A long-term prospecting study on giant viruses in terrestrial and marine Brazilian biomes.

The discovery of mimivirus in 2003 prompted the search for novel giant viruses worldwide. Despite increasing interest, the diversity and distribution of giant viruses is barely known. Here, we present data from a 2012-2022 study aimed at prospecting for amoebal viruses in water, soil, mud, and sewage samples across Brazilian biomes, using Acanthamoeba castellanii for isolation. A total of 881 aliquots from 187 samples covering terrestrial and marine Brazilian biomes were processed. Electron microscopy and PCR were used to identify the obtained isolates. Sixty-seven amoebal viruses were isolated, including mimiviruses, marseilleviruses, pandoraviruses, cedratviruses, and yaraviruses. Viruses were isolated from all tested sample types and almost all biomes. In comparison to other similar studies, our work isolated a substantial number of Marseillevirus and cedratvirus representatives. Taken together, our results used a combination of isolation techniques with microscopy, PCR, and sequencing and put highlight on richness of giant virus present in different terrestrial and marine Brazilian biomes.

Combating the causative agent of amoebic keratitis, Acanthamoeba castellanii, using Padina pavonica alcoholic extract: toxicokinetic and molecular docking approaches.

Natural products play a significant role in providing the current demand as antiparasitic agents, which offer an attractive approach for the discovery of novel drugs. The present study aimed to evaluate in vitro the potential impact of seaweed Padina pavonica (P. pavonica) extract in combating Acanthamoeba castellanii (A. castellanii). The phytochemical constituents of the extract were characterized by Gas chromatography-mass spectrometry. Six concentrations of the algal extract were used to evaluate its antiprotozoal activity at various incubation periods. Our results showed that the extract has significant inhibition against trophozoites and cysts viability, with complete inhibition at the high concentrations. The IC50 of P. pavonica extract was 4.56 and 4.89 µg/mL for trophozoites and cysts, respectively, at 24 h. Morphological alterations of A. castellanii trophozoites/cysts treated with the extract were assessed using inverted and scanning electron microscopes and showed severe damage features upon treatment with the extract at different concentrations. Molecular Docking of extracted compounds against Acanthamoeba cytochrome P450 monooxygenase (AcCYP51) was performed using Autodock vina1.5.6. A pharmacokinetic study using SwissADME was also conducted to investigate the potentiality of the identified bioactive compounds from Padina extract to be orally active drug candidates. In conclusion, this study highlights the in vitro amoebicidal activity of P. pavonica extract against A. castellanii adults and cysts and suggests potential AcCYP51 inhibition.

Examining the influence of environmental factors on Acanthamoeba castellanii and Pseudomonas aeruginosa in co-culture.

Exploration of interspecies interactions between microorganisms can have taxonomic, ecological, evolutionary, or medical applications. To better explore interactions between microorganisms it is important to establish the ideal conditions that ensure survival of all species involved. In this study, we sought to identify the ideal biotic and abiotic factors that would result in high co-culture viability of two interkingdom species, Pseudomonas aeruginosa and Acanthamoeba castellanii, two soil dwelling microbes. There have been limited studies showing long-term interactions between these two organisms as co-culture can result in high mortality for one or both organisms suggesting a predator-predator interaction may exist between them. In this study, we identified biotic and abiotic conditions that resulted in a high viability for both organisms in long-term co-culture, including optimizing temperature, nutrient concentration, choice of bacterial strains, and the initial ratio of interacting partners. These two species represent ideal partners for studying microbial interactions because amoebae act similarly to mammalian immune cells in many respects, and this can allow researchers to study host-pathogen interactions in vitro. Therefore, long-term interaction studies between these microbes might reveal the evolutionary steps that occur in bacteria when subjected to intense predation, like what occurs when pathogens enter the human body. The culture conditions characterized here resulted in high viability for both organisms for at least 14-days in co-culture suggesting that long-term experimental studies between these species can be achieved using these culture conditions.

Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis.

BACKGROUND: Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs). METHODS: We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography. RESULTS: We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba. CONCLUSIONS: This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.

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.

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.

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.

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) 0.05-0.06 µg/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 should be investigated further in vivo to determine its clinical potential.

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.

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

BACKGROUND: Acanthamoeba 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. METHODS: The 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. RESULTS: EC 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. CONCLUSION: EC 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.

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.

Phylogenomic, structural, and cell biological analyses reveal that Stenotrophomonas maltophilia replicates in acidified Rab7A-positive vacuoles of Acanthamoeba castellanii.

Acanthamoeba species are clinically relevant free-living amoebae (FLA) ubiquitously found in soil and water bodies. Metabolically active trophozoites graze on diverse microbes via phagocytosis. However, functional studies on Rab GTPases (Rabs), which are critical for controlling vesicle trafficking and maturation, are scarce for this FLA. This knowledge gap can be partly explained by the limited genetic tools available for Acanthamoeba cell biology. Here, we developed plasmids to generate fusions of A. castellanii strain Neff proteins to the N- or C-termini of mEGFP and mCherry2. Phylogenomic and structural analyses of the 11 Neff Rab7 paralogs found in the RefSeq assembly revealed that eight of them had non-canonical sequences. After correcting the gene annotation for the Rab7A ortholog, we generated a line stably expressing an mEGFP-Rab7A fusion, demonstrating its correct localization to acidified macropinocytic and phagocytic vacuoles using fluorescence microscopy live cell imaging (LCI). Direct labeling of live Stenotrophomonas maltophilia ESTM1D_MKCAZ16_6a (Sm18) cells with pHrodo Red, a pH-sensitive dye, demonstrated that they reside within acidified, Rab7A-positive vacuoles. We constructed new mini-Tn7 delivery plasmids and tagged Sm18 with constitutively expressed mScarlet-I. Co-culture experiments of Neff trophozoites with Sm18::mTn7TC1_Pc_mScarlet-I, coupled with LCI and microplate reader assays, demonstrated that Sm18 underwent multiple replication rounds before reaching the extracellular medium via non-lytic exocytosis. We conclude that S. maltophilia belongs to the class of bacteria that can use amoeba as an intracellular replication niche within a Stenotrophomonas-containing vacuole that interacts extensively with the endocytic pathway.IMPORTANCEDiverse Acanthamoeba lineages (genotypes) are of increasing clinical concern, mainly causing amoebic keratitis and granulomatous amebic encephalitis among other infections. S. maltophilia ranks among the top 10 most prevalent multidrug-resistant opportunistic nosocomial pathogens and is a recurrent member of the microbiome hosted by Acanthamoeba and other free-living amoebae. However, little is known about the molecular strategies deployed by Stenotrophomonas for an intracellular lifestyle in amoebae and other professional phagocytes such as macrophages, which allow the bacterium to evade the immune system and the action of antibiotics. Our plasmids and easy-to-use microtiter plate co-culture assays should facilitate investigations into the cellular microbiology of Acanthamoeba interactions with Stenotrophomonas and other opportunistic pathogens, which may ultimately lead to the discovery of new molecular targets and antimicrobial therapies to combat difficult-to-treat infections caused by these ubiquitous microbes.

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.

[Investigation of the Inhibition Effect of Acanthamoeba Cell-Free Supernatants Against Pseudomonas aeruginosa]. / Acanthamoeba Hücresiz Sivilarinin Pseudomonas aeruginosa Karsisinda Inhibe Edici Etkisinin Arastirilmasi.

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.

ß-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 ß-1,3-glucans. Ac specifically anchors curdlan or laminarin, indicating the presence of surface ß-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 ß-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 ß-1,3-glucan substantially inhibited this adhesion, indicating the involvement of ß-1,3-glucan recognition. Biotinylated ß-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 ß-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 ß-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 ß-1,3-glucans. These findings underscore the complexity of binding via ß-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 ß-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 ß-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.

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 48 h, followed by their overexpression at 72 h. 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.

Genetic manipulation of giant viruses and their host, Acanthamoeba castellanii.

Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology, in the spotlight for bioengineering advances. Their host, Acanthamoeba castellanii, is an accidental human pathogen that acts as a natural host and environmental reservoir of other human pathogens. Tools for genetic manipulation of viruses and host were lacking. Here, we provide a detailed method for genetic manipulation of A. castellanii and the GVs it plays host to by using CRISPR-Cas9 or homologous recombination. We detail the steps of vector preparation (4 d), transfection of amoeba cells (1 h), infection (1 h), selection (5 d for viruses, 2 weeks for amoebas) and cloning of recombinant viruses (4 d) or amoebas (2 weeks). This procedure takes ~3 weeks or 1 month for the generation of recombinant viruses or amoebas, respectively. This methodology allows the generation of stable gene modifications, which was not possible by using RNA silencing, the only previously available reverse genetic tool. We also include detailed sample-preparation steps for protein localization by immunofluorescence (4 h), western blotting (4 h), quantification of viral particles by optical density (15 min), calculation of viral lethal dose 50 (7 d) and quantification of DNA replication by quantitative PCR (4 h) to allow efficient broad phenotyping of recombinant organisms. This methodology allows the function of thousands of ORFan genes present in GVs, as well as the complex pathogen-host, pathogen-pathogen or pathogen-symbiont interactions in A. castellanii, to be studied in vivo.

Novel Drug Screening Assay for Acanthamoeba castellanii and the Anti-Amoebic Effect of Carbonic Anhydrase Inhibitors.

Acanthamoeba castellanii is an amoeba that inhabits soil and water in every part of the world. Acanthamoeba infection of the eye causes keratitis and can lead to a loss of vision. Current treatment options are only moderately effective, have multiple harmful side effects, and are tedious. In our study, we developed a novel drug screening method to define the inhibitory properties of potential new drugs against A. castellanii in vitro. We found that the clinically used carbonic anhydrase inhibitors, acetazolamide, ethoxzolamide, and dorzolamide, have promising antiamoebic properties.

Potential anti-amoebic activity of sulfonate- and sulfamate-containing carboxamide derivatives against pathogenic Acanthamoeba castellanii belonging to the genotype T4.

Acanthamoeba are ubiquitously distributed in the environment and can cause infection of the central nervous system as well a sight-threatening eye infection. Herein, the potential anti-amoebic activity of a series of sulfonate/sulfamate derivatives against pathogenic A. castellanii was evaluated. These compounds were tested using several assays namely amoebicidal, adhesion, excystation, cytotoxic, and cytopathogenicity. Amoebicidal assays revealed that the selected compounds reduced amoebae viability significantly (P < 0.05), and exhibited IC50 values at two-digit micromolar concentrations. Sulfamate derivatives 1j & 1k inhibited 50% of amoebae at 30.65 µM and 27.21 µM, respectively. The tested compounds blocked amoebae binding to host cells as well as inhibited amoebae excystation. Notably, the selected derivatives exhibited minimal human cell cytotoxicity but reduced parasite-mediated host cell damage. Overall, our study showed that sulfamate derivatives 1j & 1k have anti-amoebic potential and offer a promising avenue in the development of potential anti-amoebic drug candidates.