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Frontiers in Microbiology 2024
PubMed: 38741744
DOI: 10.3389/fmicb.2024.1401217 -
Nature Protocols Jan 2024Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology,... (Review)
Review
Giant viruses (GVs) provide an unprecedented source of genetic innovation in the viral world and are thus, besides their importance in basic and environmental virology, in the spotlight for bioengineering advances. Their host, Acanthamoeba castellanii, is an accidental human pathogen that acts as a natural host and environmental reservoir of other human pathogens. Tools for genetic manipulation of viruses and host were lacking. Here, we provide a detailed method for genetic manipulation of A. castellanii and the GVs it plays host to by using CRISPR-Cas9 or homologous recombination. We detail the steps of vector preparation (4 d), transfection of amoeba cells (1 h), infection (1 h), selection (5 d for viruses, 2 weeks for amoebas) and cloning of recombinant viruses (4 d) or amoebas (2 weeks). This procedure takes ~3 weeks or 1 month for the generation of recombinant viruses or amoebas, respectively. This methodology allows the generation of stable gene modifications, which was not possible by using RNA silencing, the only previously available reverse genetic tool. We also include detailed sample-preparation steps for protein localization by immunofluorescence (4 h), western blotting (4 h), quantification of viral particles by optical density (15 min), calculation of viral lethal dose 50 (7 d) and quantification of DNA replication by quantitative PCR (4 h) to allow efficient broad phenotyping of recombinant organisms. This methodology allows the function of thousands of ORFan genes present in GVs, as well as the complex pathogen-host, pathogen-pathogen or pathogen-symbiont interactions in A. castellanii, to be studied in vivo.
Topics: Humans; Acanthamoeba castellanii; Giant Viruses; Viruses
PubMed: 37964008
DOI: 10.1038/s41596-023-00910-y -
International Journal of Molecular... Sep 2023is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis...
is the primary causative agent of Legionnaires' disease. The mutant-type strain interrupted in the ORF7 gene region responsible for the lipopolysaccharide biosynthesis of the strain Heysham-1, lacking the -acetyl groups attached to the rhamnose of the core part, showed a higher surface polarity compared with the wild-type strain. The measurement of excitation energy transfer between fluorophores located on the surface of bacteria and eukaryotic cells showed that, at an early stage of interaction with host cells, the mutant exhibited weaker interactions with cells and THP-1-derived macrophages. The mutant displayed reduced adherence to macrophages but enhanced adherence to , suggesting that the -acetyl group of the LPS core region plays a crucial role in facilitating interaction with macrophages. The lack of core rhamnose -acetyl groups made it easier for the bacteria to multiply in amoebae and macrophages. The mutant induced TNF-α production more strongly compared with the wild-type strain. The mutant synthesized twice as many ceramides Cer(t34:0) and Cer(t38:0) than the wild-type strain. The study showed that the internal sugars of the LPS core region of sg 1 can interact with eukaryotic cell surface receptors and mediate in contacting and attaching bacteria to host cells as well as modulating the immune response to infection.
Topics: Humans; Legionella pneumophila; Lipopolysaccharides; Rhamnose; Serogroup; Bacterial Proteins; Legionnaires' Disease
PubMed: 37834049
DOI: 10.3390/ijms241914602 -
Pathogens (Basel, Switzerland) Aug 2023Free-living amoebas can impact the excretion of macroelements and microelements in urine. The aim of the present study was to examine the concentrations of...
Free-living amoebas can impact the excretion of macroelements and microelements in urine. The aim of the present study was to examine the concentrations of macroelements, including calcium (Ca), phosphorus (P), sodium (Na), potassium (K), and magnesium (Mg), as well as microelements such as manganese (Mn), zinc (Zn), copper (Cu), iron (Fe), and chromium (Cr), in the urine during acanthamoebiasis while considering the host's immunological status. This is the first study to show an increase in urinary excretion of Ca, Mn, Cu, Fe, Na, and Cr, along with a decreased excretion of K, in immunocompetent mice 16 days post sp. infection. In the final phase of infection (24 dpi), there was a further decrease in urinary K excretion and a lower level of P in sp. infected immunocompetent hosts. During acanthamoebiasis in immunosuppressed hosts, increased excretion of Zn, Fe, and Cr was observed at the beginning of the infection, and increased Na excretion only at 16 days post sp. infection. Additionally, host immunosuppression affected the concentration of Fe, Cr, Zn, Cu, Mn, and Ca in urine.
PubMed: 37623999
DOI: 10.3390/pathogens12081039 -
The Science of the Total Environment Nov 2023Acanthamoeba is an opportunistic free-living heterotrophic protist that is the most predominant amoeba in diverse ecological habitats. Acanthamoeba causes amoebic...
Acanthamoeba is an opportunistic free-living heterotrophic protist that is the most predominant amoeba in diverse ecological habitats. Acanthamoeba causes amoebic keratitis (AK), a painful and potentially blinding corneal infection. Major risk factors for AK have been linked to non-optimal contact lens hygiene practices and Acanthamoeba contamination of domestic and recreational water. This study investigated the incidence and seasonal variation of Acanthamoeba spp. within coastal lagoons located on the eastern coast of Australia and then examined the association between Acanthamoeba and water abiotic factors and bacterial species within the water. Water samples were collected from four intermittently closed and open lagoons (ICOLLs) (Wamberal, Terrigal, Avoca and Cockrone) every month between August 2019 to July 2020 except March and April. qPCR was used to target the Acanthamoeba 18S rRNA gene, validated by Sanger sequencing. Water abiotic factors were measured in situ using a multiprobe metre and 16S rRNA sequencing (V3-V4) was performed to characterise bacterial community composition. Network analysis was used to gauge putative associations between Acanthamoeba incidence and bacterial amplicon sequence variants (ASVs). Among 206 water samples analysed, 79 (38.3%) were Acanthamoeba positive and Acanthamoeba level was significantly higher in summer compared with winter, spring, or autumn (p = 0.008). More than 50% (23/45) water samples of Terrigal were positive for Acanthamoeba which is a highly urbanised area with extensive recreational activities while about 32% (16/49) samples were positive from Cockrone that is the least impacted lagoon by urban development. All sequenced strains belonged to the pathogenic genotype T4 clade except two which were of genotype clades T2 and T5. Water turbidity, temperature, intl1 gene concentration, and dissolved O were significantly associated with Acanthamoeba incidence (p < 0.05). The ASVs level of cyanobacteria, Pseudomonas spp., Candidatus spp., and marine bacteria of the Actinobacteria phylum and Acanthamoeba 18S rRNA genes were positively correlated (Pearson's r ≥ 0.14). The presence of Acanthamoeba spp. in all lagoons, except Wamberal, was associated with significant differences in the composition of bacterial communities (beta diversity). The results of this study suggest that coastal lagoons, particularly those in urbanised regions with extensive water recreational activities, may pose an elevated risk to human health due to the relatively high incidence of pathogenic Acanthamoeba in the summer. These findings underscore the importance of educating the public about the rare yet devastating impact of AK on vision and quality of life, highlighting the need for collaborative efforts between public health officials and educators to promote awareness and preventive measures, especially focusing lagoons residents and travellers.
PubMed: 37541500
DOI: 10.1016/j.scitotenv.2023.165862 -
Synthesis, anti-amoebic activity and molecular docking simulation of eugenol derivatives against sp.Saudi Pharmaceutical Journal : SPJ :... Sep 2023Amoebae of the genus can cause diseases such as amoebic keratitis and granulomatous amoebic encephalitis. Until now, treatment options for these diseases have not been...
Amoebae of the genus can cause diseases such as amoebic keratitis and granulomatous amoebic encephalitis. Until now, treatment options for these diseases have not been fully effective and have several drawbacks. Therefore, research into new drugs is needed for more effective treatment of infections. Eugenol, a phenolic aromatic compound mainly derived from cloves, has a variety of pharmaceutical properties. In this study, nine eugenol derivatives (K1-K9), consisting of five new and four known compounds, were synthesized and screened for their antiamoebic properties against sp. The structure of these compounds was characterized spectroscopically by Fourier transform infrared (FTIR), Ultraviolet-Visible (UV-Vis), H and C Nuclear Magnetic Resonance (NMR) and mass spectrometer (MS). The derived molecules were screened for antiamoebic activity by determining IC values based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and observation of amoeba morphological changes by light and fluorescence microscopy. Most of the tested compounds possessed strong to moderate cytotoxic effects against trophozoite cells with IC values ranging from 0.61 to 24.83 μg/mL. Observation of amoebae morphology by light microscopy showed that the compounds caused the transformed cells to be roundish and reduced in size. Furthermore, fluorescence microscopy observation using acridine orange (AO) and propidium iodide (PI) (AO/PI) staining showed that the cells have damaged membranes by displaying a green cytoplasm with orange-stained lysosomes. Acidification of the lysosomal structure indicated disruption of the internal structure of cells when treated with eugenol derivatives. The observed biological results were also confirmed by interaction simulations based on molecular docking between eugenol derivatives and profilin. These interactions could affect the actin-binding ability of the protein, disrupting the shape and mobility of The overall results of this study demonstrate that eugenol derivatives can be considered as potential drugs against infections caused by
PubMed: 37546528
DOI: 10.1016/j.jsps.2023.101703 -
Pathogens (Basel, Switzerland) Nov 2023Few studies have been conducted in the cooling systems of power plants; they have focused on , leaving a gap in the knowledge of other pathogenic free-living amoebae in...
Few studies have been conducted in the cooling systems of power plants; they have focused on , leaving a gap in the knowledge of other pathogenic free-living amoebae in this environment. The objective of this study was to determine the occurrence of saline-tolerant pathogenic in a geothermal power plant. The identification of isolated amoebae at genus level was carried out, observing their morphological characteristics; the determination of genotype and species of was performed via molecular biology (PCR). Water temperature ranged from 18 to 43 °C and conductivity from 4.0 × 10 to 8.7 × 10 μS/cm; this last value was greater than the seawater value. Only five amoeba genera were found. was in all the sampling sites, showing high saline tolerance. The high temperature, but mainly high conductivity, were the environmental conditions that determined the presence of pathogenic free-living amoebae in the hot water. All the strains of killed the mice, having a mortality of 40 to 100%. genotypes T10 and T5 were identified, T10 is rarely isolated from the environment, while T5 is more frequent. This is the first time that genotypes T5 and T10 have been reported in the environment in Mexico.
PubMed: 38003827
DOI: 10.3390/pathogens12111363 -
Translational Vision Science &... Feb 2024To investigate the relationship between Acanthamoeba genotypes, clinical manifestations, and outcomes in Acanthamoeba keratitis (AK) patients.
PURPOSE
To investigate the relationship between Acanthamoeba genotypes, clinical manifestations, and outcomes in Acanthamoeba keratitis (AK) patients.
METHODS
This retrospective study included 159 culture-confirmed AK patients. Patients' data were collected, including demographics, initial diagnosis, treatments, and clinical features. The genotype of Acanthamoeba was identified through sequencing the Diagnostic Fragment 3 (DF3) region in the small ribosomal subunit RNA genes. The phylogenetic tree was constructed using the ClustalW model and maximum likelihood method. Cases with "poor outcome" were defined based on specific clinical criteria, including corneal perforation, keratoplasty, other eye surgery, duration of anti-amoebic therapy ≥8.0 months, and final visual acuity ≤20/80. "Better outcome" cases were the remainder. The correlation between T4 subtypes, clinical phenotypes, and clinical prognosis were further analyzed.
RESULTS
In this study, AK was primarily attributed to the T4A genotype, with a positive correlation between geographical and genetic distances. The primary clinical associated with T4 subtypes was deep stromal infiltration. Results was also showed a significant association between T4 subtypes and clinical outcomes (P = 0.021). Further analysis revealed that T4C was closely associated with a better prognosis (P = 0.040) and T4D with worse outcomes (P = 0.013).
CONCLUSIONS
In China, AK was predominantly caused by the T4A subtype. Geographical distance positively correlated with genetic distance. Clinical prognosis varied among different subtypes, notably in T4C and T4D.
TRANSLATIONAL RELEVANCE
This study demonstrated the association between T4 subtypes and clinical phenotypes, as well as the effects of T4 subtypes on clinical prognosis.
Topics: Humans; Acanthamoeba Keratitis; Phylogeny; Retrospective Studies; Genotype; China
PubMed: 38329750
DOI: 10.1167/tvst.13.2.5 -
Nature Communications Nov 2023Legionella pneumophila (LP) secretes more than 300 effectors into the host cytosol to facilitate intracellular replication. One of these effectors, SidH, 253 kDa in...
Legionella pneumophila (LP) secretes more than 300 effectors into the host cytosol to facilitate intracellular replication. One of these effectors, SidH, 253 kDa in size with no sequence similarity to proteins of known function is toxic when overexpressed in host cells. SidH is regulated by the LP metaeffector LubX which targets SidH for degradation in a temporal manner during LP infection. The mechanism underlying the toxicity of SidH and its role in LP infection are unknown. Here, we determined the cryo-EM structure of SidH at 2.7 Å revealing a unique alpha helical arrangement with no overall similarity to known protein structures. Surprisingly, purified SidH came bound to a E. coli EF-Tu/t-RNA/GTP ternary complex which could be modeled into the cryo-EM density. Mutation of residues disrupting the SidH-tRNA interface and SidH-EF-Tu interface abolish the toxicity of overexpressed SidH in human cells, a phenotype confirmed in infection of Acanthamoeba castellani. We also present the cryo-EM structure of SidH in complex with a U-box domain containing ubiquitin ligase LubX delineating the mechanism of regulation of SidH. Our data provide the basis for the toxicity of SidH and into its regulation by the metaeffector LubX.
Topics: Humans; Legionella pneumophila; Escherichia coli; Peptide Elongation Factor Tu; Legionnaires' Disease; Ubiquitin-Protein Ligases; Bacterial Proteins
PubMed: 37923743
DOI: 10.1038/s41467-023-42683-8 -
Pathogens and Global Health Oct 2023This study examines the effects of three different drugs with metformin, acarbose and pioglitazone active ingredients used for antidiabetic purposes on cysts and...
This study examines the effects of three different drugs with metformin, acarbose and pioglitazone active ingredients used for antidiabetic purposes on cysts and trophozoites. Cultures of trophozoites and cysts were prepared to test the anti-amoebic activity of metformin, acarbose and pioglitazone. Cultures were then prepared for cyst and trophozoite forms and parasites were exposed to different concentrations (0.750 mg/mL, 0.375 mg/mL, 0.186 mg/mL and 0.093 mg/mL) of metformin, acarbose and pioglitazone. As a result of the study, the reproductive potential suppressive effects and conversion from trophozoite form to cyst form of all three substances on trophozoites and cysts were determined. Parasites were counted at 12, 24 and 48 hours in the cell counter after staining with trypan blue. In comparison of the effects of metformin, acarbose and pioglitazone used in the study on trophozoites and cysts, it was observed that all three substances were statistically effective against cysts and trophozoites at a concentration of 0.750 mg/mL. Furthermore, it was determined that all concentrations of the three active substances included in the study significantly decreased the rate of cyst formation even at the end of the 7th day. In this context, it was determined that all three substances have amebicidal effects, and they significantly inhibit the transformation of trophozoites to cyst form. It is thought that these active substances, which are currently used as anti-diabetic, can be used in combination with other drugs in infections based on our study findings.
Topics: Animals; Trophozoites; Acanthamoeba castellanii; Hypoglycemic Agents; Acarbose; Pioglitazone; Metformin
PubMed: 36436006
DOI: 10.1080/20477724.2022.2151859