 5.8S rRNA regions using specific primers. Four N . fowleri isolates were genotyped (three isolate as type 2 and one isolate as type 5) and detected for the first time from water sources in Turkey. Acanthamoeba and N . fowleri genotypes found in many natural environments are straightly related to human populations to have pathogenic potentials that may pose a risk to human health. Public health professionals should raise awareness on this issue, and public awareness education should be provided by the assistance of civil authorities. To the best of our knowledge, this is the first study on the quantitative detection and distribution of Acanthamoeba and N . fowleri genotypes in various water sources in Turkey.

Acanthamoeba , a genus that contains at least 24 species of free-living protozoa, is ubiquitous in nature. Successful treatment of Acanthamoeba infections is always very difficult and not always effective. More effective drugs must be developed, and medicinal plants may have a pivotal part in the future of drug discovery. Our research focused on investigating the in vitro anti- acanthamoebic potential of Leea indica and its constituent gallic acid in different concentrations. Water and butanol fractions exhibited significant amoebicidal activity against trophozoites and cysts. Gallic acid (100 µg/mL) revealed 83% inhibition of trophozoites and 69% inhibition of cysts. The butanol fraction induced apoptosis in trophozoites, which was observed using tunnel assay. The cytotoxicity of the fractions and gallic acid was investigated against MRC-5 and no adverse effects were observed. Gallic acid was successfully loaded within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with 82.86% encapsulation efficiency, while gallic acid showed 98.24% in vitro release at 48 hours. Moreover, the gallic acid encapsulated in the PLGA nanoparticles exhibited 90% inhibition against trophozoites. In addition, gallic acid encapsulated nanoparticles showed reduced cytotoxicity towards MRC-5 compared to gallic acid, which evidenced that natural product nanoencapsulation in polymeric nanoparticles could play an important role in the delivery of natural products.

Background Acanthamoeba species are free-living protists widely distributed in natural and artificial environments, including freshwater, soil, and water-associated facilities. Some isolates can cause opportunistic human infections, such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. The life cycle of Acanthamoeba includes an active trophozoite stage and a dormant cyst stage. The cyst is surrounded by a complex double-layered wall composed of cellulose and multiple structural proteins, including cyst wall lectins. While the architecture and molecular composition of the cyst wall have been investigated, inter-isolate sequence variation of cyst wall components remains poorly characterized. Methods We performed a genome-based comparative analysis of 31 previously characterized cyst wall lectins, analyzing the homologous sequence of each lectin across 31 publicly available Acanthamoeba genomes. Isolates were classified as clinical or environmental on the basis of their reported sources. Sequence similarity-based clustering was conducted to identify lectins associated with clinical isolates. For selected genes, sequence features at the 5′ region were further examined and experimentally validated by polymerase chain reaction using long-term axenic clinical isolates maintained at National Cheng Kung University Hospital. Results Similarity-based clustering identified eight cyst wall lectins that formed clusters enriched for clinical isolates. Among these, three lectins showed pronounced sequence variation at the 5′ region in multiple clinical isolate genomes. Polymerase chain reaction (PCR) validation confirmed that these variations were present in clinical isolates and were not attributable to genome assembly artifacts. Sequence analysis suggested that these variations may affect signal peptide features at the amino terminus of the proteins. Conclusions Our findings reveal previously underexplored sequence diversity in cyst wall lectins across Acanthamoeba isolates. This diversity may contribute to differences in cyst wall architecture between clinical and environmental isolates. These results provide new insight into cyst-associated molecular features that may be linked to pathogenic potential and offer a foundation for future studies on cyst-mediated adaptation and infection. Graphical abstract

Free-living amoebae of the genus Acanthamoeba are causative agents of keratitis and amoebic encephalitis. They are widely found in various ecological environments. Therefore, the present study brings results that can help to better understand the genotypes of the environmental isolates and their pathogenicity. This study procured 26 Acanthamoeba isolates from three recreational lakes in 2022. Polymerase chain reaction amplification was performed on positive Acanthamoeba samples. The thermotolerance, osmotolerance, and cytopathogenicity in human keratinocyte (HaCaT) cells of the samples were also evaluated. The phylogenetic analysis demonstrated that 12 isolates were of genotype T4, two (T9), six (T17), four (T8), and one each from T5 and T11. The thermo- and osmotolerance assays indicated that eight Acanthamoeba samples were potentially pathogenic. Two T4 and one T9 genotype also recorded 33-, 42-, and 133-kDa serine-type proteases, respectively. The HaCaT cell monolayer revealed that three T4 and one T9 samples achieved cytopathic effects within the 50–100% range, hence significantly cytotoxic. The lactate dehydrogenase secretion results demonstrated that three (T4) and one (T9) sample exhibited exceptional toxicity (over 40%) compared to the other samples. The responses of Acanthamoeba members with similar genotypes to pathogenicity indicator assays varied considerably, rendering correlation of pathogenicity with specific genotypes challenging.

The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. Entamoeba histolytica, Naegleria fowleri and Acanthamoeba spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.

Free-living amoebae (FLA) are prevalent in diverse environments, representing various genera and species with different pathogenicity. FLA-induced infections, such as the highly fatal amoebic encephalitis, with a mortality rate of 99%, primarily affect immunocompromised individuals while others such as Acanthamoeba keratitis (AK) and cutaneous amebiasis may affect immunocompetent individuals. Despite the prevalence of FLA, there is a lack of standardized guidelines for their detection near human habitats. To date, no studies on the isolation and identification of FLA in environmental soil samples in Warsaw have been published. The aim of this study was to determine the presence of amoebae in soil samples collected from Warsaw parks and squares frequented by humans. The isolated protozoa were genotyped. Additionally, their pathogenic potential was determined through thermophilicity tests. A total of 23 soil samples were seeded on non-nutrient agar plates (NNA) at 26 °C and monitored daily for FLA presence. From the total of 23 samples, 18 were positive for FLA growth in NNA and PCR (78.2%). Acanthamoeba spp. was the most frequently isolated genus, with a total of 13 positive samples (13/18; 72.2%), and the T4 genotype being the most common. Moreover, Platyamoeba placida (3/18; 16.7%), Stenamoeba berchidia (1/18; 5.6%) and Allovahlkampfia sp. (1/18; 5.6%), also potentially pathogenic amoebae, were isolated. To our knowledge, this is the first report of FLA presence and characterization in the Warsaw area.

Systemic infection with Acanthamoeba spp. can induce inflammatory responses within the visual axis, yet the underlying molecular mechanisms in the optic nerve remain poorly understood. The aim of the study was to determine the gene expression of Nlrp3 (encoding NOD-, LRP- and pyrin domain-containing protein 3, NLRP3), Ptgs2 (encoding cyclooxygenase-2, COX-2), Rela (encoding nuclear factor kappa B, NF-κB), and several cytokines in the optic nerve of mice during disseminated infection with Acanthamoeba sp. (T16 genotype) under various immunological conditions. In immunocompetent mice, Ptgs2 and Ifng expressions were upregulated at the beginning of infection. In the late stages, we found increased levels of Il10 and Nlrp3. In immunosuppressed mice, higher expressions of Nlrp3, Ptgs2, Rela, Il1b, Il10, Il17a, Il21, and Ifng were found in the infected mice compared to the control group. These results indicate that immunosuppression promotes prolonged inflammation by altering innate and adaptive immune responses, contributing to sustained neuroinflammatory processes affecting the optic nerve. This study provides mechanistic insight into host–pathogen interactions in the optic nerve during systemic Acanthamoeba infection. Due to the analysis being based on mRNA expression levels, direct inference regarding protein levels and the actual activity of the investigated immunological pathways is limited.

Acanthamoeba are a free-living protozoan whose pathogenic strain can cause severe human diseases, such as granulomatous encephalitis and keratitis. As such, the pathogenic mechanism between humans and Acanthamoeba is still unknown. In our previous study, we identified the secreted Acanthamoeba M28 aminopeptidase (M28AP) and then suggested that M28AP can degrade human C3b and iC3b for inhibiting the destruction of Acanthamoeba spp. with the human immune response. We constructed the produced the recombinant M28AP from a CHO cell, which is a mammalian expression system, to characterize the biochemical properties of Acanthamoeba M28AP. The recombinant M28AP more rapidly hydrolyzed Leu-AMC than Arg-AMC and could be inhibited by EDTA treatment. We show that recombinant M28AP can be delivered into the individual cell line and cause cell line apoptosis in a co-culture model. In conclusion, we successfully investigated the potential molecular characteristics of M28AP.