Herein, we investigated the anti-amoebic activity of phosphonium-chloride-based deep eutectic solvents against pathogenic Acanthamoeba castellanii of the T4 genotype. Deep eutectic solvents are ionic fluids composed of two or three substances, capable of self-association to form a eutectic mixture with a melting point lower than each substance. In this study, three distinct hydrophobic deep eutectic solvents were formulated, employing trihexyltetradecylphosphonium chloride as the hydrogen bond acceptor and aspirin, dodecanoic acid, and 4-tert-butylbenzoic acid as the hydrogen bond donors. Subsequently, all three deep eutectic solvents, denoted as DES1, DES2, DES3 formulations, underwent investigations comprising amoebicidal, adhesion, excystation, cytotoxicity, and cytopathogenicity assays. The findings revealed that DES2 was the most potent anti-amoebic agent, with a 94% elimination rate against the amoebae within 24 h at 30 °C. Adhesion assays revealed that deep eutectic solvents hindered amoebae adhesion to human brain endothelial cells, with DES2 exhibiting 88% reduction of adhesion. Notably, DES3 exhibited remarkable anti-excystation properties, preventing 94% of cysts from reverting to trophozoites. In cytopathogenicity experiments, deep eutectic solvent formulations and dodecanoic acid alone reduced amoebae-induced human brain endothelial cell death, with DES2 showing the highest effects. Lactate dehydrogenase assays revealed the minimal cytotoxicity of the tested deep eutectic solvents, with the exception of trihexyltetradecylphosphonium chloride, which exhibited 35% endothelial cell damage. These findings underscore the potential of specific deep eutectic solvents in combating pathogenic Acanthamoeba, presenting promising avenues for further research and development against free-living amoebae.
- MeSH
- Acanthamoeba castellanii * účinky léků genetika MeSH
- amébicidy farmakologie chemie MeSH
- buněčná adheze účinky léků MeSH
- endoteliální buňky účinky léků MeSH
- genotyp * MeSH
- lidé MeSH
- organofosforové sloučeniny farmakologie chemie MeSH
- rozpouštědla * chemie farmakologie MeSH
- trofozoiti účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Species of Acanthamoeba Volkonsky, 1931 are the commonest among free-living amoebae that are widespread in different water resources but with lacking phylogenetic data. This study aims at detecting molecular prevalence and genetic diversity of Acanthamoeba isolates in Kafrelsheikh Governorate, Egypt. Forty-eight water samples were collected from 12 swimming pools; four samples during each season over one year. Samples were filtered, cultivated on non-nutrient agar plates and examined microscopically. Polymerase chain reaction (PCR) and sequence analysis of positive samples targeting diagnostic fragment 3 (DF3) of the small subunit rRNA gene were done. Cultivation succeeded to detect 14 (29%) positive samples while PCR missed three positive samples. The obtained sequences were phylogenetically analysed. The phylogenetic tree was constructed for them with sequences of reference species from the NCBI database. The identified species were Acanthamoeba castellanii Douglas, 1930 (T4), A. astronyxis (Ray et Hayes, 1954) (T9) and A. hatchetti Sawyer, Visvesvara et Harke, 1977 (T11). The prevalence of species of Acanthamoeba was higher during summer and fall. Therefore, the control of the presence of Acanthmoeba spp. in swimming pools needs immediate, effective and practical measures to prevent and control infection with species of Acanthamoeba.
- MeSH
- Acanthamoeba * genetika MeSH
- fylogeneze MeSH
- genotyp MeSH
- plavecké bazény * MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Egypt MeSH
Acanthamoeba species are capable of causing amoebic keratitis (AK). As a monotherapy, alpha-mangostin is effective for the treatment of AK; however, its bioavailability is quite poor. Moreover, the efficacy of therapy is contingent on the parasite and virulent strains. To improve readiness against AK, it is necessary to find other derivatives with accurate target identification. Beta-tubulin (BT) has been used as a target for anti-Acanthamoeba (A. keratitis). In this work, therefore, a model of the BT protein of A. keratitis was constructed by homology modeling utilizing the amino acid sequence from NCBI (GenBank: JQ417907.1). Ramachandran Plot was responsible for validating the protein PDB. The verified BT PDB was used for docking with the specified ligand. Based on an improved docking score compared to alpha-mangostin (AM), two modified compounds were identified: 1,6-dihydroxy-7-methoxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (C1) and 1,6-dihydroxy-2,8-bis(3-methylbut-2-en-1-yl)-9H-xanthen-9-one (C2). In addition, molecular dynamics simulations were conducted to analyze the interaction characteristics of the two bound BT-new compound complexes. During simulations, the TRP9, ARG50, VAL52, and GLN122 residues of BT-C1 that align to the identical residues in BT-AM generate consistent hydrogen bond interactions with 0-3 and 0-2. However, the BT-C2 complex has a different binding site, TYR 258, ILE 281, and SER 302, and can form more hydrogen bonds in the range 0-4. Therefore, this study reveals that C1 and C2 inhibit BT as an additive or synergistic effect; however, further in vitro and in vivo studies are needed.
- MeSH
- Acanthamoeba * MeSH
- akantamébová keratitida * parazitologie MeSH
- lidé MeSH
- ligandy MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- tubulin MeSH
- xantony MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- MeSH
- Acanthamoeba fyziologie izolace a purifikace patogenita MeSH
- Campylobacter jejuni * patogenita růst a vývoj MeSH
- interakce hostitele a patogenu MeSH
- kampylobakterové infekce MeSH
- lidé MeSH
- mikrobiální viabilita MeSH
- protozoální infekce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
During environmental stress, the vegetative cells of the facultative pathogenic amoeba Acanthamoeba castellanii reversibly differentiate into resistant dormant stages, namely, cysts or pseudocysts. The type of resistant stage depends on the nature and duration of the stressor. Cell differentiation is accompanied by changes in morphology and cellular metabolism. Moreover, cell differentiation is also expected to be closely linked to the regulation of the cell cycle and, thus, to cellular DNA content. While the existence of the resistant stages in A. castellanii is well known, there is no consensus regarding the relationship between differentiation and cell cycle progression. In the present work, we used flow cytometry analysis to explore the changes in the DNA content during Acanthamoeba encystation and pseudocyst formation. Our results strongly indicate that A. castellanii enters encystation from the G2 phase of the cell cycle. In contrast, differentiation into pseudocysts can begin in the G1 and G2 phases. In addition, we present a phylogenetic analysis and classification of the main cell cycle regulators, namely, cyclin-dependent kinases and cyclins that are found in the genome of A. castellanii.
- MeSH
- Acanthamoeba castellanii klasifikace genetika MeSH
- buněčná diferenciace genetika MeSH
- fylogeneze MeSH
- fyziologický stres genetika MeSH
- proteiny buněčného cyklu genetika MeSH
- protozoální DNA analýza MeSH
- průtoková cytometrie MeSH
- stadia vývoje genetika MeSH
- Publikační typ
- časopisecké články MeSH
Acanthamoeba is known to interact with a plethora of microorganisms such as bacteria, fungi and viruses. In these interactions, the amoebae can be predatory in nature, transmission vehicle or an incubator. Amoebae consume microorganisms, especially bacteria, as food source to fulfil their nutritional needs by taking up bacteria through phagocytosis and lysing them in phagolysosomes and hence play an eminent role in the regulation of bacterial density in the nature and accountable for eradication of around 60% of the bacterial population in the environment. Acanthamoeba can also act as a "Trojan horse" for microbial transmission in the environment. Additionally, Acanthamoeba may serve as an incubator-like reservoir for microorganisms, including those that are pathogenic to humans, where the microorganisms use amoebae's defences to resist harsh environment and evade host defences and drugs, whilst growing in numbers inside the amoebae. Furthermore, amoebae can also be used as a "genetic melting pot" where exchange of genes as well as adaptation of microorganisms, leading to higher pathogenicity, may arise. Here, we describe bacteria, fungi and viruses that are known to interact with Acanthamoeba spp.
Acanthamoebae success as human pathogens is largely due to the highly resistant cysts which represent a crucial problem in treatment of Acanthamoeba infections. Hence, the study of cyst wall composition and encystment play an important role in finding new therapeutic strategies. For the first time, we detected high activity of cytoskeletal elements - microtubular networks and filamentous actin, in late phases of encystment. Cellulose fibrils - the main components of endocyst were demonstrated in inter-cystic space, and finally in the ectocyst, hereby proving the presence of cellulose in both layers of the cyst wall. We detected clustering of intramembranous particles (IMPs) and their density alterations in cytoplasmic membrane during encystment. We propose a hypothesis that in the phase of endocyst formation, the IMP clusters represent cellulose microfibril terminal complexes involved in cellulose synthesis that after cyst wall completion are reduced. Cyst wall impermeability, due largely to a complex polysaccharide (glycans, mainly cellulose) has been shown to be responsible for Acanthamoeba biocide resistance and cellulose biosynthesis pathway is suggested to be a potential target in treatment of Acanthamoeba infections. Disruption of this pathway would affect the synthesis of cyst wall and reduce considerably the resistance to chemotherapeutic agents.
- MeSH
- Acanthamoeba izolace a purifikace metabolismus ultrastruktura MeSH
- amébiáza parazitologie MeSH
- buněčná membrána metabolismus ultrastruktura MeSH
- buněčná stěna metabolismus ultrastruktura MeSH
- celulosa metabolismus MeSH
- cytoskelet metabolismus ultrastruktura MeSH
- elektronová mikroskopie MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- mikrotubuly metabolismus ultrastruktura MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Kontaktní čočky jsou v dnešní době široce rozšířenou a hojně používanou korekční pomůckou. Pro jejich pohodlné a bezpečné nošení je ovšem nutné o čočky správně pečovat a dodržovat určité zásady, které pomáhají bránit rozvinutí možných infekčních i neinfekčních komplikací. Pro péči o kontaktní čočky používáme různé roztoky, jejichž výběr je nutné přizpůsobit individuálním potřebám nositele.
Contact lenses are very common corrective aid that is nowadays frequently used. It is important to take care of the contact lensesproperly for their comfortable and safe wearing as to follow certain principles that help to prevent infectious and non‑infectiouscomplications. For care of the contact lenses we use different solutions and it is necessary to adapt their choice to the individualuser's needs.
Arbuscular mycorrhizal (AM) fungi can significantly contribute to plant nitrogen (N) uptake from complex organic sources, most likely in concert with activity of soil saprotrophs and other microbes releasing and transforming the N bound in organic forms. Here, we tested whether AM fungus (Rhizophagus irregularis) extraradical hyphal networks showed any preferences towards certain forms of organic N (chitin of fungal or crustacean origin, DNA, clover biomass, or albumin) administered in spatially discrete patches, and how the presence of AM fungal hyphae affected other microbes. By direct 15N labeling, we also quantified the flux of N to the plants (Andropogon gerardii) through the AM fungal hyphae from fungal chitin and from clover biomass. The AM fungal hyphae colonized patches supplemented with organic N sources significantly more than those receiving only mineral nutrients, organic carbon in form of cellulose, or nothing. Mycorrhizal plants grew 6.4-fold larger and accumulated, on average, 20.3-fold more 15N originating from the labeled organic sources than their nonmycorrhizal counterparts. Whereas the abundance of microbes (bacteria, fungi, or Acanthamoeba sp.) in the different patches was primarily driven by patch quality, we noted a consistent suppression of the microbial abundances by the presence of AM fungal hyphae. This suppression was particularly strong for ammonia oxidizing bacteria. Our results indicate that AM fungi successfully competed with the other microbes for free ammonium ions and suggest an important role for the notoriously understudied soil protists to play in recycling organic N from soil to plants via AM fungal hyphae.
- MeSH
- Acanthamoeba metabolismus MeSH
- amoniak metabolismus MeSH
- Andropogon růst a vývoj metabolismus mikrobiologie MeSH
- Bacteria metabolismus MeSH
- dusík metabolismus MeSH
- hyfy metabolismus MeSH
- mykorhiza metabolismus MeSH
- organické látky metabolismus MeSH
- oxidace-redukce MeSH
- Publikační typ
- časopisecké články MeSH
The non-reducing disaccharide trehalose can serve as a protectant against a range of environmental stressors, such as heat, cold, or dehydration, in both prokaryotes and eukaryotes, with the exception of vertebrates. Here, we analyzed trehalose metabolism in the facultatively parasitic organism Acanthamoeba castellanii, known to respond to unfavorable external conditions by forming two resistant stages: a cyst, produced in the case of chronic stress, and a pseudocyst, formed in reaction to acute stress. The possible role of trehalose in the resistant stages was investigated using a combination of bioinformatic, molecular biological and biochemical approaches. Genes for enzymes from a widespread trehalose-6-synthase-trehalose-6-phosphate phosphatase (TPS-TPP) pathway and a prokaryotic trehalose synthase (TreS) pathway were identified. The expression patterns of the genes during encystation and pseudocyst formation were analyzed and correlated with the time course of cellular trehalose content determined mass spectrometrically. The data clearly demonstrate fundamental differences between encystation and pseudocyst formation at the level of cellular metabolism.
- MeSH
- Acanthamoeba castellanii genetika metabolismus MeSH
- fylogeneze MeSH
- genom protozoální * MeSH
- messenger RNA genetika metabolismus MeSH
- metabolické sítě a dráhy MeSH
- protozoální proteiny genetika metabolismus MeSH
- RNA protozoální genetika metabolismus MeSH
- trehalosa biosyntéza MeSH
- Publikační typ
- časopisecké články MeSH
