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Acta Tropica Nov 2020Acanthamoeba spp. are free living amoeba (FLA) which are widely distributed in nature. They are opportunistic parasites and can cause severe infections to the eye, skin... (Review)
Review
Acanthamoeba spp. are free living amoeba (FLA) which are widely distributed in nature. They are opportunistic parasites and can cause severe infections to the eye, skin and central nervous system. The advances in drug discovery and modifications in the chemotherapeutic agents have shown little improvement in morbidity and mortality rates associated with Acanthamoeba infections. The mechanism-based process of drug discovery depends on the molecular drug targets present in the signaling pathways in the genome. Synthetic libraries provide a platform for broad spectrum of activities due to their desired structural modifications. Azoles, originally a class of synthetic anti-fungal drugs, disrupt the fungal cell membrane by inhibiting the biosynthesis of ergosterol through the inhibition of cytochrome P450 dependent 14α-lanosterol, a key step of the sterol pathway. Acanthamoeba and fungi share the presence of similar sterol intermediate, as ergosterol is also the major end-product in the sterol biosynthesis in Acanthamoeba. Sterols present in the eukaryotic cell membrane are one of the most essential lipids and exhibit important structural and signaling functions. Therefore, in this review we highlight the importance of specific targeting of ergosterol present in Acanthamoebic membrane by azole compounds for amoebicidal activity. Previously, azoles have also been repurposed to report antimicrobial, antiparasitic and antibacterial properties. Moreover, by loading the azoles into nanoparticles through advanced techniques in nanotechnology, such as physical encapsulation, adsorption, or chemical conjugation, the pharmacokinetics and therapeutic index of the drugs can be significantly improved. The current review proposes an important strategy to target Acanthamoeba using synthetic libraries of azoles and their conjugated nanoparticles for the first time.
Topics: Acanthamoeba; Antifungal Agents; Azoles; Ergosterol; Humans; Nanoparticles
PubMed: 32628912
DOI: 10.1016/j.actatropica.2020.105618 -
Genes Aug 2019The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed... (Review)
Review
The epithelium represents the first and most extensive line of defence against pathogens, toxins and pollutant agents in humans. In general, pathogens have developed strategies to overcome this barrier and use it as an entrance to the organism. , and spp. are amoebae mainly responsible for intestinal dysentery, meningoencephalitis and keratitis, respectively. These amoebae cause significant morbidity and mortality rates. Thus, the identification, characterization and validation of molecules participating in host-parasite interactions can provide attractive targets to timely intervene disease progress. In this work, we present a compendium of the parasite adhesins, lectins, proteases, hydrolases, kinases, and others, that participate in key pathogenic events. Special focus is made for the analysis of assorted molecules and mechanisms involved in the interaction of the parasites with epithelial surface receptors, changes in epithelial junctional markers, implications on the barrier function, among others. This review allows the assessment of initial host-pathogen interaction, to correlate it to the potential of parasite invasion.
Topics: Acanthamoeba; Animals; Entamoeba histolytica; Epithelial Cells; Host-Parasite Interactions; Humans; Naegleria fowleri; Protozoan Infections
PubMed: 31416298
DOI: 10.3390/genes10080618 -
PloS One 2022Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms...
Contact lens usage has contributed to increased incidence rates of Acanthamoeba keratitis (AK), a serious corneal infection that can lead to blindness. Since symptoms associated with AK closely resemble those incurred by bacterial or fungal keratitis, developing a diagnostic method enabling rapid detection with a high degree of Acanthamoeba-specificity would be beneficial. Here, we produced a polyclonal antibody targeting the carboxylesterase (CE) superfamily protein secreted by the pathogenic Acanthamoeba and evaluated its diagnostic potential. Western blot analysis revealed that the CE antibody specifically interacts with the cell lysates and conditioned media of pathogenic Acanthamoeba, which were not observed from the cell lysates and conditioned media of human corneal epithelial (HCE) cells, Fusarium solani, Staphylococcus aureus, and Pseudomonas aeruginosa. High titers of A. castellanii-specific antibody production were confirmed sera of immunized mice via ELISA, and these antibodies were capable of detecting A. castellanii from the cell lysates and their conditioned media. The specificity of the CE antibody was further confirmed on A. castellanii trophozoites and cysts co-cultured with HCE cells, F. solani, S. aureus, and P. aeruginosa using immunocytochemistry. Additionally, the CE antibody produced in this study successfully interacted with 7 different Acanthamoeba species. Our findings demonstrate that the polyclonal CE antibody specifically detects multiple species belong to the genus Acanthamoeba, thus highlighting its potential as AK diagnostic tool.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Animals; Antibodies, Protozoan; Antibody Specificity; Carboxylesterase; Cell Line; Cells, Cultured; Contact Lenses; Culture Media, Conditioned; Early Diagnosis; Epithelial Cells; Epithelium, Corneal; Humans; Immunization; Male; Mice; Protozoan Proteins
PubMed: 34986189
DOI: 10.1371/journal.pone.0262223 -
Investigative Ophthalmology & Visual... Jan 2022To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii.
PURPOSE
To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii.
METHODS
A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration.
RESULTS
The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL.
CONCLUSIONS
pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.
Topics: Acanthamoeba Keratitis; Acanthamoeba castellanii; Amebicides; Animals; Cells, Cultured; Contact Lens Solutions; Cornea; Disease Models, Animal; Epithelium, Corneal; Eye Infections, Parasitic; Humans; Hydrogels; Microscopy, Fluorescence; Polylysine; Swine; Trophozoites
PubMed: 34994769
DOI: 10.1167/iovs.63.1.11 -
Experimental Parasitology Dec 2023The free-living protozoan Acanthamoeba can cause severe keratitis known as Acanthamoeba Keratitis (AK) and granulomatous amoebic encephalitis (GAE). The pathogenesis of...
INTRODUCTION
The free-living protozoan Acanthamoeba can cause severe keratitis known as Acanthamoeba Keratitis (AK) and granulomatous amoebic encephalitis (GAE). The pathogenesis of Acanthamoeba includes intricate interactions between the organism and the host's immune system. The downstream analysis of a well-annotated genome assembly along with proteomic analysis can unravel several biological processes and aid in the identification of potential genes involved in pathogenicity.
METHODS
Based on the next-generation sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein were selected as probable pathogenic targets that were validated by conventional PCR in a total of 30 Acanthamoeba isolates. This was followed by real-time PCR for the evaluation of relative gene expression in the keratitis and amoebic encephalitis animal model induced using keratitis (CHA5), encephalitis (CHA24) and non-pathogenic environmental isolate (CHA36). In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate before and after treatment with polyhexamethylene biguanide (PHMB).
RESULTS
The conventional PCR demonstrated the successful amplification of lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein genes in clinical and environmental isolates. The expression analysis revealed phospholipase, lysophospholipase, and mannose-binding genes to be significantly upregulated in the keratitis isolate (CHA 5) during AK in the animal model. In the case of the amoebic encephalitis model, phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase were significantly upregulated in the encephalitis isolate compared to the keratitis isolate. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates in the pre and post-treatment with PHMB.
CONCLUSION
The gene expression data suggests that lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) could play a role in the contact-dependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.
Topics: Animals; Acanthamoeba; Mannose-Binding Lectin; Lysophospholipase; Chromatography, Liquid; Proteomics; Tandem Mass Spectrometry; Acanthamoeba Keratitis; Amebiasis; Encephalitis; Real-Time Polymerase Chain Reaction; Gene Expression; Peptide Hydrolases
PubMed: 37820893
DOI: 10.1016/j.exppara.2023.108630 -
Indian Journal of Medical Microbiology 2021Acanthamoeba is increasingly implicated in causing keratitis in patients wearing contact lens or ocular trauma and has a poor prognosis. Establishment of an animal model...
CONTEXT
Acanthamoeba is increasingly implicated in causing keratitis in patients wearing contact lens or ocular trauma and has a poor prognosis. Establishment of an animal model is critical to study the disease pathology, pathogenesis and to evaluate anti-amoebic drugs. Some studies have used contact lenses to establish Acanthamoeba keratitis (AK) in a mouse model, which is expensive and not very successful as lenses get dislodged.
OBJECTIVE
To assess the feasibility of using parafilm (Bemis Company Inc., USA) as an alternative to contact lens for the establishment of AK in the mouse model.
METHODS
Thirty-six Balb/c mice in three groups of six mice each for two strains of Acanthamoeba were used to induce AK. Three experimental approaches used were; i) Acanthamoeba impregnated contact lens, ii) Acanthamoeba impregnated parafilm and iii) scratching followed by inoculation of Acanthamoeba suspension. In all three models, tarsorrhaphy was performed. Infection was evaluated by clinical examination and also through microscopic examination of corneal scrapings and corneal sections.
RESULTS
AK model was successfully established with parafilm whereas only one mouse developed AK with the use of contact lens and none with scratching and Acanthamoeba inoculation.
CONCLUSION
The use of parafilm is convenient, reliable and cheaper and can be considered an alternative to contact lenses to induce AK in a mouse model.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Animals; Contact Lenses; Disease Models, Animal; Humans; Mice; Mice, Inbred BALB C; Paraffin
PubMed: 33508396
DOI: 10.1016/j.ijmmb.2021.01.005 -
Parasitology Research Oct 2020The evolutionary history of Acanthamoeba has been substantially resolved by the 18S rDNA phylogeny which made it possible to delimit the main lines associated with some...
The evolutionary history of Acanthamoeba has been substantially resolved by the 18S rDNA phylogeny which made it possible to delimit the main lines associated with some classical species. Some of them have proven to be polyphyletic, but the inappropriate use of treating under the same names unrelated strains persists. In this study, phylogenies based on the complete genes of nuclear and mitochondrial rDNA were compared, in order to verify the congruence of the different lines. Various groups can thus be identified, some of which associated with the type strains of given species. Recognizing them only by their species names would significantly reduce the current confusion, in addition to logically following basic taxonomic rules. In this manner, the well-known polyphyletic taxa A. castellanii and A. polyphaga, are restricted to the two lines specified by their type strains, while other widely used strains like Neff and Linc-AP1 that are often confused with the previous ones, can be assigned to their own lines. New species are potentially present in other groups and additional efforts are needed to delimit them.
Topics: Acanthamoeba; Animals; DNA, Mitochondrial; DNA, Ribosomal; Genes, Protozoan; Genotype; Phylogeny
PubMed: 32789533
DOI: 10.1007/s00436-020-06843-9 -
Acta Parasitologica Sep 2022This study was aimed to investigate the presence of pathogenic free-living amoebae (FLA) in suspected cases of meningoencephalitis with unknown causes of death in Turkey.
Genotyping and Molecular Identification of Acanthamoeba Genotype T4 and Naegleria fowleri from Cerebrospinal Fluid Samples of Patients in Turkey: Is it the Pathogens of Unknown Causes of Death?
PURPOSE
This study was aimed to investigate the presence of pathogenic free-living amoebae (FLA) in suspected cases of meningoencephalitis with unknown causes of death in Turkey.
METHOD
A total of 92 patients, who were diagnosed as meningoencephalitis, were enrolled. All cerebrospinal fluid (CSF) samples were directly microscopically examined and cultured. Acanthamoeba, N. fowleri and B. mandrillaris were further investigated using molecular diagnostic tools including real-time PCR, sequencing, and phylogenetic analyses.
RESULTS
The examined CSF samples were not found positive for the presence of FLA by microscopic examination and culture method. However, two CSF samples were detected positive by real-time PCR assay. Of the positive CSF samples, one was identified as Acanthamoeba genotype T4 and the second positive sample was identified as N. fowleri belonging to genotype II. Furthermore, the pathogens diagnoses was verified through Sanger sequencing.
CONCLUSION
This study was significant to report the presence of Acanthamoeba genotype T4 and N. fowleri genotype II in CSF samples by real-time PCR assay. The present study shows the significance of primary amoebic meningoencephalitis (PAM) and granulomatous amoebic encephalitis (GAE) as one of the differential diagnoses to be considered by clinicians during the evaluation of suspected meningoencephalitis or cases of unknown cause in Turkey. Using real-time PCR, this has made the rapid detection, in a short time-frame, of Acanthamoeba and N. fowleri in CSF samples from patients. The problems with qPCR is that it is not available in every laboratory, reagents are expensive, and it requires skilled and expert personnel to set up these assays.
Topics: Acanthamoeba; Amebiasis; Amoeba; Cause of Death; Genotype; Humans; Meningoencephalitis; Naegleria fowleri; Phylogeny; Turkey
PubMed: 35864411
DOI: 10.1007/s11686-022-00597-3 -
Frontiers in Microbiology 2022keratitis is often caused when contaminate contact lenses and infect the cornea. is pervasive in the environment as a motile, foraging trophozoite or...
INTRODUCTION
keratitis is often caused when contaminate contact lenses and infect the cornea. is pervasive in the environment as a motile, foraging trophozoite or biocide-resistant and persistent cyst. As contact lens contamination is a potential first step in infection, we studied behavior and interactions on different contact lens materials. We hypothesized that contact lenses may induce aggregation, which is a precursor to encystment, and that aggregated encystment would be more difficult to disinfect than motile trophozoites.
METHODS
Six clinically and/or scientifically relevant strains of (ATCC 30010, ATCC 30461, ATCC 50370, ATCC 50702, ATCC 50703, and ATCC PRA-115) were investigated on seven different common silicone hydrogel contact lenses, and a no-lens control, for aggregation and encystment for 72 h. Cell count and size were used to determine aggregation, and fluorescent staining was used to understand encystment. RNA seq was performed to describe the genome of which was individually motile or aggregated on different lens materials. Disinfection efficacy using three common multi-purpose solutions was calculated to describe the potential disinfection resistance of trophozoites, individual cysts, or spheroids.
RESULTS
trophozoites of all strains examined demonstrated significantly more aggregation on specific contact lens materials than others, or the no-lens control. Fluorescent staining demonstrated encystment in as little as 4 hours on contact lens materials, which is substantially faster than previously reported in natural or laboratory settings. Gene expression profiles corroborated encystment, with significantly differentially expressed pathways involving actin arrangement and membrane complexes. High disinfection resistance of cysts and spheroids with multi-purpose solutions was observed.
DISCUSSION
Aggregation/encystment is a protective mechanism which may enable to be more disinfection resistant than individual trophozoites. This study demonstrates that some contact lens materials promote aggregation and encystment, and spheroids obstruct multi-purpose solutions from disinfecting .
PubMed: 36601401
DOI: 10.3389/fmicb.2022.1089092 -
Parasitology Research Sep 2021Acanthamoeba spp. are among the most worldwide prevalent protozoa. It is the causative agent of a disease known as Acanthamoeba keratitis, a painful and severe... (Review)
Review
Acanthamoeba spp. are among the most worldwide prevalent protozoa. It is the causative agent of a disease known as Acanthamoeba keratitis, a painful and severe sight-threatening corneal infection that can lead to blindness. In recent years, the prevalence of Acanthamoeba keratitis has rapidly increased, growing its importance to human health. This systematic review aims to assess the distribution of Acanthamoeba sp. genotypes causing keratitis around the world, considering the sample collected type and the used identification method. Most of the cases were found in Asia and Europe. Not surprisingly, the T4 genotype was the most prevalent worldwide, followed by T3, T15, T11, and T5. Furthermore, the T4 genotype contains a higher number of species. Given the differences in pathology, susceptibility to treatment, and clinical outcome between distinct genotypes, it is essential to genotype isolates from Acanthamoeba keratitis cases to help to establish a better correlation between in vitro and in vivo activities, resulting in better drug therapies and successful treatment in cases of this important ocular infection.
Topics: Acanthamoeba; Acanthamoeba Keratitis; Cornea; Genotype; Humans
PubMed: 34351492
DOI: 10.1007/s00436-021-07261-1