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Biomedicine & Pharmacotherapy =... Feb 2023Free Living Amoeba (FLA) infections caused by Acanthamoeba genus include chronic nervous system diseases such as Granulomatous Amoebic Encephalitis (GAE), or a severe...
Free Living Amoeba (FLA) infections caused by Acanthamoeba genus include chronic nervous system diseases such as Granulomatous Amoebic Encephalitis (GAE), or a severe eye infection known as Acanthamoeba keratitis (AK). Current studies focused on therapy against these diseases are aiming to find novel compounds with amoebicidal activity and low toxicity to human tissues. Brown algae, such as Gongolaria abies-marina (previously known as Cystoseira abies-marina, S.G. Gmelin), presents bioactive molecules of interest, including some with antiprotozoal activity. In this study, six meroterpenoids were isolated and purified from the species Gongolaria abies-marina. Gongolarones A (1), B (2) and C (3) were identified as new compounds. Additionally, cystomexicone B (4), 1'-methoxyamentadione (5) and 6Z-1'-methoxyamentadione (6) were isolated. All compounds exhibited amoebicidal activity against Acanthamoeba castellanii Neff, A. polyphaga and A. griffini strains. Gongolarones A (1) and C (3) showed the lowest IC values against the two stages of these amoebae (trophozoite and cyst). Structure-activity relationship revealed that the cyclization by ether formation from C-12 to C-15 of 1, and the isomerization Δ to Δ of 3, increased the antiamoeboid activity of both compounds. Furthermore, gongolarones A (1) and C (3) triggered chromatin condensation, mitochondrial malfunction, oxidative stress, and disorganization of the tubulin-actin cytoskeleton in treated trophozoites. Moreover, transmission electron microscopy (TEM) images analysis revealed that compounds 1 and 3 induced autophagy process and inhibited the encystation process. All those results suggest that both compounds could induce programmed cell death (PCD) in Acanthamoeba.
Topics: Animals; Humans; Acanthamoeba castellanii; Amebicides; Trophozoites; Actin Cytoskeleton
PubMed: 36916403
DOI: 10.1016/j.biopha.2022.114185 -
Bioengineering (Basel, Switzerland) Feb 2019Extracellular vesicles (EVs) are membranous compartments of distinct cellular origin and biogenesis, displaying different sizes and include exosomes, microvesicles, and... (Review)
Review
Extracellular vesicles (EVs) are membranous compartments of distinct cellular origin and biogenesis, displaying different sizes and include exosomes, microvesicles, and apoptotic bodies. The EVs have been described in almost every living organism, from simple unicellular to higher evolutionary scale multicellular organisms, such as mammals. Several functions have been attributed to these structures, including roles in energy acquisition, cell-to-cell communication, gene expression modulation and pathogenesis. In this review, we described several aspects of the recently characterized EVs of the protozoa a free-living amoeba (FLA) of emerging epidemiological importance, and compare their features to other parasites' EVs. These EVs are comprised of small microvesicles and exosomes and carry a wide range of molecules involved in many biological processes like cell signaling, carbohydrate metabolism and proteolytic activity, such as kinases, glucanases, and proteases, respectively. Several biomedical applications of these EVs have been proposed lately, including their use in vaccination, biofuel production, and the pharmaceutical industry, such as platforms for drug delivery.
PubMed: 30717103
DOI: 10.3390/bioengineering6010013 -
International Journal of Molecular... Aug 2023Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the...
Uncoupling proteins (UCPs) are mitochondrial inner membrane transporters that mediate free-fatty-acid-induced, purine-nucleotide-inhibited proton leak into the mitochondrial matrix, thereby uncoupling respiratory substrate oxidation from ATP synthesis. The aim of this study was to provide functional evidence that the putative gene of the free-living protozoan amoeba, , encodes the mitochondrial protein with uncoupling activity characteristic of UCPs and to investigate its role during oxidative stress. We report the sequencing and cloning of a complete coding sequence, its phylogenetic analysis, and the heterologous expression of AcUCP in the strain Measurements of mitochondrial respiratory activity and membrane potential indicate that the heterologous expression of AcUCP causes AcUCP-mediated uncoupling activity. In addition, in a model of oxidative stress with increased reactive oxygen species levels (superoxide dismutase 1 knockout yeasts), AcUCP expression strongly promotes cell survival and growth. The level of superoxide anion radicals is greatly reduced in the Δ strain expressing AcUCP. These results suggest that AcUCP targeted to yeast mitochondria causes uncoupling and may act as an antioxidant system. Phylogenetic analysis shows that the UCP diverges very early from other UCPs, but clearly locates within the UCP subfamily rather than among other mitochondrial anion carrier proteins.
PubMed: 37569876
DOI: 10.3390/ijms241512501 -
Proceedings of the National Academy of... Aug 2022Predatory protozoa play an essential role in shaping microbial populations. Among these protozoa, are ubiquitous in the soil and aqueous environments inhabited by ....
Predatory protozoa play an essential role in shaping microbial populations. Among these protozoa, are ubiquitous in the soil and aqueous environments inhabited by . Observations of predator-prey interactions between these two microorganisms revealed a predation strategy in which assemble in aggregates, termed backpacks, on their posterior. The rapid formation and specific location of backpacks led to the assumption that may recruit by releasing an attractant. However, this hypothesis has not been validated, and the mechanisms driving this process remained unknown. Here, we combined video microscopy, microfluidics, single-cell image analyses, and theoretical modeling to characterize predator-prey interactions of and and determined whether bacterial chemotaxis contributes to the backpack formation. Our results indicate that captures are not driven by chemotaxis. Instead, random encounters of bacteria with amoebae initialize bacterial capture and aggregation. This is supported by the strong correlation between experimentally derived capture rates and theoretical encounter models at the single-cell level. Observations of the spatial rearrangement of trapped by revealed that bacterial aggregation into backpacks is mainly driven by amoeboid locomotion. Overall, we show that two nonspecific, independent mechanisms, namely random encounters enhanced by bacterial motility and predator surface-bound locomotion, drive backpack formation, resulting in a bacterial aggregate on the amoeba ready for phagocytosis. Due to the prevalence of these two processes in the environment, we expect this strategy to be widespread among amoebae, contributing to their effectiveness as predators.
Topics: Acanthamoeba castellanii; Chemotaxis; Listeria monocytogenes; Locomotion; Microfluidics; Microscopy, Video; Phagocytosis; Single-Cell Analysis
PubMed: 35914149
DOI: 10.1073/pnas.2122659119 -
The Korean Journal of Parasitology Oct 2018Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their...
Acanthamoeba spp. are free-living protozoa that are opportunistic pathogens for humans. Cysteine proteases of Acanthamoeba have been partially characterized, but their biochemical and functional properties are not clearly understood yet. In this study, we isolated a gene encoding cysteine protease of A. castellanii (AcCP) and its biochemical and functional properties were analyzed. Sequence analysis of AcCP suggests that this enzyme is a typical cathepsin L family cysteine protease, which shares similar structural characteristics with other cathepsin L-like enzymes. The recombinant AcCP showed enzymatic activity in acidic conditions with an optimum at pH 4.0. The recombinant enzyme effectively hydrolyzed human proteins including hemoglobin, albumin, immunoglobuins A and G, and fibronectin at acidic pH. AcCP mainly localized in lysosomal compartment and its expression was observed in both trophozoites and cysts. AcCP was also identified in cultured medium of A. castellanii. Considering to lysosomal localization, secretion or release by trophozoites and continuous expression in trophozoites and cysts, the enzyme could be a multifunctional enzyme that plays important biological functions for nutrition, development and pathogenicity of A. castellanii. These results also imply that AcCP can be a promising target for development of chemotherapeutic drug for Acanthamoeba infections.
Topics: Acanthamoeba castellanii; Amino Acid Sequence; Base Sequence; Cysteine Proteases; Hydrogen-Ion Concentration; Lysosomes; Trophozoites
PubMed: 30419726
DOI: 10.3347/kjp.2018.56.5.409 -
Molecular Pharmacology Dec 2020Cytochromes P450 (P450, CYP) metabolize a wide variety of endogenous and exogenous lipophilic molecules, including most drugs. Sterol 14-demethylase (CYP51) is a target...
Cytochromes P450 (P450, CYP) metabolize a wide variety of endogenous and exogenous lipophilic molecules, including most drugs. Sterol 14-demethylase (CYP51) is a target for antifungal drugs known as conazoles. Using X-ray crystallography, we have discovered a domain-swap homodimerization mode in CYP51 from a human pathogen, CYP51 (AcCYP51). Recombinant AcCYP51 with a truncated transmembrane helix was purified as a heterogeneous mixture corresponding to the dimer and monomer units. Spectral analyses of these two populations have shown that the CO-bound ferrous form of the dimeric protein absorbed at 448 nm (catalytically competent form), whereas the monomeric form absorbed at 420 nm (catalytically incompetent form). AcCYP51 dimerized head-to-head via N-termini swapping, resulting in formation of a nonplanar protein-protein interface exceeding 2000 Å with a total solvation energy gain of -35.4 kcal/mol. In the dimer, the protomers faced each other through the F and G -helices, thus blocking the substrate access channel. In the presence of the drugs clotrimazole and isavuconazole, the AcCYP51 drug complexes crystallized as monomers. Although clotrimazole-bound AcCYP51 adopted a typical CYP monomer structure, isavuconazole-bound AcCYP51 failed to refold 74 N-terminal residues. The failure of AcCYP51 to fully refold upon inhibitor binding in vivo would cause an irreversible loss of a structurally aberrant enzyme through proteolytic degradation. This assumption explains the superior potency of isavuconazole against The dimerization mode observed in this work is compatible with membrane association and may be relevant to other members of the CYP family of biologic, medical, and pharmacological importance. SIGNIFICANCE STATEMENT: We investigated the mechanism of action of antifungal drugs in the human pathogen . We discovered that the enzyme target [ sterol 14-demethylase (AcCYP51)] formed a dimer via an N-termini swap, whereas drug-bound AcCYP51 was monomeric. In the AcCYP51-isavuconazole complex, the protein target failed to refold 74 N-terminal residues, suggesting a fundamentally different mechanism of AcCYP51 inactivation than only blocking the active site. Proteolytic degradation of a structurally aberrant enzyme would explain the superior potency of isavuconazole against .
Topics: 14-alpha Demethylase Inhibitors; Acanthamoeba castellanii; Amebiasis; Crystallography, X-Ray; Humans; Molecular Dynamics Simulation; Nitriles; Protein Binding; Protein Domains; Protein Multimerization; Proteolysis; Protozoan Proteins; Pyridines; Recombinant Proteins; Sterol 14-Demethylase; Triazoles
PubMed: 33008918
DOI: 10.1124/molpharm.120.000092 -
Antibiotics (Basel, Switzerland) Nov 2022is a ubiquitous free-living amoeba capable of instigating keratitis and granulomatous amoebic encephalitis in humans. Treatment remains limited and inconsistent....
is a ubiquitous free-living amoeba capable of instigating keratitis and granulomatous amoebic encephalitis in humans. Treatment remains limited and inconsistent. Accordingly, there is a pressing need for novel compounds. Nanotechnology has been gaining attention for enhancing drug delivery and reducing toxicity. Previous work has shown that various antibiotic classes displayed antiamoebic activity. Herein, we employed two antibiotics: ampicillin and ceftriaxone, conjugated with the nanocarrier zinc oxide and β-cyclodextrin, and tested them against via amoebicidal, amoebistatic, encystment, excystment, cytopathogenicity, and cytotoxicity assays at a concentration of 100 μg/mL. Notably, zinc oxide β-cyclodextrin ceftriaxone significantly inhibited growth and cytopathogenicity. Additionally, both zinc oxide β-cyclodextrin ceftriaxone and ceftriaxone markedly inhibited encystment. Furthermore, all the tested compounds displayed negligible cytotoxicity. However, minimal anti-excystment or amoebicidal effects were observed for the compounds. Accordingly, this novel nanoconjugation should be employed in further studies in hope of discovering novel anti- compounds.
PubMed: 36551378
DOI: 10.3390/antibiotics11121721 -
Antimicrobial Agents and Chemotherapy Jun 2022Infection with pathogenic free-living amoebae, including Naegleria fowleri, spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because...
Infection with pathogenic free-living amoebae, including Naegleria fowleri, spp., and Balamuthia mandrillaris, can lead to life-threatening illnesses, primarily because of catastrophic central nervous system involvement. Efficacious treatment options for these infections are lacking, and the mortality rate due to infection is high. Previously, we evaluated the N. fowleri glucokinase (Glck) as a potential target for therapeutic intervention, as glucose metabolism is critical for viability. Here, we extended these studies to the glucokinases from two other pathogenic free-living amoebae, including Acanthamoeba castellanii (Glck) and (Glck). While these enzymes are similar (49.3% identical at the amino acid level), they have distinct kinetic properties that distinguish them from each other. For ATP, Glck and Glck have apparent values of 472.5 and 41.0 μM, while Homo sapiens Glck (Glck) has a value of 310 μM. Both parasite enzymes also have a higher apparent affinity for glucose than the human counterpart, with apparent values of 45.9 μM (Glck) and 124 μM (Glck) compared to ~8 mM for Glck. Additionally, Glck and Glck differ from each other and other Glcks in their sensitivity to small molecule inhibitors, suggesting that inhibitors with pan-amoebic activity could be challenging to generate.
Topics: Acanthamoeba; Amebiasis; Amoeba; Balamuthia mandrillaris; Glucokinase; Humans; Naegleria fowleri
PubMed: 35604214
DOI: 10.1128/aac.02373-21 -
Biomedicine & Pharmacotherapy =... Jun 2022The genus Acanthamoeba is characterized by being a group of ubiquitous and free-living amoebae that inhabit a variety of environments. Generally, human infections by...
The genus Acanthamoeba is characterized by being a group of ubiquitous and free-living amoebae that inhabit a variety of environments. Generally, human infections by this parasite are associated with Acanthamoeba keratitis, especially in contact lens wearers, and with chronic but fatal granulomatous amoebic meningoencephalitis. Current treatments used for eradication of amoeba from infection sites represent a challenge for pharmacotherapy, due to the lack of effective treatment and the amoebae highly resistant to anti-amoebic drugs. In this study, we describe the results of the assessment of the IC of 10 isobenzofuran-1(3H)-one derivatives (QOET) against four Acanthamoeba strains. The compounds QOET-3 and QOET-9 were the selected derivatives with the lowest IC in A. castellanii Neff trophozoites (73.71 ± 0.25 and 69.99 ± 15.32 µM, respectively). Interestingly, analysis of the compound effects on the cell apoptosis-like features showed that both active molecules triggered programmed cell death (PCD) in A. castellanii Neff. The results obtained in this study highlights that isobenzofuranone derivatives could represent an interesting source for developing novel antiamoebic drugs.
Topics: Acanthamoeba Keratitis; Acanthamoeba castellanii; Amebicides; Animals; Cell Death; Humans; Trophozoites
PubMed: 35658232
DOI: 10.1016/j.biopha.2022.113062 -
Biocontrol Science 2022Acanthamoeba castellanii is a ubiquitous organism found in environmental water. The amoeba is pathogenic to toward humans and is also a reservoir of bacteria of the...
Acanthamoeba castellanii is a ubiquitous organism found in environmental water. The amoeba is pathogenic to toward humans and is also a reservoir of bacteria of the genus Legionella, a causative agent of legionellosis. Oakmoss, a source of natural fragrance ingredients, and its components are antibacterial agents that are specifically active against the genus Legionella. In the present study, oakmoss and its components were investigated for their inhibitory effects on total (extra- and intracellular) Legionella pneumophila within A. castellanii and on L. pneumophila within A. castellanii. Among the oakmoss components, 3-hydroxy-5-methylphenyl 2,4-dihydroxy-6-methylbenzoate (1), 3-methoxy-5-methylphenyl 2,4-dihydroxy-6-methylbenzoate (2), and 8-(2,4-dihydroxy-6-(2-oxoheptyl)phenoxy)-6-hydroxy-3-pentyl-1H-isochromen-1-one (8) reduced the number of total bacteria (extra- and intracellular) in a test culture and also exhibited high amoebicidal activity against L. pneumophila within A. castellanii at concentrations lower than their IC values for A. castellanii. In contrast, 6,8-dihydroxy-3-pentyl-1H-isochromen-1-one (5) reduced the total number of L. pneumophila and, also that of total bacteria after 24 h of treatment (P < 0.05), whereas the compound did not exhibit amoebicidal activity against L. pneumophila within A. castellanii at concentrations lower than its IC value against A. castellanii. Thus, it is suggested that these oakmoss components could be good candidates for disinfectants to protect from Legionella infection.
Topics: Acanthamoeba castellanii; Humans; Legionella pneumophila; Resins, Plant; Terpenes
PubMed: 35314557
DOI: 10.4265/bio.27.21