Abstract:BACKGROUND
Acanthamoeba is the genus of free-living amoebae that is most frequently isolated in nature. To date, 20 Acanthamoeba genotypes have been described. Genotype T4 is responsible for approximately 90% of encephalitis and keratitis cases. Due to the ubiquitous presence of amoebae, isolation from environmental sources is not uncommon; to determine the clinical importance of an isolation, it is necessary to have evidence of the pathogenic potential of amoebae.OBJECTIVEThe aim of this study was to physiolo… Show more
“…Acanthamoeba isolated from a water sample of a hospital was axenically grown in 25 cm 2 cell culture flasks with 0.75% proteose peptone, 0.75% yeast extract and 1.5% glucose (PYG) medium. Amoebae were cultured and maintained as reported by Castro Artavia et al in 2017 [63].…”
Section: Axenic Culture Of Acanthamoebamentioning
confidence: 99%
“…Areas of gelatin digestion, which indicate protease activity, were seen as nonstained regions in the gel. To characterize the type of proteases produced, we followed the methodology described by Castro Artavia et al [63], using phenylmethylsulfonyl fluoride (PMSF) 1 mM (Sigma Aldrich Co., St.…”
Section: Determination and Characterization Of Protease Secretion By mentioning
Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases in humans. The wide distribution of Acanthamoeba, the characteristic of being amphizoic and the severity of the disease motivate researchers to focus on the isolation of these organisms, but also in demonstrating direct and indirect factors that could indicate a possible pathogenic potential. Here, we performed the characterization of the pathogenic potential of an Acanthamoeba T5 isolate collected from a water source in a hospital. Osmo- and thermotolerance, the secretion of proteases and the effect of trophozoites over cell monolayers were analyzed by different methodologies. Additionally, we confirm the secretion of extracellular vesicles (EVs) of this isolate incubated at two different temperatures, and the presence of serine and cysteine proteases in these vesicles. Finally, using atomic force microscopy, we determined some nanomechanical properties of the secreted vesicles and found a higher value of adhesion in the EVs obtained at 37 °C, which could have implications in the parasite´s survival and damaging potential in two different biological environments.
“…Acanthamoeba isolated from a water sample of a hospital was axenically grown in 25 cm 2 cell culture flasks with 0.75% proteose peptone, 0.75% yeast extract and 1.5% glucose (PYG) medium. Amoebae were cultured and maintained as reported by Castro Artavia et al in 2017 [63].…”
Section: Axenic Culture Of Acanthamoebamentioning
confidence: 99%
“…Areas of gelatin digestion, which indicate protease activity, were seen as nonstained regions in the gel. To characterize the type of proteases produced, we followed the methodology described by Castro Artavia et al [63], using phenylmethylsulfonyl fluoride (PMSF) 1 mM (Sigma Aldrich Co., St.…”
Section: Determination and Characterization Of Protease Secretion By mentioning
Acanthamoeba is a genus of free-living amoebae widely distributed in nature, associated with the development of encephalitis and keratitis. Despite the fact that it is common to find genotype T5 in environmental samples, only a few cases have been associated with clinical cases in humans. The wide distribution of Acanthamoeba, the characteristic of being amphizoic and the severity of the disease motivate researchers to focus on the isolation of these organisms, but also in demonstrating direct and indirect factors that could indicate a possible pathogenic potential. Here, we performed the characterization of the pathogenic potential of an Acanthamoeba T5 isolate collected from a water source in a hospital. Osmo- and thermotolerance, the secretion of proteases and the effect of trophozoites over cell monolayers were analyzed by different methodologies. Additionally, we confirm the secretion of extracellular vesicles (EVs) of this isolate incubated at two different temperatures, and the presence of serine and cysteine proteases in these vesicles. Finally, using atomic force microscopy, we determined some nanomechanical properties of the secreted vesicles and found a higher value of adhesion in the EVs obtained at 37 °C, which could have implications in the parasite´s survival and damaging potential in two different biological environments.
“…Based on the 18S rRNA gene, the genus Acanthamoeba is defined and distributed into 22 different ribo-genotypes (T1-T22) [5,8]. Most of the human infections due to Acanthamoeba are related to ribo-genotype T4 [9]. Indeed, several studies have estimated that 90% of AK amoebic keratitis cases have been linked to this genotype [9].…”
Section: Introductionmentioning
confidence: 99%
“…Most of the human infections due to Acanthamoeba are related to ribo-genotype T4 [9]. Indeed, several studies have estimated that 90% of AK amoebic keratitis cases have been linked to this genotype [9]. Acanthamoeba triangularis strain SH 621 is a ubiquitous FLA belonging to genotype T4, the most pathogenic group of Acanthamoeba species [10].…”
Acanthamoeba triangularis strain SH 621 is a free-living amoeba belonging to Acanthamoeba ribo-genotype T4. This ubiquitous protist is among the free-living amoebas responsible for Acanthamoeba keratitis, a severe infection of human cornea. Genome sequencing and genomic comparison were carried out to explore the biological functions and to better understand the virulence mechanism related to the pathogenicity of Acanthamoeba keratitis. The genome assembly harbored a length of 66.43 Mb encompassing 13,849 scaffolds. The analysis of predicted proteins reported the presence of 37,062 ORFs. A complete annotation revealed 33,168 and 16,605 genes that matched with NCBI non-redundant protein sequence (nr) and Cluster of Orthologous Group of proteins (COG) databases, respectively. The Kyoto Encyclopedia of Genes and Genomes Pathway (KEGG) annotation reported a great number of genes related to carbohydrate, amino acid and lipid metabolic pathways. The pangenome performed with 8 available amoeba genomes belonging to genus Acanthamoeba revealed a core genome containing 843 clusters of orthologous genes with a ratio core genome/pangenome of less than 0.02. We detected 48 genes related to virulent factors of Acanthamoeba keratitis. Best hit analyses in nr database identified 99 homologous genes shared with amoeba-resisting microorganisms. This study allows the deciphering the genome of a free-living amoeba with medical interest and provides genomic data to better understand virulence-related Acanthamoeba keratitis.
“…Current molecular classification is based on the sequences of the nuclear small ribosomal subunit gene (18S rDNA) that classifies Acanthamoeba strains within 22 genotypes known as T1 to T22 [4]. Although genotype T4 is the most common in both clinical and environmental samples, other genotypes-namely T2, T3, T5, T6, T11, T13, and T15, among others-have been also related to AK and amoebic encephalitis [5,6].…”
The establishment of an effective therapeutic agent against Acanthamoeba keratitis (AK), remains until present, an issue to be solved due to the existence of a cyst stage in the life cycle of Acanthamoeba. Moreover, the effectiveness of the current standard therapeutic agents varies depending on the tested Acanthamoeba strains and its resistance pattern. In the present study, two 10-point augmented simplex-centroid designs were used to formulate a three-component mixture system using water, atorvastatin, and Diclofenaco-lepori or Optiben. The amoebicidal effects and in vitro-induced toxicity in a eukaryotic cell line were determined for all experiments. The optimal mixture to inhibit the parasite without inducing toxicity was established in the first plan as 30% Optiben, 63.5% atorvastatin, and 3.1% water. As for the second experimental design, the optimal mixture to inhibit Acanthamoeba with lower toxicity effect was composed of 17.6% Diclofenaco-lepori and 82.4% atorvastatin.
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