Acanthamoeba Castellanii Research Articles

Comprehensive characterization of extracellular vesicles produced by environmental (Neff) and clinical (T4) strains of Acanthamoeba castellanii.

We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of A. castellanii EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for Acanthamoeba-related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two Acanthamoeba castellanii strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of A. castellanii EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of Acanthamoeba-related diseases.

Continuous year-round isolation of giant viruses from brackish shoreline soils

Giant viruses, categorized under Nucleocytoviricota, are believed to exist ubiquitously in natural environments. However, comprehensive reports on isolated giant viruses remain scarce, with limited information available on unrecoverable strains, viral proliferation sites, and natural hosts. Previously, the author highlighted Pandoravirus hades, Pandoravirus persephone, and Mimivirus sp. styx, isolated from brackish water soil, as potential hotspots for giant virus multiplication. This study presents findings from nearly a year of monthly sampling within the same brackish water region after isolating the three aforementioned strains. This report details the recurrent isolation of a wide range of giant viruses. Each month, four soil samples were randomly collected from an approximately 5 × 10 m plot, comprising three soil samples and one water sample containing sediment from the riverbed. Acanthamoeba castellanii was used as a host for virus isolation. These efforts consistently yielded at least one viral species per month, culminating in a total of 55 giant virus isolates. The most frequently isolated species was Mimiviridae (24 isolates), followed by Marseilleviridae (23 isolates), Pandoravirus (6 isolates), and singular isolates of Pithovirus and Cedratvirus. Notably, viruses were not consistently isolated from any of the four samples every month, with certain sites yielding no viruses. Cluster analysis based on isolate numbers revealed that soil samples from May and water and sediment samples from January produced the highest number of viral strains. These findings underscore brackish coastal soil as a significant site for isolating numerous giant viruses, highlighting the non-uniform distribution along coastlines.

Anomalous dynamics of a passive droplet in active turbulence

Motion of a passive deformable object in an active environment serves as a representative of both in-vivo systems such as intracellular particle motion in Acanthamoeba castellanii, or in-vitro systems such as suspension of beads inside dense swarms of Escherichia coli. Theoretical modeling of such systems is challenging due to the requirement of well resolved hydrodynamics which can explore the spatiotemporal correlations around the suspended passive object in the active fluid. We address this critical lack of understanding using coupled hydrodynamic equations for nematic liquid crystals with finite active stress to model the active bath, and a suspended nematic droplet with zero activity. The droplet undergoes deformation fluctuations and its movement shows periods of “runs” and “stays”. At relatively low interfacial tension, the droplet begins to break and mix with the outer active bath. We establish that the motion of the droplet is influenced by the interplay of spatial correlations of the flow and the size of the droplet. The mean square displacement shows a transition from ballistic to normal diffusion which depends on the droplet size. We discuss this transition in relation to spatiotemporal scales associated with velocity correlations of the active bath and the droplet.

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target.

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC50 0.45 μM), VT1598 (0.25 μM), and VT1161 (0.20 μM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

The ecto-3′-nucleotidase activity of Acanthamoeba castellanii trophozoites increases their adhesion to host cells through the generation of extracellular adenosine

Acanthamoeba castellanii, a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein (MBP) is well established as the major factor related to Acanthamoeba pathogenesis. However, additional factors that participate in the adhesion process and protect trophozoites from cytolytic effects caused by host immune responses remain unknown. Ectonucleotidases, including 3′-nucleotidase/nuclease (3′-NT/NU), a bifunctional enzyme that was recently reported in A. castellanii, are frequently related to the establishment of parasitic infections. We verified that trophozoites can hydrolyze 3′-AMP, and this activity is similar to that observed in other protists. The addition of 3′-AMP increases the adhesion of trophozoites to LLC-MK2 epithelial cells, and this stimulation is completely reversed by DTT, an inhibitor of ecto-3′-nucleotidase activity. Lesions in corneal cells caused by AK infection may elevate the extracellular level of 3′-AMP. We believe that ecto-3′-nucleotidase activity can modulate the host immune response, thus facilitating the establishment of parasitic infection. This activity results from the generation of extracellular adenosine, which can bind to purinergic receptors present in host immune cells. Positive feedback may occur in this cascade of events once the ecto-3′-nucleotidase activity of trophozoites is increased by the adhesion of trophozoites to LLC-MK2 cells.

Microeukaryotic communities diversity with a special emphasis on protozoa taxa in an integrated wastewater treatment system

This study developed an integrated wastewater treatment system that combines an upflow anaerobic sludge blanket (UASB), downflow hanging non-woven fabric (DHNW), and anaerobic baffled reactor (ABR) to explore the effect of treatment stages on the diversity of microeukaryotic communities. This study aimed to bridge the knowledge gap regarding the influence of integrated system stages on microeukaryotic community diversity. Through 18S rRNA amplicon sequencing, we identified unique microeukaryotic communities across different stages, with the aerobic phase hosting 35.77% of unique amplicon sequence variants (ASVs). The results of principal component analysis (PCA) and non-multidimensional scale analysis (nMDS) demonstrated the significant influence of wastewater treatment on both environmental factors and the microeukaryotic communities. Ciliophora was notably abundant in the effluent (42.09%) and sludge (17.11%). The aerobic stage was dominated by Ochrophyta, a diverse group of algae instrumental in nutrient removal, such as nitrogen and phosphorus, through biological processes. A redundancy analysis (RDA) revealed a positive correlation between chemical and biochemical oxygen demand and Cryptomycotina, highlighting its potential as a bioindicator for treatment efficacy. The detection of protozoan species, such as Acanthamoeba castellanii and Vermamoeba vermiformis, in the outlet stage poses health risks, whereas Cryptosporidium sp. was found in both the inlet and aerobic stages but not in the outlet. Our study reveals the complex nature of microeukaryotic diversity in the wastewater treatment system and its implications for treatment performance and public health.

Anti-Acanthamoeba metallopharmaceuticals: Amoebicidal activity and synergistic effect of copper(II)coordination compound.

Acanthamoeba spp. emerged as a clinically important pathogen related to amoebic keratitis. It is among the main causes of corneal transplantation and vision loss in ophthalmology. The treatment protocols have a low cure rate, high toxicity, and need for drug combination. Transition metal compounds have shown promising antiprotozoal effects. This study evaluates the amoebicidal activity of copper(II) coordination compounds in combination with chlorhexidine and the cytotoxicity to topical ocular application. These copper(II) coordination compounds were screened against Acanthamoeba castellanii trophozoites (ATCC 50492). The cytotoxicity on rabbit corneal cell line (ATCC-CCL 60) was performed. The compounds showed high amoebicidal potential, with inhibition of trophozoite viability above 80%. The Cp12 and Cp13 compounds showed Minimal Inhibitory Amoebicidal Concentration (MIAC) at 200µM and mean inhibitory concentration (IC50) values lower than 10µM. Against the cysts, Cp12 showed a reduction in viability (48%) in the longest incubation period. A synergistic effect for Cp12 with chlorhexidine was observed. The compounds have a dose-dependent effect against rabbit corneal cells. Compound Cp12 haspotential for future application in developing ophthalmic formulations against Acanthamoeba keratitis and its use in multipurpose solutions is highlighted.

Isolation of viruses, including mollivirus, with the potential to infect Acanthamoeba from a Japanese warm temperate zone.

Acanthamoeba castellanii is infected with diverse nucleocytoplasmic large DNA viruses. Here, we report the co-isolation of 12 viral strains from marine sediments in Uranouchi Inlet, Kochi, Japan. Based on the morphological features revealed by electron microscopy, these isolates were classified into four viral groups including Megamimiviridae, Molliviridae, Pandoraviridae, and Pithoviridae. Genomic analyses indicated that these isolates showed high similarities to the known viral genomes with which they are taxonomically clustered, and their phylogenetic relationships were also supported by core gene similarities. It is noteworthy that Molliviridae was isolated from the marine sediments in the Japanese warm temperate zone because other strains have only been found in the subarctic region. Furthermore, this strain has 19 and 4 strain-specific genes found in Mollivirus sibericum and Mollivirus kamchatka, respectively. This study extends our knowledge about the habitat and genomic diversity of Molliviridae.

Enhanced transport of bacteria along root systems by protists can impact plant health.

Soil protists have been shown to contribute to the structure and function of the rhizosphere in a variety of ways. Protists are key contributors to nutrient cycling through the microbial loop, where biomass is digested by protists and otherwise stored nutrients are returned to the environment. Protists have also been shown to feed on plant pathogenic bacteria and alter root microbiomes in ways that may benefit plants. Recently, a mechanism involving bacterial transport, facilitated by protists, has been hypothesized to contribute to the spatial distribution of bacteria in the rhizosphere. Here, we observe the differential abilities of three soil protists: a ciliate (Colpoda sp.), a flagellate (Cercomonas sp.), and a naked amoeba (Acanthamoeba castellanii) to transport nitrogen-fixing Sinorhizobium meliloti to infectible root tips. Co-inoculation of protists plus S. meliloti resulted in the movement of bacteria, as measured by the presence of nitrogen-fixing nodules, up to 15 cm farther down the root systems when compared to plants inoculated with S. meliloti alone. Co-inoculation of the ciliate, Colpoda sp., with S. meliloti, resulted in shoot weights that were similar to plants that grew in nitrogen-replete potting mix. Colpoda sp.-feeding style and motility likely contributed to their success at transporting bacteria through the rhizosphere. We observed that the addition of protists alone without the co-inoculum of S. meliloti resulted in plants with larger shoot weights than control plants. Follow-up experiments showed that protists plus their associated microbiomes were aiding in plant health, likely through means of nutrient cycling.IMPORTANCEProtists represent a significant portion of the rhizosphere microbiome and have been shown to contribute to plant health, yet they are understudied compared to their bacterial and fungal counterparts. This study elucidates their role in the rhizosphere community and suggests a mechanism by which protists can be used to move bacteria along plant roots. We found that the co-inoculation of protists with nitrogen-fixing beneficial bacteria, Sinorhizobium meliloti, resulted in nodules farther down the roots when compared to plants inoculated with S. meliloti alone, and shoot weights similar to plants that received nitrogen fertilizer. These data illustrate the ability of protists to transport viable bacteria to uninhabited regions of the root system.

A comparative study on the chemical composition, anti-microbial, antiparasitic, and cytotoxic activities of Rhanterium adpressum Coss. & Dur. and Rhanterium suaveolens Desf. essential oils from Algeria

The aim of the present study was to evaluate and compare the chemical composition, antimicrobial, antiparasitic, and cytotoxic properties of the essential oils extracted from Rhanterium adpressum Coss. & Dur. (EORA) and Rhanterium suaveolens Desf. (EORS). The main compounds of EORA were myrcene (18.51%), α-pinene (14.36%), sabinene (13.55%), β-pinene (5.67%), and camphene (5.39%). While camphene (25.06%), α-pinene (13.14%), myrcene (9.74%), sabinene (8.27%), D-limonene (7.86%), and β-pinene (7.34%) constituted the major components of EORS. A potent antibacterial activity was also recorded against Escherichia coli with MIC = 5.72 mg/mL for EORA and MIC = 5.96 mg/mL for EORS. Concerning the antifungal effect, a remarkable efficiency was exerted against the two tested Candida albicans strains (MIC < 5.96 mg/mL) for EORS and against Candida albicans 26 (MIC = 5.72 mg/mL) for EORA. Furthermore, a promising antiparasitic ability was observed against Acanthamoeba castellanii (IC50 = 6.42±0.21 μg/mL) for EORS and against Leishmania amazonensis (IC50=9.66±1.46 μg/mL) for EORA. However, low cytotoxicity was recorded against macrophages J774-A1 (LC50 = 67.68±4.02 μg/mL) only for EORS. Based on the present findings, it may be concluded that the two tested EOs possess significant antimicrobial and antiparasitic activities with minimal cytotoxicity, so they can be used as new sources of antimicrobial and antiparasitic drugs.

Self-assembled micelles loaded with itraconazole as anti-Acanthamoeba nano-formulation.

Acanthamoeba castellanii are opportunistic pathogens known to cause infection of the central nervous system termed: granulomatous amoebic encephalitis, that mostly effects immunocompromised individuals, and a sight threatening keratitis, known as Acanthamoeba keratitis, which mostly affects contact lens wearers. The current treatment available is problematic, and is toxic. Herein, an amphiphilic star polymer with AB2 miktoarms [A = hydrophobic poly(ℇ-Caprolacton) and B = hydrophilic poly (ethylene glycol)] was synthesized by ring opening polymerization and CuI catalyzed azide-alkyne cycloaddition. Characterization by 1H and 13C NMR spectroscopy, size-exclusion chromatography and fluorescence spectroscopy was accomplished. The hydrophobic drug itraconazole (ITZ) was incorporated in self-assembled micellar structure of AB2 miktoarms through co-solvent evaporation. The properties of ITZ loaded (ITZ-PCL-PEG2) and blank micelles (PCL-PEG2) were investigated through zeta sizer, scanning electron microscopy and Fourier-transform infrared spectroscopy. Itraconazole alone (ITZ), polymer (DPB-PCL), empty polymeric micelles (PCL-PEG2) alone, and itraconazole loaded in polymeric micelles (ITZ-PCL-PEG2) were tested for anti-amoebic potential against Acanthamoeba, and the cytotoxicity on human cells were determined. The polymer was able to self-assemble in aqueous conditions and exhibited low value for critical micelle concentration (CMC) 0.05-0.06µg/mL. The maximum entrapment efficiency of ITZ was 68%. Of note, ITZ, DPB, PCL-PEG2 and ITZ-PCL-PEG2 inhibited amoebae trophozoites by 37.34%, 36.30%, 35.77%, and 68.24%, respectively, as compared to controls. Moreover, ITZ-PCL-PEG2 revealed limited cytotoxicity against human keratinocyte cells. These results are indicative that ITZ-PCL-PEG2 micelle show significantly better anti-amoebic effects as compared to ITZ alone and thus shouldbe investigated further in vivo to determine its clinical potential.

(‒)-Epicatechin reveals amoebicidal activity against Acanthamoeba castellanii by activating the programmed cell death pathway

BackgroundAcanthamoeba is an opportunistic pathogen that can cause human infections such as granulomatous amebic encephalitis and acanthamoeba keratitis. However, no specific drug to treat the diseases has been developed. Therefore, the discovery or development of novel drugs for treating Acanthamoeba infections is urgently needed. The anti-protozoan activity of (‒)-epicatechin (EC) has been reported, suggesting it is an attractive anti-protozoal drug candidate. In this study, the amoebicidal activity of EC against A. castellanii was assessed and its mechanism of action was unveiled. MethodsThe amoebicidal activity of EC against A. castellanii trophozoites and the cytotoxicity of EC in HCE-2 and C6 cells were determined with cell viability assay. The underlying amoebicidal mechanism of EC against A. castellanii was analyzed by the apoptosis/necrosis assay, TUNEL assay, mitochondrial dysfunction assay, caspase-3 assay, and quantitative reverse transcription polymerase chain reaction. The cysticidal activity of EC was also investigated. ResultsEC revealed amoebicidal activity against A. castellanii trophozoites with an IC50 of 37.01 ± 3.96 µM, but was not cytotoxic to HCE-2 or C6 cells. EC induced apoptotic events such as increases in DNA fragmentation and intracellular reactive oxygen species production in A. castellanii. EC also caused mitochondrial dysfunction in the amoebae, as evidenced by the loss of mitochondrial membrane potential and reductions in ATP production. Caspase-3 activity, autophagosome formation, and the expression levels of autophagy-related genes were also increased in EC-treated amoebae. EC led to the partial death of cysts and the inhibition of excystation. ConclusionEC revealed promising amoebicidal activity against A. castellanii trophozoites via programmed cell death events. EC could be a candidate drug or supplemental compound for treating Acanthamoeba infections.

Interaction between Acanthamoeba and Staphylococcus.

Free-living amoebae of the genus Acanthamoeba are infected by various bacteria in nature, and thus bacteria can protect themselves from adverse environmental conditions. Contrary to this ameba-bacteria relationship whether Acanthamoeba has antibacterial effects on bacteria is the different aspect of the relationship between these microorganisms. In this study, we investigate various Acanthamoeba strains have antibacterial effects on various Staphylococcus strains. Three environmental Acanthamoeba strains, isolated from various aquatic environments in Turkey, and Acanthamoeba castellanii ATCC 50373 standard strains were used in the study. The antistaphylococcal effect of cell-free supernatant (CFS) obtained from these amoebae against 12 different Staphylococcus bacteria was investigated by colony counting method. In addition, the pathogenicity of the tested Acanthamoeba strains was determined using osmotolerance and thermotolerance tests. CFSs obtained from Acanthamoeba were found to have varying degrees of antistaphylococcal effects on various Staphylococcus strains (0%-100%). It was determined that the CFS of the standard Acanthamoeba strain showed 100% inhibitory effect against one clinical methicillin-resistant Staphylococcus aureus strain (M2). Also, CFS of Ugöl strain showed 99.97% inhibitory effect against one clinical methicillin-sensitive Staphylococcus epidermidis strain (L3). It was determined that all Acanthamoeba isolates had no pathogenic potential. According to the results, it has been observed that Acanthamoeba produces antibacterial substance(s) against Staphylococcus bacteria and that the ameba-bacteria relationship may also result in the detriment of the bacteria. Furthermore, the current study indicates that new and natural antimicrobial agents from Acanthamoeba can be used as an alternative to infections caused by Staphylococcus.

Influence of the Age of Free-Living Amoeba Cysts on Their Vertical Distribution in a Water Column.

Free-living amoebae (FLA) are widely distributed protozoa in both natural and artificial environments such as drinking water. In addition to the ability of all FLA to transport various pathogenic microorganisms, certain species, such as Acanthamoeba spp. or Balamuthia mandrillaris, have intrinsic pathogenic abilities and cause severe cerebral infections. Previous work has shown an enrichment of FLA cysts in biofilm developed in upper levels of Drinking Water Storage Towers (DWSTs), suggesting that differences in densities of FLA cysts may play a role in their unequal distribution in the water column. To evaluate this hypothesis, a model of a water column was created for this study and used to analyze the vertical distribution of cysts of the FLA Acanthamoeba castellanii, Vermamoeba vermiformis, and Balamuthia mandrillaris from 0 to 23 weeks. Interestingly, our data showed that the cysts of both A. castellanii and V. vermiformis were enriched in upper water levels during their aging. However, B. mandrillaris cysts were equally distributed in the water column during the entire study. These results show that, in addition to the role of water level variation in the DWST, some FLA cysts can become less dense during their aging, which contributes to their enrichment in upper water and therefore biofilm levels.

Amoebicidal effect of synthetic indoles against Acanthamoeba spp.: a study of cell death.

Organic and synthetic chemistry plays a crucial role in drug discovery fields. Moreover, chemical modifications of available molecules to enhance their efficacy, selectivity and safety have been considered as an attractive approach for the development of new bioactive agents. Indoles, a versatile group of natural heterocyclic compounds, have been widely used in pharmaceutical industry due to their broad spectrum of activities including antimicrobial, antitumoral and anti-inflammatory among others. Herein, we report the amoebicidal activity of different indole analogs on Acanthamoeba castellanii Neff. Among the 40 tested derivatives, eight molecules were able to inhibit this protistan parasite. The structure-activity relationship (SAR) analysis of their anti-Acanthamoeba activity would suggest that a carboxylation of C-3 position and the incorporation of halogen as chlorine/fluorine would enhance their biological profile, presumably by increasing their lipophilicity and therefore their ability to cross the cell membrane. Fluorescence image base system was used to investigate the effect of indole 6o c-6 on the cytoskeleton network and various programmed cell death features. We were able to highlight that the methyl 6-chloro-1H-indole-3-carboxylate could induce program cell death by the mitochondrial dysfunction.

Could giant viruses be considered as a biotechnological tool for preventing and controlling Acanthamoeba infections?

The aim of the study was to evaluate the efficiency of mimivirus as a potential therapeutic and prophylactic tool against Acanthamoeba castellanii, the etiological agent of Acanthamoeba keratitis, a progressive corneal infection, that is commonly associated with the use of contact lenses and can lead to blindness if not properly treated. Mimivirus particles were tested in different multiplicity of infection, along with commercial multipurpose contact lenses' solutions, aiming to assess their ability to prevent encystment and excystment of A. castellanii. Solutions were evaluated for their amoebicidal potential and cytotoxicity in MDCK cells, as well as their effectiveness in preventing A. castellanii damage in Madin-Darby canine kidney (MDCK) cells. Results indicated that mimivirus was able to inhibit the formation of A. castellanii cysts, even in the presence of Neff encystment solution. Mimivirus also showed greater effectiveness in controlling A. castellanii excystment compared to commercial solutions. Additionally, mimivirus solution was more effective in preventing damage caused by A. castellanii, presented greater amoebicidal activity, and were less cytotoxic to MDCK cells than commercial MPS. Mimivirus demonstrates a greater ability to inhibit A. castellanii encystment and excystment compared to commercial multipurpose contact lens solutions. Additionally, mimivirus is less toxic to MDCK cells than those commercial solutions. New studies utilizing in vivo models will be crucial for confirming safety and efficacy parameters.

Lactase can target cellular differentiation of Acanthamoeba castellanii belonging to the T4 genotype.

The free living Acanthamoeba spp. are ubiquitous amoebae associated with potentially blinding disease known as Acanthamoeba keratitis (AK) and a fatal central nervous system infection granulomatous amoebic encephalitis (GAE). With the inherent ability of cellular differentiation, it can phenotypically transform to a dormant cyst form from an active trophozoite form. Acanthamoeba cysts are highly resistant to therapeutic agents as well as contact lens cleaning solutions. One way to tackle drug resistance against Acanthamoeba is by inhibiting the formation of cysts from trophozoites. The biochemical analysis showed that the major component of Acanthamoeba cyst wall is composed of carbohydrate moieties such as galactose and glucose. The disaccharide of galactose and glucose is lactose. In this study, we analyzed the potential of lactase enzyme to target carbohydrate moieties of cyst walls. Amoebicidal assessment showed that lactase was ineffective against trophozoite of A. castellanii but enhanced amoebicidal effects of chlorhexidine. The lactase enzyme did not show any toxicity against normal human keratinocyte cells (HaCaT) at the tested range. Hence, lactase can be used for further assessment for development of potential therapeutic agents in the management of Acanthamoeba infection as well as formulation of effective contact lens disinfectants.

Oxidase enzyme genes are differentially expressed during Acanthamoeba castellanii encystment.

Acanthamoeba castellanii, a ubiquitous protozoan, is responsible for significant diseases such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. A crucial survival strategy of A. castellanii involves the formation of highly resistant cysts during adverse conditions. This study delves into the cellular processes underpinning encystment, focusing on gene expression changes related to reactive oxygen species (ROS) balance, with a particular emphasis on mitochondrial processes. Our findings reveal a dynamic response within the mitochondria during encystment, with the downregulation of key enzymes involved in oxidative phosphorylation (COX, AOX, and NADHalt) during the initial 48h, followed by their overexpression at 72h. This orchestrated response likely creates a pro-oxidative environment, facilitating encystment. Analysis of other ROS processing enzymes across the cell reveals differential expression patterns. Notably, antioxidant enzymes, such as catalases, glutaredoxins, glutathione S-transferases, peroxiredoxins, and thioredoxins, mirror the mitochondrial trend of downregulation followed by upregulation. Additionally, glycolysis and gluconeogenesis are downregulated during the early stages in order to potentially balance the metabolic requirement of the cyst. Our study underscores the importance of ROS regulation in Acanthamoeba encystment. Understanding these mechanisms offers insights into infection control and identifies potential therapeutic targets. This work contributes to unraveling the complex biology of A. castellanii and may aid in combatting Acanthamoeba-related infections. Further research into ROS and oxidase enzymes is warranted, given the organism's remarkable respiratory versatility.

β-1,3-Glucan recognition by Acanthamoeba castellanii as a putative mechanism of amoeba-fungal interactions.

In this study, we conducted an in-depth analysis to characterize potential Acanthamoeba castellanii (Ac) proteins capable of recognizing fungal β-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface β-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to Ac was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, Histoplasma capsulatum (Hc) G217B, possessing a β-1,3-glucan outer layer, showed significant adhesion to Ac compared to a Hc G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble β-1,3-glucan substantially inhibited this adhesion, indicating the involvement of β-1,3-glucan recognition. Biotinylated β-1,3-glucan-binding proteins from Ac exhibited higher binding to Hc G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the β-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several Ac proteins capable of binding β-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to β-1,3-glucans. These findings underscore the complexity of binding via β-1,3-glucan and suggest the existence of alternative fungal recognition pathways in Ac.IMPORTANCEAcanthamoeba castellanii (Ac) and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of Ac's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of Ac that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of β-1,3-glucan binding by A. castellanii and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for β-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

[Investigation of the Inhibition Effect of Acanthamoeba Cell-Free Supernatants Against Pseudomonas aeruginosa].

Free-living amoebae belonging to the genus Acanthamoeba are microorganisms that live in air, soil and aquatic environments. In humans, they cause infections such as amoebic keratitis, graulamotous amoebic encephalitis that are difficult to treat and can be fatal. In addition, it is known that they contribute to the replication of bacteria and increase their pathogenicity by being a host for various bacteria. However, information on its inhibitory properties against bacteria and its production of antimicrobial agents is very limited. In this context, in this study, it was aimed to investigate whether cell-free supernatants of Acanthamoeba strains have antibacterial effects against Pseudomonas aeruginosa isolates. Four different Acanthamoeba strains (A10, A13, A14, U.GÖL) isolated from aquatic environments in our country were selected and used in the study, P.aeruginosa isolates (PA2, PA3, PA4, PA5) were selected from clinical strains belonging to patients in our country. Acanthamoeba castellanii ATCC 50373 and P.aeruginosa ATCC 27853 were used as standard strains. P.aeruginosa isolates were grown on nutrient agar at 37 °C and Acanthamoeba strains were grown on E.coli spread non-nutrient agar at 30 °C under aerobic conditions. Pepton yeast extract glucose (PYG) medium supplemented with penicillin and streptomycin was used to obtain axenic cultures of Acanthamoeba strains. After the centrifugation of axenic cultures at 3000 rpm for five minutes, Acanthamoeba-cell-free supernatants were obtained by filtering the supernatant part through a sterile filter with a pore diameter of 0.22 µm. The antibacterial activities of these supernatants against P.aeruginosa isolates were determined using the colony counting method. Analysis of each Acanthamoeba-cell-free supernatants was performed according to the GC-MS method. Acanthamoeba-cell-free supernatants were found to have varying degrees of inhibitory effects (3.9-91.5%) against tested P.aeruginosa isolates. It was determined that the cell-free supernatant of A.castellanii ATCC 50373 strain showed the highest antibacterial effect (91.5%) against PA5 isolate. A14 strain showed similar inhibitory effects (89.4%) against the same Pseudomonas isolate. The average inhibitory effect of most of the Acanthamoeba strains of our country was found to be higher than that of the reference strain A.castellanii ATCC 50492. It is thought that the compounds responsible for the anti-Pseudomonas activity of the tested Acanthamoeba strains may be fructose, phosphoric acid, galactose, N-Acetylphenylalanine and glucopyranose determined as major compounds. This is the first study showing the anti-Pseudomonas activity of microorganisms of the genus Acanthamoeba living in the waters of our country. Acanthamoeba, which is widely found in nature, appears to be a good source for new antimicrobial agents.