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Parasites & Vectors Jun 2019Species of Acanthamoeba are facultative pathogens which can cause sight threatening Acanthamoeba keratitis and a rare but deadly brain infection, granulomatous amoebic...
BACKGROUND
Species of Acanthamoeba are facultative pathogens which can cause sight threatening Acanthamoeba keratitis and a rare but deadly brain infection, granulomatous amoebic encephalitis. Due to conversion of Acanthamoeba trophozoites to resistant cyst stage, most drugs are found to be ineffective at preventing recurrence of infection. This study was designed to test the antiacanthamoebic effects of different cobalt nanoparticles (CoNPs) against trophozoites and cysts, as well as parasite-mediated host cell cytotoxicity.
METHODS
Three different varieties of CoNPs were synthesized by utilizing hydrothermal and ultrasonication methods and were thoroughly characterized by X-ray diffraction and field emission scanning electron microscopy. Amoebicidal, encystation, excystation, and host cell cytopathogenicity assays were conducted to study the antiacanthamoebic effects of CoNPs.
RESULTS
The results of the antimicrobial evaluation revealed that cobalt phosphate Co(PO) hexagonal microflakes, and 100 nm large cobalt hydroxide (Co(OH)) nanoflakes showed potent amoebicidal activity at 100 and 10 µg/ml against Acanthamoeba castellanii as compared to granular cobalt oxide (CoO) of size 35-40 nm. Furthermore, encystation and excystation assays also showed consistent inhibition at 100 µg/ml. CoNPs also inhibited amoebae-mediated host cell cytotoxicity as determined by lactate dehydrogenase release without causing significant damage to human cells when treated alone.
CONCLUSIONS
To our knowledge, these findings determined, for the first time, the effects of composition, size and morphology of CoNPs against A. castellanii. Co(PO) hexagonal microflakes showed the most promising antiamoebic effects as compared to Co(OH) nanoflakes and granular CoO. The results reported in the present study hold potential for the development of antiamoebic nanomedicine.
Topics: Acanthamoeba castellanii; Amebiasis; Amebicides; Cells, Cultured; Cobalt; Microscopy, Electron, Scanning; Nanoparticles; Trophozoites
PubMed: 31159839
DOI: 10.1186/s13071-019-3528-2 -
Microbiology Spectrum Dec 2021Acanthamoeba castellanii is a free-living, pathogenic ameba found in the soil and water. It invades the body through ulcerated skin, the nasal passages, and eyes and can...
Acanthamoeba castellanii is a free-living, pathogenic ameba found in the soil and water. It invades the body through ulcerated skin, the nasal passages, and eyes and can cause blinding keratitis and granulomatous encephalitis. However, the mechanisms underlying the opportunistic pathogenesis of A. castellanii remain unclear. In this study, we observed that commensal bacteria significantly reduced the cytotoxicity of the ameba on mammalian cells. This effect occurred in the presence of both Gram-positive and Gram-negative commensals. Additionally, commensals mitigated the disruption of cell junctions. experiments on mouse eyeballs further showed that the commensals protected the corneal epithelial layer. Together, these findings indicate that A. castellanii is pathogenic to individuals with a dysbiosis of the microbiota at infection sites, further highlighting the role of commensals as a natural barrier during parasite invasion. Acanthamoeba castellanii, an opportunistic protozoan widely present in the environment, can cause keratitis and encephalitis in humans. However, only a few reports describe how the ameba acts as an opportunistic pathogen. Our study showed that the normal microbiota interfered with the cytotoxicity of , persevered during invasion, and reduced corneal epithelium peeling in the mouse eyeball model. This suggests that commensals may act as a natural barrier against invasion. In future, individuals who suffer from keratitis should be examined for microbiota absence or dysbiosis to reduce the incidence of infection in clinical settings.
Topics: Acanthamoeba Keratitis; Acanthamoeba castellanii; Animals; Cornea; Epithelium; Female; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; In Vitro Techniques; Male; Mice; Mice, Inbred BALB C; Symbiosis
PubMed: 34935418
DOI: 10.1128/Spectrum.00512-21 -
Experimental Parasitology Dec 2017Acanthamoeba castellanii is a free-living amoeba commonly found in aquatic environment. It feeds on bacteria even if some bacteria resist amoebal digestion. Thus,...
Acanthamoeba castellanii is a free-living amoeba commonly found in aquatic environment. It feeds on bacteria even if some bacteria resist amoebal digestion. Thus, A. castellanii is described as a Trojan horse able to harbor pathogenic bacteria. L. pneumophila is one of the amoeba-resisting bacteria able to avoid host degradation by phagocytosis and to multiply inside the amoeba. When infecting its host, L. pneumophila injects hundreds of effectors via a type IV secretion system that change physiology of the amoeba to its profit. In this study, we assess mobility of A. castellanii upon infection with L. pneumophila. Electron-microscopy analysis of amoebae revealed a reduction of acanthopodia on cells infected with L. pneumophila. Analysis of velocity showed that migration of A. castellanii infected with L. pneumophila was significantly impaired compare to uninfected cells. Taken together, infection with L. pneumophila could prevent formation of cytoplasmic extensions such as acanthopodia with consequences on the shape, adherence and mobility of A. castellanii.
Topics: Acanthamoeba castellanii; Cell Adhesion; Legionella pneumophila; Microscopy, Electron, Scanning; Movement; Phagocytosis; Time-Lapse Imaging; Trophozoites
PubMed: 28778742
DOI: 10.1016/j.exppara.2017.07.013 -
International Journal of Antimicrobial... Feb 2020β-Lactams are well known as the best antibiotics for inhibiting the cross-linking between adjacent polysaccharide chains and peptides in the peptidoglycan layer of...
β-Lactams are well known as the best antibiotics for inhibiting the cross-linking between adjacent polysaccharide chains and peptides in the peptidoglycan layer of bacterial cell walls, causing bacterial cell lysis. There are no reports on the action of and resistance mechanisms to β-lactams in protozoa. Acanthamoeba castellanii is a free-living protozoan pathogen capable of causing blinding keratitis and fatal granulomatous encephalitis. When Acanthamoeba is exposed to harsh conditions, it differentiates into the cyst stage to avoid environmental stresses, such as drug treatment. In this study, it was shown that the mature encystation rate of A. castellanii is decreased by treatment with cefotaxime (CTX) and clavulanic acid (CLA); however, the drugs do not kill the amoeba. We hypothesise that β-lactam antibiotics may disturb synthesis of the double cell wall during the encystation process of Acanthamoeba. Interestingly, CTX is considered a powerful β-lactam, whereas CLA is considered a weak β-lactam but an efficient β-lactamase inhibitor. It was demonstrated that Acanthamoeba expresses β-lactamases to prevent inhibition of the encystation process by β-lactams. To reveal the functions of Acanthamoeba β-lactamases, a recombinant Acanthamoeba β-lactamase was produced in Escherichia coli that conferred resistance to β-lactams such as CTX, cefuroxime, penicillin and meropenem. Consequently, we suggest that Acanthamoeba produces enzymes similar to β-lactamases to avoid interference from the environment. Here we provide a new point of view on an important gene responsible for drug resistance and advocate for the development of more efficient treatment against Acanthamoeba infection.
Topics: Acanthamoeba castellanii; Antiprotozoal Agents; Immunodiffusion; Phylogeny; RNA, Messenger; beta-Lactam Resistance; beta-Lactamases; beta-Lactams
PubMed: 31622653
DOI: 10.1016/j.ijantimicag.2019.10.004 -
Investigative Ophthalmology & Visual... Jul 2018Acanthamoeba keratitis is a well-known intractable corneal infectious disease. We investigated the anti-Acanthamoeba effect of exogenous nitric oxide (NO).
PURPOSE
Acanthamoeba keratitis is a well-known intractable corneal infectious disease. We investigated the anti-Acanthamoeba effect of exogenous nitric oxide (NO).
METHODS
Acanthamoeba castellanii was axenically cultured and exposed to various concentrations of NO donors, such as sodium nitrite, sodium nitroprusside (SNP), and NO-releasing silica nanoparticles (coated in branched polyethylene imine, size:100 nm), for 1 to 7 days (sodium nitrite and SNP: 0, 0.1, 1, 10, 100, and 1000 μM; silica nanoparticles: 0, 6.25, 12.5, 25, 50, and 100 μg/mL). Human corneal epithelial cells (HCECs) were cultured and exposed to sodium nitrite, SNP (0, 0.1, 1, 10, 100, and 1000 μM), and silica nanoparticles for 1, 2, and 3 days.
RESULTS
Sodium nitrite and SNP showed a dose-dependent inhibitory effect on A. castellanii viability. A more prominent inhibitory effect was observed with SNP (less than 10% of organisms survived at 7-day culture with 1000 μM) compared with sodium nitrite. However, more cytotoxicity on HCEC was observed with SNP. NO-releasing silica nanoparticles were successfully internalized into the amoebic cytoplasm and accumulated in large vacuoles. Although blank silica nanoparticles had no inhibitory effect on A. castellanii viability, NO-releasing silica nanoparticles showed a dose-dependent amoebicidal effect. Furthermore, no cystic transformation of A. castellanii was observed under a phase contrast microscope or transmission electron microscope after exogenous NO treatment.
CONCLUSIONS
Our results demonstrated the anti-Acanthamoeba effect of exogenous NO. This finding suggests that NO-releasing drug platforms, including nano-carriers, can be a promising therapeutic strategy for Acanthamoeba keratitis.
Topics: Acanthamoeba castellanii; Animals; Antiprotozoal Agents; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Epithelium, Corneal; Free Radical Scavengers; Humans; Microscopy, Electron, Transmission; Microscopy, Phase-Contrast; Nitric Oxide; Nitric Oxide Donors
PubMed: 29971441
DOI: 10.1167/iovs.18-23786 -
Experimental Parasitology Nov 2020Acanthamoeba castellanii is a protist that has a high predation efficiency for bacteria in a number of monoxenic culture experiments. However, the role of A. castellanii...
Acanthamoeba castellanii is a protist that has a high predation efficiency for bacteria in a number of monoxenic culture experiments. However, the role of A. castellanii in the microbial community is still unknown because of the lack of studies on multiple-species interactions. The aim of this study was to investigate the change of bacterial composition after A. castellanii emerges in a water environment. We added A. castellanii to an environmental water sample and incubated it for two days. Then, we performed 16S ribosomal RNA sequencing techniques to analyze the changes in bacterial composition. In this study, A. castellanii slightly increased the relative abundance of a few opportunistic pathogens, such as Legionella, Roseomonas, and Haemophilus. This result may be related to the training ground hypothesis. On the other hand, the growth of some bacteria was inhibited, such as Cyanobacteria and Firmicutes. Although A. castellanii did not drastically change the whole bacterial community, we surprisingly found the dissolved oxygen concentration was increased after incubation with A. castellanii. We applied environmental water at the laboratory scale to investigate the interactions among A. castellanii, complex microbial communities and the environment. We identified the bacteria that are sensitive to A. castellanii and further found the novel relationship between dissolved oxygen and microbial interaction. Our results helped to clarify the role of A. castellanii in microbial communities.
Topics: Acanthamoeba castellanii; Bdellovibrio; DNA; Legionella; Microbiota; Oxygen; Ponds; RNA, Ribosomal, 16S; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Virulence
PubMed: 32918877
DOI: 10.1016/j.exppara.2020.107985 -
Experimental Parasitology Dec 2017Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They are frequently found in water and soil samples, but they have also been...
Free-living amoebae (FLA) are opportunistic protozoa widely distributed in the environment. They are frequently found in water and soil samples, but they have also been reported to be associated with bacterial human pathogens such as Legionella spp. Campylobacter spp or Vibrio cholerae among others. Including within Vibrio spp. V. harveyi (Johnson and Shunk, 1936) is a bioluminescent marine bacteria which has been found swimming freely in tropical marine waters, being part of the stomach and intestine microflora of marine animals, and as both a primary and opportunistic pathogen of marine animals. Our aim was to study the interactions between Vibrio harveyi and Acanthamoeba castellanii Neff. Firstly, in order to analyze changes in it cultivability, V. harveyi was coincubated with A. castellanii Neff axenic culture and with Acanthamoeba Conditioned Medium (ACM) at different temperatures in aerobic conditions. Interestingly, at 4 °C and 18-20 °C bacteria were still cultivable in marine agar, at 28 °C, in aerobic conditions, but there weren't significant differences comparing with the controls. We also noted an enhanced migration of Acanthamoeba toward V. harveyi on non-nutrient agar plates compared to controls with no bacteria.
Topics: Acanthamoeba castellanii; Anti-Bacterial Agents; Aquaculture; Coculture Techniques; Microbial Sensitivity Tests; Movement; Vibrio
PubMed: 28917709
DOI: 10.1016/j.exppara.2017.09.003 -
The Korean Journal of Parasitology Feb 2021Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular...
Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term 'integral component of membrane' were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.
Topics: Acanthamoeba castellanii; Acetyltransferases; Catalysis; Gene Ontology; Genes, Protozoan; Hydrolases; Legionella pneumophila; Methyltransferases; Oxidoreductases; Sequence Analysis, RNA; Symbiosis; Transcription, Genetic; Transcriptome
PubMed: 33684989
DOI: 10.3347/kjp.2021.59.1.67 -
Parasitology Research Jan 2024Acanthamoeba castellanii, a ubiquitous protozoan, is responsible for significant diseases such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. A...
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.
Topics: Humans; Acanthamoeba castellanii; Reactive Oxygen Species; Catalase; Acanthamoeba Keratitis; Amebiasis; Cysts
PubMed: 38289423
DOI: 10.1007/s00436-024-08138-9 -
Parasites & Vectors Nov 2020Acanthamoeba spp. are free-living amoeba that are ubiquitously distributed in the environment. This study examines pathogenic Acanthamoeba cysteine proteases (AcCPs)...
BACKGROUND
Acanthamoeba spp. are free-living amoeba that are ubiquitously distributed in the environment. This study examines pathogenic Acanthamoeba cysteine proteases (AcCPs) belonging to the cathepsin L-family and explores the mechanism of AcCP3 interaction with host cells.
METHODS
Six AcCP genes were amplified by polymerase chain reaction (PCR). Quantitative real-time PCR was used to analyse the relative mRNA expression of AcCPs during the encystation process and between pre- and post-reactivated trophozoites. To further verify the role of AcCP3 in these processes, AcCP3 recombinant proteins were expressed in Escherichia coli, and the hydrolytic activity of AcCP3 was determined. The influence of the AcCP3 on the hydrolytic activity of trophozoites and the toxicity of trophozoites to human corneal epithelial cells (HCECs) was examined by inhibiting AcCP3 expression using siRNA. Furthermore, the levels of p-Raf and p-Erk were examined in HCECs following coculture with AcCP3 gene knockdown trophozoites by Western blotting.
RESULTS
During encystation, five out of six AcCPs exhibited decreased expression, and only AcCP6 was substantially up-regulated at the mRNA level, indicating that most AcCPs were not directly correlated to encystation. Furthermore, six AcCPs exhibited increased expression level following trophozoite reactivation with HEp-2 cells, particularly AcCP3, indicating that these AcCPs might be virulent factors. After refolding of recombinant AcCP3 protein, the 27 kDa mature protein from the 34 kDa pro-protein hydrolysed host haemoglobin, collagen and albumin and showed high activity in an acidic environment. After AcCP3 knockdown, the hydrolytic activity of trophozoite crude protein against gelatin was decreased, suggesting that these trophozoites had decreased toxicity. Compared with untreated trophozoites or negative control siRNA-treated trophozoites, AcCP3-knockdown trophozoites were less able to penetrate and damage monolayers of HCECs. Western blot analysis showed that the activation levels of the Ras/Raf/Erk/p53 signalling pathways in HCECs decreased after inhibiting the expression of trophozoite AcCP3.
CONCLUSIONS
AcCP6 was correlated to encystation. Furthermore, AcCP3 was a virulent factor in trophozoites and participated in the activation of the Ras/Raf/Erk/p53 signalling pathways of host cells.
Topics: Acanthamoeba castellanii; Cathepsin L; Cysteine Proteases; Gene Expression; HeLa Cells; Host-Parasite Interactions; Humans; Parasite Encystment; Protozoan Proteins; Recombinant Proteins; Sequence Alignment; Trophozoites
PubMed: 33228764
DOI: 10.1186/s13071-020-04474-8