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Parasites, Hosts and Diseases May 2024Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based...
Free-living amoebae (FLA) are found in diverse environments, such as soils, rivers, and seas. Hence, they can be used as bioindicators to assess the water quality based solely on their presence. In this study, we determined the presence of FLA in river water by filtering water samples collected from various sites and culturing the resulting filtrates. FLA were detected in all the water samples with varying quality grades (Grades Ι-V). The significant increase in the size of the amoebae population with the deterioration in the water quality. Monoxenic cultures of the amoebae were performed, and genomic DNAs were isolated, among which 18S rDNAs were sequenced to identify the amoeba species. Of the 12 species identified, 10 belonged to the Acanthamoeba genus; of the remaining 2 species, one was identified as Vannella croatica and the other as a species of Vermamoeba. Acanthamoeba was detected in samples with Grades Ι to VI quality, whereas the Vermamoeba species was present only in Grade Ι water. V. croatica was found exclusively in water with Grade ΙΙ quality. Following morphological observations, genomic DNA was sequenced using 16S rDNA to determine whether the species of Acanthamoeba harbored endosymbionts. Most of the isolated Acanthamoeba contained endosymbionts, among which 4 species of endogenous bacteria were identified and examined using transmission electron microscopy. This study provides evidence that the distribution of amoebae other than Acanthamoeba may be associated with water quality. However, further confirmation will be required based on accurate water quality ratings and assessments using a more diverse range of FLA.
Topics: Amoeba; Water Quality; Phylogeny; Rivers; DNA, Protozoan; Acanthamoeba; RNA, Ribosomal, 18S; DNA, Ribosomal; Biodiversity; Sequence Analysis, DNA; RNA, Ribosomal, 16S
PubMed: 38835259
DOI: 10.3347/PHD.24020 -
Microbiome Jul 2023Legionella are parasites of freshwater protozoa, responsible for Legionellosis. Legionella can be found in a variety of aquatic environments, including rivers, lakes,...
BACKGROUND
Legionella are parasites of freshwater protozoa, responsible for Legionellosis. Legionella can be found in a variety of aquatic environments, including rivers, lakes, and springs, as well as in engineered water systems where they can potentially lead to human disease outbarks. Legionella are considered to be predominantly freshwater organisms with a limited ability to proliferate in saline environments. Exposure of Legionella to high sodium concentrations inhibits growth and virulence of laboratory strains, particularly under elevated temperatures. Nonetheless, Legionella have been identified in some saline environments where they likely interact with various protozoan hosts. In this work, we examine how these selection pressures, sodium and grazing, help shape Legionella ecology within natural environments. Utilizing Legionella-specific primers targeting a variable region of the Legionella 16S rRNA gene, we characterized Legionella abundance, diversity, and community composition in natural spring clusters of varying sodium concentrations, focusing on high sodium concentrations and elevated temperatures.
RESULTS
We observed the highest abundance of Legionella in spring clusters of high salinity, particularly in combination with elevated temperatures. Legionella abundance was strongly related to sodium concentrations. The Legionella community structure in saline environments was characterized by relatively low diversity, compared to spring clusters of lower salinity. The community composition in high salinity was characterized by few dominant Legionella genotypes, not related to previously described species. Protozoan microbial community structure and composition patterns resembled those of Legionella, suggesting a common response to similar selection pressures. We examined Legionella co-occurrence with potential protozoan hosts and found associations with Ciliophora and Amoebozoa representatives.
CONCLUSIONS
Our results indicate that selection forces in saline environments favor a small yet dominant group of Legionella species that are not closely related to known species. These novel environmental genotypes interact with various protozoan hosts, under environmental conditions of high salinity. Our findings suggest that alternative survival mechanisms are utilized by these species, representing mechanisms distinct from those of well-studied laboratory strains. Our study demonstrate how salinity can shape communities of opportunistic pathogens and their hosts, in natural environments, shedding light on evolutionary forces acting within these complex environments. Video Abstract.
Topics: Humans; Legionella; RNA, Ribosomal, 16S; Ecology; Water Microbiology; Fresh Water
PubMed: 37518067
DOI: 10.1186/s40168-023-01611-0 -
Genes Feb 2024, the causative agent of amebiasis, is the third leading cause of death among parasitic diseases globally. Its life cycle includes encystation, which has been mostly...
, the causative agent of amebiasis, is the third leading cause of death among parasitic diseases globally. Its life cycle includes encystation, which has been mostly studied in , responsible for reptilian amebiasis. However, the molecular mechanisms underlying this process are not fully understood. Therefore, we focused on the identification and characterization of Myb proteins, which regulate the expression of encystation-related genes in various protozoan parasites. Through bioinformatic analysis, we identified 48 genes in encoding MYB-domain-containing proteins. These were classified into single-repeat 1R (20), 2R-MYB proteins (27), and one 4R-MYB protein. The in-silico analysis suggests that these proteins are multifunctional, participating in transcriptional regulation, chromatin remodeling, telomere maintenance, and . Transcriptomic data analysis revealed expression signatures of genes, suggesting a potential orchestration in the regulation of early and late encystation-excystation genes. Furthermore, we identified probable target genes associated with reproduction, the meiotic cell cycle, ubiquitin-dependent protein catabolism, and endosomal transport. In conclusion, our findings suggest that Myb proteins regulate stage-specific proteins and a wide array of cellular processes. This study provides a foundation for further exploration of the molecular mechanisms governing encystation and unveils potential targets for therapeutic intervention in amebiasis.
Topics: Humans; Entamoeba; Entamoeba histolytica; Gene Expression Profiling; Gene Expression Regulation; Amebiasis
PubMed: 38397191
DOI: 10.3390/genes15020201 -
Current Biology : CB Aug 2023Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water...
Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments, such as freshwater, must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify the basic principles governing contractile vacuole function, we investigate here the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages: the discoban Naegleria gruberi and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology, we find that although these species respond differently to osmotic challenges, they both use vacuolar-type proton pumps for filling contractile vacuoles and actin for osmoregulation, but not to power water expulsion. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. These analyses show that cytoplasmic pressure is sufficient to drive contractile vacuole emptying for a wide range of cellular pressures and vacuolar geometries. Because vacuolar-type proton-pump-dependent contractile vacuole filling and pressure-dependent emptying have now been validated in three eukaryotic lineages that diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.
Topics: Cytosol; Dictyostelium; Osmolar Concentration; Water-Electrolyte Balance; Vacuoles; Eukaryota; Water
PubMed: 37478864
DOI: 10.1016/j.cub.2023.06.061 -
BioRxiv : the Preprint Server For... Dec 2023This study investigates the genomic characteristics of , a small-sized amoeba within the Tubulinea clade of the Amoebozoa supergroup. Despite Tubulinea's significance...
This study investigates the genomic characteristics of , a small-sized amoeba within the Tubulinea clade of the Amoebozoa supergroup. Despite Tubulinea's significance in various fields, genomic data for this clade have been scarce presents the smallest free-living amoeba genome within Tubulinea and Amoebozoa to date. Comparative analysis reveals intriguing parallels with parasitic lineages in terms of genome size and predicted gene numbers, emphasizing the need to understand the consequences of reduced genomes in free-living amoebae. Functional categorization of predicted genes in shows similar percentages of ortholog groups to other amoebae in various categories, but a distinctive feature is the extensive gene contraction in orphan (ORFan) genes and those involved in biological processes. Notably, among the few genes that underwent expansion, none are related to cellular components, suggesting adaptive processes that streamline biological processes and cellular components for efficiency and energy conservation. The investigation delves into genomic structural evidence, including gene content and repetitive elements, illuminating the distinctive genomic traits of and providing reinforcement for its compact genome size. Overall, this research underscores the diversity within Tubulinea, highlights knowledge gaps in Amoebozoa genomics, and positions as a valuable addition to genomic datasets, prompting further exploration of complexities in Amoebozoa diversity and genome evolution.
PubMed: 38106205
DOI: 10.1101/2023.12.07.570689 -
Frontiers in Cellular and Infection... 2024Acanthamoeba infection is a serious public health concern, necessitating the development of effective and safe anti-Acanthamoeba chemotherapies. Poly (ADP-ribose)...
INTRODUCTION
Acanthamoeba infection is a serious public health concern, necessitating the development of effective and safe anti-Acanthamoeba chemotherapies. Poly (ADP-ribose) polymerases (PARPs) govern a colossal amount of biological processes, such as DNA damage repair, protein degradation and apoptosis. Multiple PARP-targeted compounds have been approved for cancer treatment. However, repurposing of PARP inhibitors to treat Acanthamoeba is poorly understood.
METHODS
In the present study, we attempted to fill these knowledge gaps by performing anti-Acanthamoeba efficacy assays, cell biology experiments, bioinformatics, and transcriptomic analyses.
RESULTS
Using a homology model of Acanthamoeba poly (ADP-ribose) polymerases (PARPs), molecular docking of approved drugs revealed three potential inhibitory compounds: olaparib, venadaparib and AZ9482. In particular, venadaparib exhibited superior docking scores (-13.71) and favorable predicted binding free energy (-89.28 kcal/mol), followed by AZ9482, which showed a docking score of -13.20 and a binding free energy of -92.13 kcal/mol. Notably, the positively charged cyclopropylamine in venadaparib established a salt bridge (through E535) and a hydrogen bond (via N531) within the binding pocket. For comparison, AZ9482 was well stacked by the surrounding aromatic residues including H625, Y652, Y659 and Y670. In an assessment of trophozoites viability, AZ9482 exhibited a dose-and time-dependent anti-trophozoite effect by suppressing Acanthamoeba PARP activity, unlike olaparib and venadaparib. An Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis assay revealed AZ9482 induced trophozoite necrotic cell death rather than apoptosis. Transcriptomics analyses conducted on Acanthamoeba trophozoites treated with AZ9482 demonstrated an atlas of differentially regulated proteins and genes, and found that AZ9482 rapidly upregulates a multitude of DNA damage repair pathways in trophozoites, and intriguingly downregulates several virulent genes. Analyzing gene expression related to DNA damage repair pathway and the rate of apurinic/apyrimidinic (AP) sites indicated DNA damage efficacy and repair modulation in Acanthamoeba trophozoites following AZ9482 treatment.
DISCUSSION
Collectively, these findings highlight AZ9482, as a structurally unique PARP inhibitor, provides a promising prototype for advancing anti-Acanthamoeba drug research.
Topics: Molecular Docking Simulation; Poly(ADP-ribose) Polymerase Inhibitors; Humans; Piperazines; Phthalazines; Drug Repositioning; Poly(ADP-ribose) Polymerases; Acanthamoeba; Computational Biology; Apoptosis; Gene Expression Profiling; Antiprotozoal Agents; Trophozoites
PubMed: 38863831
DOI: 10.3389/fcimb.2024.1414135 -
Scientific Reports Oct 2023Naegleria gruberi is a free-living amoeboflagellate commonly found in freshwater and in soils around the world. It is a non-pathogenic relative of Naegleria fowleri,...
Naegleria gruberi is a free-living amoeboflagellate commonly found in freshwater and in soils around the world. It is a non-pathogenic relative of Naegleria fowleri, which is the etiologic agent of Primary Amoebic Meningoencephalitis (PAM). PAM occurs world-wide and it is considered a rare disease, but its fatality rate is high (96%) mainly because of delay in initiation of treatment due to misdiagnosis and lack of a specific treatment. The analysis of gene expression by quantitative real-time PCR in N. gruberi could be a highly efficient means to understand the pathogenicity of N. fowleri and also to find drug targets. Accurate RT-qPCR analysis requires correct normalization of gene expression data using reference genes (RG), whose expression should be constant under different experimental conditions. In this study, six genes, representing the most frequently used housekeeping genes, were selected for evaluation as reference genes in N. gruberi. The expression and stability of these genes was evaluated employing four algorithms (geNorm, NormFinder, BestKeeper and RefFinder). This work shows significant variations of the stability of RGs depending on the algorithms employed and on the experimental conditions (i.e. logarithmic, stationary, heat-shock and oxidative stress). The geNorm, NormFinder and RefFinder analysis of all the experimental conditions in combination revealed that ACT and G6PD were the most stable RGs. While BestKeeper analysis showed that 18S and TBP were the most stable RGs. Moreover, normalization of HSP90 gene expression with the most stable RGs resulted in an upregulation whereas when the normalization was done with the unstable RGs, the gene expression was not reliable. Hence, the implications of this study are relevant to gene expression studies in N. gruberi.
Topics: Naegleria; Gene Expression Profiling; Algorithms; Naegleria fowleri; Real-Time Polymerase Chain Reaction; Reference Standards; Gene Expression
PubMed: 37798308
DOI: 10.1038/s41598-023-43892-3 -
Parasites & Vectors Dec 2023Acanthamoeba spp. are opportunistic pathogens that cause inflammation, mostly in the brain, lungs and cornea. Recent reports indicate kidney dysfunction in hosts with...
BACKGROUND
Acanthamoeba spp. are opportunistic pathogens that cause inflammation, mostly in the brain, lungs and cornea. Recent reports indicate kidney dysfunction in hosts with systemic acanthamoebiasis. The aim of the study was to analyze the gene expression and protein concentration of NADPH oxidase 2 and 4 (NOX2 and NOX4, respectively) and nuclear erythroid 2-related factor (Nrf2) in the kidneys of hosts with systemic acanthamoebiasis. We also aimed to determine the protein and gene expressions of Bcl2, Bax, caspases 3 and 9.
METHODS
Mice were divided into four groups based on their immunological status and Acanthamoeba sp. infection: A, immunocompetent Acanthamoeba sp.-infected mice; AS, immunosuppressed Acanthamoeba sp.- infected mice; C, immunocompetent uninfected mice; CS, immunosuppressed uninfected mice. NOX2, NOX4 and Nrf2 were analyzed by quantitative reverse transcription PCR (qRT-PCR) and ELISA methods, while pro-apoptotic and anti-apoptotic proteins (Bax and Bcl-2, respectively), Cas9, Cas3 were analyzed by qRT-PCR and western blot methods. RESULTS: Increased gene expression and/or protein concentration of NOX2 and NOX4 were found in both immunocompetent and immunosuppressed mice infected with Acanthamoeba sp. (groups A and AS, respectively). Gene expression and/or protein concentration of Nrf2 were higher in group A than in control animals. Compared to control mice, in the AS group the expression of the Nrf2 gene was upregulated while the concentration of Nrf2 protein was decreased. Additionally in A group, higher gene and protein expression of Bcl-2, and lower gene as well as protein expression of Bax, caspases 3 and 9 were noted. In contrast, the AS group showed lower gene and protein expression of Bcl-2, and higher gene as well as protein expression of Bax, caspases 3 and 9.
CONCLUSIONS
This study is the first to address the mechanisms occurring in the kidneys of hosts infected with Acanthamoeba sp. The contact of Acanthamoeba sp. with the host cell surface and/or the oxidative burst caused by elevated levels of NOXs lead to an antioxidant response enhanced by the Nrf2 pathway. Acanthamoeba sp. have various strategies concerning apoptosis. In immunocompetent hosts, amoebae inhibit the apoptosis of kidney cells, and in immunosuppressed hosts, they lead to increased apoptosis by the intrinsic pathway and thus to a more severe course of the disease.
Topics: Mice; Animals; NF-E2-Related Factor 2; bcl-2-Associated X Protein; Kidney; Oxidative Stress; Apoptosis; Amebiasis; Acanthamoeba; Proto-Oncogene Proteins c-bcl-2; Caspases
PubMed: 38041167
DOI: 10.1186/s13071-023-06052-0 -
Journal of Ayub Medical College,... 2023A group of eukaryotic organisms that are either free-living or parasitic and feed on organic matter, such as microorganisms or organic detritus. This group is referred...
BACKGROUND
A group of eukaryotic organisms that are either free-living or parasitic and feed on organic matter, such as microorganisms or organic detritus. This group is referred to as a polyphyletic classification. They can be found in a wide variety of patterns and sizes, ranging from an amoeba, which can vary its shape, to a paramecium, which has a fixed shape and a complicated structure. The objective was to find out the occurrence of Entamoeba gingivalis and Trichomonas among Dental Patients visiting the Periodontology Department Dental Section, Sandeman, Provincial Hospital Quetta.
METHODS
The study design was randomized cross-sectional and conducted in the Department of Periodontology Dental Section, Sandeman Provincial Hospital Quetta, from March 2022 to February 2023. A total of 110 known cases of periodontitis and gingivitis were recruited in this study, and further to laboratory work the bacterial biofilm samples were collected from both gingivitis and periodontitis patients. The dental plaque was placed on individual glass microscope slides and a drop of saline and mixed and covered with a coverslip. The wet smear was examined immediately under 40X, and the objective for the presence and absence of motile amoebae or flagellated protozoa was recorded. .
RESULTS
out of 110 known cases of periodontitis and gingivitis 91 (83%) patients were males and 19 (17%) were females among 110 patients, 40 (36%) patients were periodontitis and 70 (64%) patients were gingivitis. Among 40 (36%) periodontitis patients 3(7.5%) were Entamoeba gingivalis and 4(10%) were Trichomonas Tenax while 70 (64%) cases of Gingivitis 20 (28%) of Entamoeba gingivalis and 1(1%) Trichomonas tenax was recorded. No patient had both species of protozoa were seen in this study.
CONCLUSION
The results of this study revealed the frequency of occurrence of E. gingivalis and T. tenax and the state of periodontitis and gingivitis. The prevalence of E. gingivalis was higher than T. tenax. We recommended further research with a higher number of patients and may use more advanced and reliable laboratory techniques such as PCR and electron microscopy.
Topics: Male; Female; Humans; Trichomonas; Entamoeba; Cross-Sectional Studies; Gingivitis; Periodontitis
PubMed: 38406902
DOI: 10.55519/JAMC-S4-12660 -
International Journal For Parasitology.... Aug 2023Naegleria fowleri is an opportunistic protozoan, belonging to the free-living amoeba group, that can be found in warm water bodies. It is causative agent the primary...
Naegleria fowleri is an opportunistic protozoan, belonging to the free-living amoeba group, that can be found in warm water bodies. It is causative agent the primary amoebic meningoencephalitis, a fulminant disease with a rapid progression that affects the central nervous system. However, no 100% effective treatments are available and those that are currently used involve the appearance of severe side effects, therefore, there is an urgent need to find novel antiamoebic compounds with low toxicity. In this study, the in vitro activity of six oxasqualenoids obtained from the red algae Laurencia viridis was evaluated against two different strains of N. fowleri (ATCC® 30808 and ATCC® 30215) as well as their cytotoxicity against murine macrophages. Yucatecone was the molecule with the highest selectivity index (>2.98 and 5.23 respectively) and it was selected to continue with the cell death type determination assays. Results showed that yucatone induced programmed cell death like responses in treated amoebae causing DNA condensation and cellular membrane damage among others. In this family of oxasqualenoids, it seems that the most significative structural feature to induce activity against N. fowleri is the presence of a ketone at C-18. This punctual oxidation transforms an inactive compound into a lead compound as the yucatecone and 18-ketodehydrotyrsiferol with IC values of 16.25 and 12.70 μM, respectively. The assessment of in silico ADME/Tox analysis revealed that the active compounds showed good Human Oral Absorption and demonstrate that are found to be within the limit of approved drug parameter range. Hence, the study highlights promising potential of yucatone to be tested for therapeutic use against primary amoebic meningoencephalitis.
Topics: Humans; Animals; Mice; Naegleria fowleri; Central Nervous System Protozoal Infections; Amoeba
PubMed: 37270868
DOI: 10.1016/j.ijpddr.2023.05.004