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The Science of the Total Environment Feb 2024Despite extensive research, little is known about the composition of eukaryotic protists in environmental samples. This is due to low parasite concentrations, the...
Despite extensive research, little is known about the composition of eukaryotic protists in environmental samples. This is due to low parasite concentrations, the complexity of parasite diversity, and a lack of suitable reference databases and standardized protocols. To bridge this knowledge gap, this study used 18S rRNA short amplicon and shotgun metagenomic sequencing approaches to profile protozoan microbial communities as well as their functional pathways in treated and untreated wastewater samples collected from different regions of South Africa. Results demonstrated that protozoan diversity (Shannon index P-value = 0.03) and taxonomic composition (PERMANOVA, P-value = 0.02) was mainly driven by the type of wastewater samples (treated & untreated) and geographic location. However, these WWTPs were also found to contain a core community of protozoan parasites. The untreated wastewater samples revealed a predominant presence of free-living, parasitic, and potentially pathogenic protists typically found in humans and animals, ranging from Alveolata (27 %) phylum (Apicomplexa and Ciliophora) to Excavata (3.88 %) (Discoba and Parasalia) and Amoebozoa (2.84 %) (Entamoeba and Acanthamoeba). Shotgun metagenomics analyses in a subset of the untreated wastewater samples confirmed the presence of public health-importance protozoa, including Cryptosporidium species (3.48 %), Entamoeba hystolitica (6.58 %), Blastocystis hominis (2.91 %), Naegleria gruberi (2.37 %), Toxoplasma gondii (1.98 %), Cyclospora cayetanensis (1.30 %), and Giardia intestinalis (0.31 %). Virulent gene families linked to pathogenic protozoa, such as serine/threonine protein phosphatase and mucin-desulfating sulfatase were identified. Additionally, enriched pathways included thiamine diphosphate biosynthesis III, heme biosynthesis, Methylerythritol 4-Phosphate Pathway, methyl erythritol phosphate (MEP), and pentose phosphate pathways. These findings suggest that protozoan pathogens may possess metabolic and growth potential within WWTPs, posing a severe risk of transmission to humans and animals if inadequately disinfected before release. This study provides a baseline for the future investigation of diverse protozoal communities in wastewater, which are of public health importance.
Topics: Animals; Humans; Wastewater; Cryptosporidium; Cryptosporidiosis; RNA, Ribosomal, 18S; Parasites; Eukaryota; Entamoeba; Metagenomics; Phosphates
PubMed: 38154626
DOI: 10.1016/j.scitotenv.2023.169602 -
Diagnostic Microbiology and Infectious... Oct 2023Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and...
Acanthamoeba keratitis is a devastating infectious disease of the cornea caused by an opportunistic amoeba, Acanthamoeba castellanii. It is poorly recognized, and diagnostic delays can lead to irreversible damage to the vision. The gold standard for diagnosis has been a sample culture that lasts approximately 2 weeks. Nevertheless, the essence of time has led to the need for an accurate and fast technique to detect A. castellanii from a sample. We developed both traditional and quantitative real-time-PCR-based methods to detect A. castellanii in less than 3 hours and with the sensitivity of one amoeba. Diagnostic laboratories can select the best-suited method for their purposes from 2 comparable methods. The correct treatment can be initiated from the emergency room when the diagnosis has been made quickly within a few hours, hence saving the patient from long-term complications.
Topics: Humans; Acanthamoeba castellanii; Rapid Diagnostic Tests; Acanthamoeba Keratitis; Cornea; Real-Time Polymerase Chain Reaction
PubMed: 37506594
DOI: 10.1016/j.diagmicrobio.2023.116014 -
European Journal of Cell Biology Dec 2023Major facilitator superfamily domain-containing protein 8 (MFSD8) is a transmembrane protein that has been reported to function as a lysosomal chloride channel. In...
Major facilitator superfamily domain-containing protein 8 (MFSD8) is a transmembrane protein that has been reported to function as a lysosomal chloride channel. In humans, homozygous mutations in MFSD8 cause a late-infantile form of neuronal ceroid lipofuscinosis (NCL) called CLN7 disease. In the social amoeba Dictyostelium discoideum, Mfsd8 localizes to cytoplasmic puncta and vesicles, and regulates conserved processes during the organism's life cycle. Here, we used D. discoideum to examine the effect of mfsd8-deficiency on the secretome during the early stages of multicellular development. Mass spectrometry revealed 61 proteins that were differentially released by cells after 4 and 8 h of starvation. Most proteins were present in increased amounts in mfsd8 conditioned buffer compared to WT indicating that loss of mfsd8 deregulates protein secretion and/or causes the release of proteins not normally secreted by WT cells. GO term enrichment analyses showed that many of the proteins aberrantly released by mfsd8 cells localize to compartments and regions of the cell associated with the endo-lysosomal and secretory pathways. Mass spectrometry also revealed proteins previously known to be impacted by the loss of mfsd8 (e.g., cathepsin D), as well as proteins that may underlie mfsd8-deficiency phenotypes during aggregation. Finally, we show that mfsd8-deficiency reduces intracellular proteasome 20S activity due to the abnormal release of at least one proteasomal subunit. Together, this study reveals the impact of mfsd8 loss on the secretome during D. discoideum aggregation and lays the foundation for follow up work that investigates the role of altered protein release in CLN7 disease.
Topics: Humans; Dictyostelium; Secretome; Membrane Proteins; Mutation; Lysosomes; Membrane Transport Proteins
PubMed: 37742391
DOI: 10.1016/j.ejcb.2023.151361 -
Microbial Pathogenesis Dec 2023The prevalence of Blastocystis sp., its genetic diversity and the distribution of circulating subtypes (STs) were molecularly investigated in a cohort of autochthonous...
The prevalence of Blastocystis sp., its genetic diversity and the distribution of circulating subtypes (STs) were molecularly investigated in a cohort of autochthonous and immigrant patients with gastrointestinal symptoms hospitalized over the period February 2022-June 2023 at the Policlinico Ospedaliero-Universitario "Riuniti", Foggia, in Southern Italy. The population variables, including patient geographical origin, gender and age classes were reported. Out of the 927 investigated patients, 36 (3.9%) were positive for Blastocystis sp. A statistically significant association with African origin and age classes >18 years old was found. ST1 (allele 4), ST2 (alleles 9, 13), ST3 (alleles 34, 36) and ST4 (allele 92) were the subtypes detected with a different distribution between autochthonous and immigrant patients. Co-infections with enteric protozoa such as Giardia duodenalis and Dientamoeba fragilis, pathogenic bacteria as Clostridioides difficile, Campylobacter jejuni and Aeromonas sp. and viral infections such as Norovirus were found in 33% of cases. This is the first study of Blastocystis sp., its circulating subtypes and allele variability among patients with different geographical origin in an area of Southern Italy, in the Central Mediterranean, characterized by high immigrant pressure. These results provide baseline data to better investigate a potential interaction between Blastocystis sp. and other risk factors in patients with gastrointestinal symptoms.
Topics: Humans; Adolescent; Blastocystis; Blastocystis Infections; Prevalence; Genetic Variation; Italy; Emigrants and Immigrants; Feces; Phylogeny
PubMed: 37839760
DOI: 10.1016/j.micpath.2023.106377 -
International Journal of Molecular... Dec 2023In this work, we established, validated, and optimized a novel computational framework for tracing arbitrarily oriented actin filaments in cryo-electron tomography maps....
In this work, we established, validated, and optimized a novel computational framework for tracing arbitrarily oriented actin filaments in cryo-electron tomography maps. Our approach was designed for highly complex intracellular architectures in which a long-range cytoskeleton network extends throughout the cell bodies and protrusions. The irregular organization of the actin network, as well as cryo-electron-tomography-specific noise, missing wedge artifacts, and map dimensions call for a specialized implementation that is both robust and efficient. Our proposed solution, , accumulates densities along paths of a specific length in various directions, starting from locally determined seed points. The highest-density paths originating from the seed points form short linear candidate filament segments, which are further scrutinized and classified by users via inspection of a novel , which visualizes the likelihood of being a part of longer filaments. The pruned linear candidate filament segments are then iteratively fused into continuous, longer, and curved filaments based on their relative orientations, gap spacings, and extendibility. When applied to the simulated phantom tomograms of a filopodium under experimental conditions, demonstrated high efficacy, with F1-scores ranging from 0.85 to 0.90, depending on the noise level. Furthermore, when applied to a previously untraced experimental tomogram of mouse fibroblast lamellipodia, the filaments predicted by exhibited a good visual agreement with the experimental map. The framework is highly time efficient and can complete the tracing process in just a few minutes. The source code is publicly available with version 3.2 of the free and open-source software package.
Topics: Mice; Animals; Dictyostelium; Actin Cytoskeleton; Cytoskeleton; Actins; Electron Microscope Tomography
PubMed: 38139012
DOI: 10.3390/ijms242417183 -
Biophysical Journal Jul 2023The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric...
The actin filament network is in part remodeled by the action of a family of filament severing proteins that are responsible for modulating the ratio between monomeric and filamentous actin. Recent work on the protein actophorin from the amoeba Acanthamoeba castellani identified a series of site-directed mutations that increase the thermal stability of the protein by 22°C. Here, we expand this observation by showing that the mutant protein is also significantly stable to both equilibrium and kinetic chemical denaturation, and employ computer simulations to account for the increase in thermal or chemical stability through an accounting of atomic-level interactions. Specifically, the potential of mean force (PMF) can be obtained from steered molecular dynamics (SMD) simulations in which a protein is unfolded. However, SMD can be inefficient for large proteins as they require large solvent boxes, and computationally expensive as they require increasingly many SMD trajectories to converge the PMF. Adaptive steered molecular dynamics (ASMD) overcomes the second of these limitations by steering the particle in stages, which allows for convergence of the PMF using fewer trajectories compared with SMD. Use of the telescoping water scheme within ASMD partially overcomes the first of these limitations by reducing the number of waters at each stage to only those needed to solvate the structure within a given stage. In the PMFs obtained from ASMD, the work of unfolding Acto-2 was found to be higher than the Acto-WT by approximately 120 kCal/mol and reflects the increased stability seen in the chemical denaturation experiments. The evolution of the average number of hydrogen bonds and number of salt bridges during the pulling process provides a mechanistic view of the structural changes of the actophorin protein as it is unfolded, and how it is affected by the mutation in concert with the energetics reported through the PMF.
Topics: Acanthamoeba; Actins; Amoeba; Molecular Dynamics Simulation; Solvents; Protein Denaturation
PubMed: 36461639
DOI: 10.1016/j.bpj.2022.11.2941 -
Scientific Reports Aug 2023Balamuthia mandrillaris is a pathogenic protozoan that causes a rare but almost always fatal infection of the central nervous system and, in some cases, cutaneous...
Balamuthia mandrillaris is a pathogenic protozoan that causes a rare but almost always fatal infection of the central nervous system and, in some cases, cutaneous lesions. Currently, the genomic data for this free-living amoeba include the description of several complete mitochondrial genomes. In contrast, two complete genomes with draft quality are available in GenBank, but none of these have a functional annotation. In the present study, the complete genome of B. mandrillaris isolated from a freshwater artificial lagoon was sequenced and assembled, obtaining an assembled genome with better assembly quality parameter values than the currently available genomes. Afterward, the genome mentioned earlier, along with strains V039 and 2046, were subjected to functional annotation. Finally, comparative genomics analysis was performed, and it was found that homologous genes in the core genome potentially involved in the virulence of Acanthamoeba spp. and Trypanosoma cruzi. Moreover, eleven of fifteen genes were identified in the three strains described as potential target genes to develop new treatment approaches for B. mandrillaris infections. These results describe proteins in this protozoan's complete genome and help prioritize which target genes could be used to develop new treatments.
Topics: Balamuthia mandrillaris; Virulence; Comparative Genomic Hybridization; Acanthamoeba; Genomics
PubMed: 37653073
DOI: 10.1038/s41598-023-41657-6 -
Infection, Genetics and Evolution :... Sep 2023Blastocystis sp., is an intestinal protist with a broad host range and a high prevalence in human populations worldwide, even in developed Western countries. The...
Blastocystis sp., is an intestinal protist with a broad host range and a high prevalence in human populations worldwide, even in developed Western countries. The publication of conflicting evidence has divided the scientific community about the pathogenic role of this parasite. Even though, genetic studies on Blastocystis sp. revealed associations between genotypes and different pathogenic profiles. Conventionally, the detection of this parasite is based on microscopic or PCR methods, which offer meager or null performance in detecting mixed infections. In this work, we applied a metataxonomic NGS approach targeting the V4 region of the eukaryotic SSU-rRNA gene and classical phylogenetic methods. This approach allowed us to detect Blastocystis sp. in stool samples from infected children living in an urban setting in the city of Medellin attending the same daycare center. Phylogenetic analysis identified the subtypes present in the children as ST1, ST2, and ST3. Besides, mixed infections of subtypes ST1 + ST3 were spotted in 16% of the analyzed stool samples.
Topics: Humans; Child; Blastocystis; Phylogeny; Coinfection; Colombia; Genetic Variation; Feces; Blastocystis Infections; Prevalence; DNA, Protozoan
PubMed: 37406785
DOI: 10.1016/j.meegid.2023.105478 -
Parasites, Hosts and Diseases Nov 2023Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae....
Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.
Topics: Acanthamoeba castellanii; Escherichia coli; Phagocytosis; Legionella pneumophila; Phagosomes
PubMed: 38043535
DOI: 10.3347/PHD.23088 -
Turkiye Parazitolojii Dergisi Mar 2024The aim of this study was to evaluate the pathogenicity of strains with T4, T5, T11, and T12 genotypes by comparing the osmotolerance and thermotolerance...
OBJECTIVE
The aim of this study was to evaluate the pathogenicity of strains with T4, T5, T11, and T12 genotypes by comparing the osmotolerance and thermotolerance characteristics of strains isolated from genotype groups, within species with the same genotype, and from environmental and keratitis cases.
METHODS
In this study, after axenic cultures of 22 strains with T4 (Neff, A, B, D, E), T5, T11, and T12 genotypes isolated from clinical and environmental samples, thermotolerance (37 °C, 39 °C and 41 °C) and osmotolerance (0.5 M, 1 M) tests were performed.
RESULTS
All strains showed growth ability at 37 °C and 0.5 M osmolarity. While all five strains isolated from patients with keratitis showed growth ability at 37 °C and 0.5 M osmolarity, no growth was detected at 41 °C and 1 M osmolarity. When the tolerance characteristics of the strains with the same genotype were evaluated, the strains with the T5 and T4E genotypes showed the same characteristics. When strains with the T4 genotype were evaluated in general, 31.25% of the strains were found to grow at 39 °C and 6.25% at 41 °C. Of the T4Neff strains, only one strain did not show the ability to reproduce at 39 °C and showed a different feature from the other strains. While the strain with the T11 genotype grew at all temperatures, the strain with the T12 genotype did not grow at 41 °C.
CONCLUSION
According to our research results, we believe that tolerance to 39 °C and 1 M mannitol is not an indicator of pathogenicity. More studies with strains are required to clarify this issue.
Topics: Humans; Acanthamoeba; Virulence; Thermotolerance; Genotype; Mannitol
PubMed: 38449362
DOI: 10.4274/tpd.galenos.2024.92408