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1.
J Med Microbiol ; 69(1): 87-95, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31846414

RESUMO

Introduction. Acanthamoeba keratitis is a sight-threatening corneal infection that is commonly reported among contact lens users and those suffering from corneal trauma. The prevalence of Acanthamoeba species or genotypes in causing keratitis infection is not well known.Aim. This study was conducted to identify and genotype Acanthamoeba isolates from keratitis patients, targeting the ribosomal nuclear subunit (Rns) region, and describe the associated clinical presentation and treatment outcome.Methodology. Thirty culture-confirmed patients with Acanthamoeba keratitis, identified in a tertiary eye care centre in South India during the period from December 2016 to December 2018, were included in this study. The data collected from patient records include demographic details, history of illness, mode of trauma, treatment history and follow-up status. The genotype and species were identified based on the Rns sequence and phylogenetic tree analysis.Results. Acanthamoeba culbertsoni was the most predominant keratitis-causing species, followed by Acanthamoeba quina, Acanthamoeba castellanii, Acanthamoeba healyi, Acanthamoeba hatchetti, Acanthamoeba polyphaga and Acanthamoeba stevensoni. Three major genotypes were identified (T4, T11 and T12), with the T4 genotype being the most predominant, with four subclusters, i.e. T4A, T4B, T4D and T4E. This is the first report on corneal infection by the A. stevensoni T11 genotype and the A. healyi T12 genotype. No significant correlation was observed between the clinical outcomes of corneal disease and the genotypes or species.Conclusion. Rns genotyping is very effective in identifying the Acanthamoeba species and genotype in keratitis. Genotyping of Acanthamoeba spp. will help to advance our understanding of genotype-specific pathogenesis and geographical distribution.


Assuntos
Ceratite por Acanthamoeba/patologia , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/classificação , Acanthamoeba castellanii/genética , Genótipo , Filogenia , Acanthamoeba castellanii/isolamento & purificação , Genes de RNAr , Técnicas de Genotipagem , Humanos , Índia , Centros de Atenção Terciária
2.
PLoS Negl Trop Dis ; 13(5): e0007352, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31095564

RESUMO

BACKGROUND: Acanthamoeba castellanii, which causes keratitis and blindness in under-resourced countries, is an emerging pathogen worldwide, because of its association with contact lens use. The wall makes cysts resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. METHODOLOGY/PRINCIPAL FINDINGS: Transmission electron microscopy and structured illumination microscopy (SIM) showed purified cyst walls of A. castellanii retained an outer ectocyst layer, an inner endocyst layer, and conical ostioles that connect them. Mass spectrometry showed candidate cyst wall proteins were dominated by three families of lectins (named here Jonah, Luke, and Leo), which bound well to cellulose and less well to chitin. An abundant Jonah lectin, which has one choice-of-anchor A (CAA) domain, was made early during encystation and localized to the ectocyst layer of cyst walls. An abundant Luke lectin, which has two carbohydrate-binding modules (CBM49), outlined small, flat ostioles in a single-layered primordial wall and localized to the endocyst layer and ostioles of mature walls. An abundant Leo lectin, which has two unique domains with eight Cys residues each (8-Cys), localized to the endocyst layer and ostioles. The Jonah lectin and glycopolymers, to which it binds, were accessible in the ectocyst layer. In contrast, Luke and Leo lectins and the glycopolymers, to which they bind, were mostly inaccessible in the endocyst layer and ostioles. CONCLUSIONS/SIGNIFICANCE: The most abundant A. castellanii cyst wall proteins are three sets of lectins, which have carbohydrate-binding modules that are conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique to Acanthamoebae (8-Cys of Leo). Cyst wall formation is a tightly choreographed event, in which lectins and glycopolymers combine to form a mature wall with a protected endocyst layer. Because of its accessibility in the ectocyst layer, an abundant Jonah lectin is an excellent diagnostic target.


Assuntos
Acanthamoeba castellanii/crescimento & desenvolvimento , Acanthamoeba castellanii/metabolismo , Amebíase/parasitologia , Celulose/metabolismo , Lectinas/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba castellanii/química , Acanthamoeba castellanii/genética , Sequência de Aminoácidos , Humanos , Ceratite/parasitologia , Lectinas/química , Lectinas/genética , Estágios do Ciclo de Vida , Ligação Proteica , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Alinhamento de Sequência
3.
Biochim Biophys Acta Gen Subj ; 1863(3): 521-527, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30578833

RESUMO

BACKGROUND: During standard gene cloning, the recombinant protein appearing in bacteria as the result of expression leakage very often inhibits cell proliferation leading to blocking of the cloning procedure. Although different approaches can reduce transgene basal expression, the recombinant proteins, which even in trace amounts inhibit bacterial growth, can completely prevent the cloning process. METHODS: Working to solve the problem of DNase II-like cDNA cloning, we developed a novel cloning approach. The method is based on separate cloning of the 5' and 3' fragments of target cDNA into a vector in such a way that the short Multiple Cloning Site insertion remaining between both fragments changes the reading frame and prevents translation of mRNA arising as a result of promoter leakage. Subsequently, to get the vector with full, uninterrupted Open Reading Frame, the Multiple Cloning Site insertion is removed by in vitro restriction/ligation reactions, utilizing the unique restriction site present in native cDNA. RESULTS: Using this designed method, we cloned a coding sequence of AcDNase II that is extremely toxic for bacteria cells. Then, we demonstrated the usefulness of the construct prepared in this way for overexpression of AcDNase II in eukaryotic cells. CONCLUSIONS: The designed method allows cloning of toxic protein coding sequences that cannot be cloned by standard methods. GENERAL SIGNIFICANCE: Cloning of cDNAs encoding toxic proteins is still a troublesome problem that hinders the progress of numerous studies. The method described here is a convenient solution to cloning problems that are common in research on toxic proteins.


Assuntos
Clonagem Molecular/métodos , Citotoxinas/genética , Citotoxinas/metabolismo , Proteínas Recombinantes , Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Acanthamoeba castellanii/metabolismo , DNA Complementar/genética , DNA Complementar/metabolismo , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/toxicidade , Transgenes/genética
4.
Parasit Vectors ; 11(1): 604, 2018 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-30477573

RESUMO

BACKGROUND: Acanthamoeba spp. can cause serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis and cutaneous acanthamoebiasis. Cysteine biosynthesis and the L-serine metabolic pathway play important roles in the energy metabolism of Acanthamoeba spp. However, no study has confirmed the functions of cysteine synthase (AcCS) in the cysteine pathway and phosphoglycerate dehydrogenase (AcGDH) or phosphoserine aminotransferase (AcSPAT) in the non-phosphorylation serine metabolic pathway of Acanthamoeba. METHODS: The AcCS, AcGDH and AcSPAT genes were amplified by PCR, and their recombinant proteins were expressed in Escherichia coli. Polyclonal antibodies against the recombinant proteins were prepared in mice and used to determine the subcellular localisation of each native protein by confocal laser scanning microscopy. The enzymatic activity of each recombinant protein was also analysed. Furthermore, each gene expression level was analysed by quantitative PCR after treatment with different concentrations of cysteine or L-serine. RESULTS: The AcCS gene encodes a 382-amino acid protein with a predicted molecular mass of 43.1 kDa and an isoelectric point (pI) of 8.11. The AcGDH gene encodes a 350-amino acid protein with a predicted molecular mass of 39.1 kDa and a pI of 5.51. The AcSPAT gene encodes a 354-amino acid protein with a predicted molecular mass of 38.3 kDa and a pI of 6.26. Recombinant AcCS exhibited a high cysteine synthesis activity using O-acetylserine and Na2S as substrates. Both GDH and SPAT catalysed degradation, rather than synthesis, of serine. Exogenous L-serine or cysteine inhibited the expression of all three enzymes in a time- and dose-dependent manner. CONCLUSIONS: This study demonstrated that AcCS participates in cysteine biosynthesis and serine degradation via the non-phosphorylation serine metabolic pathway, providing a molecular basis for the discovery of novel anti-Acanthamoeba drugs.


Assuntos
Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Cisteína/metabolismo , Redes e Vias Metabólicas/genética , Serina/metabolismo , Acanthamoeba castellanii/efeitos dos fármacos , Acanthamoeba castellanii/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Cisteína/biossíntese , Cisteína/farmacologia , Cisteína Sintase/genética , Cisteína Sintase/imunologia , Cisteína Sintase/metabolismo , Sistemas de Liberação de Medicamentos , Escherichia coli/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Glicólise , Camundongos , Microscopia Confocal , Fosfoglicerato Desidrogenase/genética , Fosfoglicerato Desidrogenase/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Serina/biossíntese , Serina/farmacologia , Desidrogenase do Álcool de Açúcar/genética , Desidrogenase do Álcool de Açúcar/imunologia , Desidrogenase do Álcool de Açúcar/metabolismo , Transaminases/genética , Transaminases/imunologia , Transaminases/metabolismo
5.
Int J Mol Sci ; 19(11)2018 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-30413017

RESUMO

In our previous report, we had shown that the free-living amoeba Acanthamoeba castellanii influenced the abundance, competiveness, and virulence of Mesorhizobium loti NZP2213, the microsymbiont of agriculturally important plants of the genus Lotus. The molecular basis of this phenomenon; however, had not been explored. In the present study, we demonstrated that oatB, the O-acetyltransferase encoding gene located in the lipopolysaccharide (LPS) synthesis cluster of M. loti, was responsible for maintaining the protective capacity of the bacterial cell envelope, necessary for the bacteria to fight environmental stress and survive inside amoeba cells. Using co-culture assays combined with fluorescence and electron microscopy, we showed that an oatB mutant, unlike the parental strain, was efficiently destroyed after rapid internalization by amoebae. Sensitivity and permeability studies of the oatB mutant, together with topography and nanomechanical investigations with the use of atomic force microscopy (AFM), indicated that the incomplete substitution of lipid A-core moieties with O-polysaccharide (O-PS) residues rendered the mutant more sensitive to hydrophobic compounds. Likewise, the truncated LPS moieties, rather than the lack of O-acetyl groups, made the oatB mutant susceptible to the bactericidal mechanisms (nitrosative stress and the action of lytic enzymes) of A. castellanii.


Assuntos
Acanthamoeba castellanii/microbiologia , Acetiltransferases/genética , Proteínas de Bactérias/genética , Mesorhizobium/genética , Acanthamoeba castellanii/genética , Acanthamoeba castellanii/patogenicidade , Parede Celular/microbiologia , Mutação
6.
Parasitol Res ; 117(9): 2957-2962, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29987413

RESUMO

Acanthamoeba has 22 genotypes with the T4 genotype being the main causative agent of amoebic granulomatous encephalitis and keratitis. Because the molecular mechanisms of the immune defenses of neutrophils and macrophages against histoparasites are based on oxidative stress, parasites may rely on their antioxidant systems to preclude immune defenses. Therefore, understanding of the effect of oxidative stress on vital characteristics of Acanthamoeba castellanii (T4 genotype) and the antioxidant defense responses of Acanthamoeba to oxidative status will cast light on immune cell-parasite interactions. Acanthamoeba T4 cells were cultured in RPMI-1640 medium containing different concentrations of hydrogen peroxide (H2O2). The survival of Acanthamoeba was evaluated by MTT assay and the IC50 concentration was calculated. The total antioxidant capacity (TAC) of the parasite was determined by the cupric reducing antioxidant capacity (CUPRAC) method. Malondialdehyde (MDA) as a marker of lipid peroxidation, protein carbonyl content as a measure of oxidized protein, total thiol (-SH) groups present on proteins as a major source of cellular antioxidants, and total oxidant status (TOS) were evaluated by colorimetric methods. The reactive oxygen species level increased markedly after induction of oxidative stress by the treatment of Acanthamoeba T4 with H2O2. Exposure to H2O2 also significantly increased the MDA and protein carbonyl content. The TOS level and total thiol groups also increased in the treated group compared to those in untreated parasites, although the results were not statistically significant. The TAC level was found to be significantly higher in H2O2-treated parasites, confirming that the parasite fosters its total antioxidant capacity to overcome oxidative conditions. This study showed that under oxidative stress, the defense reactions of the parasite are in part mediated by increasing its antioxidant activity, which is important for the survival of the parasite.


Assuntos
Acanthamoeba castellanii/metabolismo , Antioxidantes/metabolismo , Peróxido de Hidrogênio/farmacologia , Estresse Oxidativo/fisiologia , Acanthamoeba castellanii/genética , Biomarcadores , Genótipo , Humanos , Peroxidação de Lipídeos/fisiologia , Malondialdeído/análise , Oxirredução/efeitos dos fármacos , Carbonilação Proteica/fisiologia , Proteólise
7.
Parasitol Res ; 117(7): 2283-2289, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29797083

RESUMO

Pathogenic bacteria share their natural habitat with many other organisms such as animals, plants, insects, parasites and amoeba. Interactions between these organisms influence not only the life style of the host organisms, but also modulate bacterial physiology. Adaptation can include biofilm formation, capsule formation, and production of virulence factors. Although biofilm formation is a dominant mode of bacterial life in environmental settings, its role in host-pathogen interactions is not extensively studied. In this work, we investigated the role of molecular pathways involved in rdar biofilm formation in the interaction of Salmonella typhimurium with the Acanthamoeba castellanii genotype T4. Genes coding for the rdar biofilm activator CsgD, the cellulose synthase BcsA, and curli fimbriae subunits CsgBA were deleted from the genome of S. typhimurium. Assessment of interactions of wild-type and mutant strains of S. typhimurium with A. castellanii revealed that deletion of the cellulose synthase BcsA promoted association and uptake by A. castellanii, whereas the interactions with csgD and csgBA mutants were not changed. Our findings suggest that cellulose synthase BcsA inhibits the capabilities of S. typhimurium to associate with and invade into A. castellanii.


Assuntos
Acanthamoeba castellanii/genética , Acanthamoeba castellanii/microbiologia , Biofilmes/crescimento & desenvolvimento , Glucosiltransferases/genética , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Celulose , Regulação Bacteriana da Expressão Gênica , Genótipo , Interações Microbianas/genética , Interações Microbianas/fisiologia , Salmonella typhimurium/metabolismo , Transativadores/genética
8.
Int J Biol Sci ; 14(3): 306-320, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29559848

RESUMO

Amoebozoans are in many aspects interesting research objects, as they combine features of single-cell organisms with complex signaling and defense systems, comparable to multicellular organisms. Acanthamoeba castellanii is a cosmopolitan species and developed diverged feeding abilities and strong anti-bacterial resistance; Entamoeba histolytica is a parasitic amoeba, who underwent massive gene loss and its genome is almost twice smaller than that of A. castellanii. Nevertheless, both species prosper, demonstrating fitness to their specific environments. Here we compare transcriptomes of A. castellanii and E. histolytica with application of orthologs' search and gene ontology to learn how different life strategies influence genome evolution and restructuring of physiology. A. castellanii demonstrates great metabolic activity and plasticity, while E. histolytica reveals several interesting features in its translational machinery, cytoskeleton, antioxidant protection, and nutritional behavior. In addition, we suggest new features in E. histolytica physiology that may explain its successful colonization of human colon and may facilitate medical research.


Assuntos
Acanthamoeba castellanii/genética , Acanthamoeba castellanii/fisiologia , Adaptação Fisiológica , Hibridização Genômica Comparativa , Entamoeba histolytica/genética , Entamoeba histolytica/fisiologia , Actinas/genética , Expressão Gênica , Genes de Protozoários , Análise de Sequência de RNA , Transcriptoma
9.
Virulence ; 9(1): 818-836, 2018 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-29560793

RESUMO

Acanthamoeba castellanii (Ac) are ubiquitously distributed in nature, and by contaminating medical devices such as heart valves and contact lenses, they cause a broad range of clinical presentations to humans. Although several molecules have been described to play a role in Ac pathogenesis, including parasite host-tissue invasion and escaping of host-defense, little information is available on their mechanisms of secretion. Herein, we describe the molecular components secreted by Ac, under different protein availability conditions to simulate host niches. Ac extracellular vesicles (EVs) were morphologically and biochemically characterized. Dynamic light scattering analysis of Ac EVs identified polydisperse populations, which correlated to electron microscopy measurements. High-performance thin liquid chromatography of Ac EVs identified phospholipids, steryl-esters, sterol and free-fatty acid, the last two also characterized by GC-MS. Secretome composition (EVs and EVs-free supernatants) was also determined and proteins biological functions classified. In peptone-yeast-glucose (PYG) medium, a total of 179 proteins were identified (21 common proteins, 89 exclusive of EVs and 69 in EVs-free supernatant). In glucose alone, 205 proteins were identified (134 in EVs, 14 common and 57 proteins in EVs-free supernatant). From those, stress response, oxidative and protein and amino acid metabolism proteins prevailed. Qualitative differences were observed on carbohydrate metabolism enzymes from Krebs cycle and pentose phosphate shunt. Serine proteases and metalloproteinases predominated. Analysis of the cytotoxicity of Ac EVs (upon uptake) and EVs-free supernatant to epithelial and glioblastoma cells revealed a dose-dependent effect. Therefore, the Ac secretome differs depending on nutrient conditions, and is also likely to vary during infection.


Assuntos
Acanthamoeba castellanii/metabolismo , Amebíase/parasitologia , Vesículas Extracelulares/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Acanthamoeba castellanii/genética , Animais , Linhagem Celular , Vesículas Extracelulares/genética , Homeostase , Humanos , Transporte Proteico , Proteoma/genética , Proteômica , Proteínas de Protozoários/genética , Via Secretória
10.
Future Microbiol ; 13: 195-207, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29322818

RESUMO

AIM: This study intended to trace the evolution of acid-base buffers and water homeostasis in eukaryotes. Acanthamoeba castellanii  was selected as a model unicellular eukaryote for this purpose. Homologies of proteins involved in pH and water regulatory mechanisms at cellular levels were compared between humans and A. castellanii. MATERIALS & METHODS: Amino acid sequence homology, structural homology, 3D modeling and docking prediction were done to show the extent of similarities between carbonic anhydrase 1 (CA1), aquaporin (AQP), band-3 protein and H+ pump. Experimental assays were done with acetazolamide (AZM), brinzolamide and mannitol to observe their effects on the trophozoites of  A. castellanii. RESULTS: The human CA1, AQP, band-3 protein and H+-transport proteins revealed similar proteins in Acanthamoeba. Docking showed the binding of AZM on amoebal AQP-like proteins.  Acanthamoeba showed transient shape changes and encystation at differential doses of brinzolamide, mannitol and AZM.  Conclusion: Water and pH regulating adapter proteins in Acanthamoeba and humans show significant homology, these mechanisms evolved early in the primitive unicellular eukaryotes and have remained conserved in multicellular eukaryotes.


Assuntos
Acanthamoeba castellanii/genética , Acanthamoeba castellanii/fisiologia , Evolução Molecular , Homeostase/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Anidrases Carbônicas/química , Anidrases Carbônicas/genética , Biologia Computacional , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Protozoários/química , Homologia de Sequência de Aminoácidos , Água
11.
Parasitol Res ; 117(1): 265-271, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29218442

RESUMO

Infectious diseases are the leading cause of morbidity and mortality, killing more than 15 million people worldwide. This is despite our advances in antimicrobial chemotherapy and supportive care. Nanoparticles offer a promising technology to enhance drug efficacy and formation of effective vehicles for drug delivery. Here, we conjugated amphotericin B, nystatin (macrocyclic polyenes), and fluconazole (azole) with silver nanoparticles. Silver-conjugated drugs were synthesized successfully and characterized by ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, and atomic force microscopy. Conjugated and unconjugated drugs were tested against Acanthamoeba castellanii belonging to the T4 genotype using amoebicidal assay and host cell cytotoxicity assay. Viability assays revealed that silver nanoparticles conjugated with amphotericin B (Amp-AgNPs) and nystatin (Nys-AgNPs) exhibited significant antiamoebic properties compared with drugs alone or AgNPs alone (P < 0.05) as determined by Trypan blue exclusion assay. In contrast, conjugation of fluconazole with AgNPs had limited effect on its antiamoebic properties. Notably, AgNP-coated drugs inhibited amoebae-mediated host cell cytotoxicity as determined by measuring lactate dehydrogenase release. Overall, here we present the development of a new formulation of more effective antiamoebic agents based on AgNPs coated with drugs that hold promise for future applications.


Assuntos
Amebíase/tratamento farmacológico , Amebicidas/farmacologia , Anfotericina B/farmacologia , Fluconazol/farmacologia , Nanopartículas Metálicas/química , Nistatina/farmacologia , Acanthamoeba castellanii/efeitos dos fármacos , Acanthamoeba castellanii/genética , Amebíase/parasitologia , Amebicidas/química , Anfotericina B/química , Fluconazol/química , Humanos , Microscopia de Força Atômica , Nistatina/química , Prata/química , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier
12.
J Eukaryot Microbiol ; 65(2): 191-199, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-28787535

RESUMO

Acanthamoeba spp. can be parasitic in certain situations and are responsible for serious human infections, including Acanthamoeba keratitis, granulomatous amoebic encephalitis, and cutaneous acanthamoebiasis. We analyzed the fatty acid composition of Acanthamoeba castellanii trophozoites and tested the inhibitory activity of the main fatty acids, oleic acid and arachidonic acid, in vitro. Oleic acid markedly inhibited the growth of A. castellanii, with trophozoite viability of 57.4% at a concentration of 200 µM. Caspase-3 staining and annexin V assays showed that apoptotic death occurred in A. castellanii trophozoites. Quantitative PCR and dot blot analysis showed increased levels of metacaspase and interleukin-1ß converting enzyme, which is also an indication of apoptosis. In contrast, arachidonic acid showed negligible inhibition of growth of A. castellanii trophozoites. Stimulated expression of Atg3, Atg8 and LC3A/B genes and monodansylcadaverine labeling suggested that oleic acid induces apoptosis by triggering autophagy of trophozoites.


Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ácido Oleico/farmacologia , Trofozoítos/efeitos dos fármacos , Acanthamoeba castellanii/genética , Autofagia , Caspase 3/genética
13.
BMC Genomics ; 18(1): 997, 2017 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-29284403

RESUMO

BACKGROUND: The existence of mitochondria-related organelles (MROs) is proposed for eukaryotic organisms. The Amoebozoa includes some organisms that are known to have mitosomes but also organisms that have aerobic mitochondria. However, the mitochondrial protein apparatus of this supergroup remains largely unsampled, except for the mitochondrial outer membrane import complexes studied recently. Therefore, in this study we investigated the mitochondrial inner membrane and intermembrane space complexes, using the available genome and transcriptome sequences. RESULTS: When compared with the canonical cognate complexes described for the yeast Saccharomyces cerevisiae, amoebozoans with aerobic mitochondria, display lower differences in the number of subunits predicted for these complexes than the mitochondrial outer membrane complexes, although the predicted subunits appear to display different levels of diversity in regard to phylogenetic position and isoform numbers. For the putative mitosome-bearing amoebozoans, the number of predicted subunits suggests the complex elimination distinctly more pronounced than in the case of the outer membrane ones. CONCLUSION: The results concern the problem of mitochondrial and mitosome protein import machinery structural variability and the reduction of their complexity within the currently defined supergroup of Amoebozoa. This results are crucial for better understanding of the Amoebozoa taxa of both biomedical and evolutionary importance.


Assuntos
Amebozoários/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Acanthamoeba castellanii/genética , Amebozoários/classificação , Células Cultivadas , Dictyostelium/genética , Perfilação da Expressão Gênica , Genômica , Proteínas de Transporte da Membrana Mitocondrial/classificação , Filogenia , Subunidades Proteicas/genética
14.
Protist ; 168(6): 649-662, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29100111

RESUMO

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.


Assuntos
Acanthamoeba castellanii/genética , Genoma de Protozoário , Proteínas de Protozoários/genética , Trealose/biossíntese , Acanthamoeba castellanii/metabolismo , Redes e Vias Metabólicas , Filogenia , Proteínas de Protozoários/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Protozoário/genética , RNA de Protozoário/metabolismo
15.
Korean J Parasitol ; 55(3): 233-238, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28719947

RESUMO

Pathogenic Naegleria fowleri, Acanthamoeba castellanii, and Acanthamoeba polyphaga, are distributed worldwide. They are causative agents of primary amoebic meningoencephalitis or acanthamoebic keratitis in humans, respectively. Trophozoites encyst in unfavorable environments, such as exhausted food supply and desiccation. Until recently, the method of N. fowleri encystation used solid non-nutrient agar medium supplemented with heat-inactivated Escherichia coli; however, for the amoebic encystment of Acanthamoeba spp., a defined, slightly modified liquid media is used. In this study, in order to generate pure N. fowleri cysts, a liquid encystment medium (buffer 1) modified from Page's amoeba saline was applied for encystation of N. fowleri. N. fowleri cysts were well induced after 24 hr with the above defined liquid encystment medium (buffer 1). This was confirmed by observation of a high expression of differential mRNA of nfa1 and actin genes in trophozoites. Thus, this liquid medium can replace the earlier non-nutrient agar medium for obtaining pure N. fowleri cysts. In addition, for cyst formation of Acanthamoeba spp., buffer 2 (adjusted to pH 9.0) was the more efficient medium. To summarize, these liquid encystment media may be useful for further studies which require axenic and pure amoebic cysts.


Assuntos
Acanthamoeba castellanii/fisiologia , Meios de Cultura , Mimiviridae/fisiologia , Naegleria fowleri/fisiologia , Encistamento de Parasitas , Acanthamoeba castellanii/genética , Tampões (Química) , Meios de Cultura/química , Concentração de Íons de Hidrogênio , Mimiviridae/genética , Naegleria fowleri/genética , RNA Mensageiro , RNA de Protozoário , Cloreto de Sódio
16.
Korean J Parasitol ; 55(2): 109-114, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28506031

RESUMO

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.


Assuntos
Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Encistamento de Parasitas/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/isolamento & purificação , Acanthamoeba castellanii/citologia , Acanthamoeba castellanii/crescimento & desenvolvimento , Citoplasma/genética , Citoplasma/metabolismo , DNA de Protozoário/genética , Expressão Gênica/genética , Fusão Gênica , Proteínas de Fluorescência Verde , Encistamento de Parasitas/fisiologia , Proteína-Arginina N-Metiltransferases/química
17.
Korean J Parasitol ; 55(2): 115-120, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28506032

RESUMO

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1-3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.


Assuntos
Acanthamoeba castellanii/crescimento & desenvolvimento , Acanthamoeba castellanii/genética , Cisteína Proteases/genética , Metilação de DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Expressão Gênica/genética , Encistamento de Parasitas/genética , Acanthamoeba castellanii/enzimologia , Ilhas de CpG/genética , Cisteína Proteases/fisiologia , Epigênese Genética/genética , Metilação , Encistamento de Parasitas/fisiologia , Regiões Promotoras Genéticas/genética , Trofozoítos
18.
J Basic Microbiol ; 57(7): 574-579, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28466971

RESUMO

Here we describe features of apoptosis in unicellular Acanthamoeba castellanii belonging to the T4 genotype. When exposed to apoptosis-inducing compounds such as doxorubicin, A. castellanii trophozoites exhibited cell shrinkage and membrane blebbing as observed microscopically, DNA fragmentation using agarose gel electrophoresis, and phosphatidylserine (PS) externalization using annexin V immunostaining. Overall, these findings suggest the existence of apoptosis in A. castellanii possibly mediated by intrinsic apoptotic cascade. Further research in this field could provide avenues to selectively induce apoptosis in A. castellanii by triggering intrinsic apoptotic cascade.


Assuntos
Acanthamoeba castellanii/citologia , Acanthamoeba castellanii/fisiologia , Apoptose , Acanthamoeba castellanii/efeitos dos fármacos , Acanthamoeba castellanii/genética , Animais , Anexina A5/análise , Fragmentação do DNA , Doxorrubicina/farmacologia , Genótipo , Trofozoítos/efeitos dos fármacos
19.
J Negat Results Biomed ; 16(1): 6, 2017 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-28366172

RESUMO

BACKGROUND: Gold compounds have shown promise in the treatment of non-communicable diseases such as rheumatoid arthritis and cancer, and are considered of value as anti-microbial agents against Gram-negative and Gram-positive bacteria, and have anti-parasitic properties against Schistosoma mansoni, Trypanosoma brucei, Plasmodium falciparum, Leishmania infantinum, Giardia lamblia, and Entamoeba histolytica. They are known to affect enzymatic activities that are required for the cellular respiration processes. METHODS: Anti-amoebic effects of phosphanegold(I) thiolates were tested against clinical isolate of A. castellanii belonging to the T4 genotype by employing viability assays, growth inhibition assays, encystation assays, excystation assays, and zymographic assays. RESULTS: The treatment of A. castellanii with the phosphanegold(I) thiolates tested (i) had no effect on the viability of A. castellanii as determined by Trypan blue exclusion test, (ii) did not affect amoebae growth using PYG growth medium, (iii) did not inhibit cellular differentiation, and (iv) had no effect on the extracellular proteolytic activities of A. castellanii. CONCLUSION: Being free-living amoeba, A. castellanii is a versatile respirator and possesses respiratory mechanisms that adapt to various aerobic and anaerobic environments to avoid toxic threats and adverse conditions. For the first time, our findings showed that A. castellanii exhibits resistance to the toxic effects of gold compounds and could prove to be an attractive model to study mechanisms of metal resistance in eukaryotic cells.


Assuntos
Acanthamoeba castellanii/genética , Compostos de Ouro/farmacologia , Compostos de Sulfidrila/farmacologia , Acanthamoeba castellanii/efeitos dos fármacos , Acanthamoeba castellanii/crescimento & desenvolvimento , Sobrevivência Celular/efeitos dos fármacos , Genótipo , Compostos de Ouro/química , Encistamento de Parasitas/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Compostos de Sulfidrila/química , Trofozoítos/efeitos dos fármacos , Trofozoítos/fisiologia
20.
Sci Rep ; 6: 36448, 2016 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-27805070

RESUMO

Legionella pneumophila is a ubiquitous, pathogenic, Gram-negative bacterium responsible for legionellosis. Like many other amoeba-resistant microorganisms, L. pneumophila resists host clearance and multiplies inside the cell. Through its Dot/Icm type IV secretion system, the bacterium injects more than three hundred effectors that modulate host cell physiology in order to promote its own intracellular replication. Here we report that L. pneumophila prevents proliferation of its natural host Acanthamoeba castellanii. Infected amoebae could not undergo DNA replication and no cell division was observed. The Dot/Icm secretion system was necessary for L. pneumophila to prevent the eukaryotic proliferation. The absence of proliferation was associated with altered amoebal morphology and with a decrease of mRNA transcript levels of CDC2b, a putative regulator of the A. castellanii cell cycle. Complementation of CDC28-deficient Saccharomyces cerevisiae by the CDC2b cDNA was sufficient to restore proliferation of CDC28-deficient S. cerevisiae and suggests for the first time that CDC2b from A. castellanii could be functional and a bona fide cyclin-dependent kinase. Hence, our results reveal that L. pneumophila impairs proliferation of A. castellanii and this effect could involve the cell cycle protein CDC2b.


Assuntos
Acanthamoeba castellanii/microbiologia , Legionella pneumophila/fisiologia , Acanthamoeba castellanii/genética , Acanthamoeba castellanii/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína Quinase CDC28 de Saccharomyces cerevisiae/deficiência , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/classificação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Tamanho Celular , Replicação do DNA , Escherichia coli/fisiologia , Humanos , Microscopia de Vídeo , Mutagênese , Filogenia , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência
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