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International Journal For Parasitology.... May 2024Acanthamoeba, a free-living amoeba, is commonly found in various natural environments, such as rivers and soil, as well as in public baths, swimming pools, and sewers....
Acanthamoeba, a free-living amoeba, is commonly found in various natural environments, such as rivers and soil, as well as in public baths, swimming pools, and sewers. Acanthamoeba can cause severe illness such as granulomatous amoebic encephalitis and Acanthamoeba keratitis (AK) in humans. AK, the most recognized disease, can cause permanent visual impairment or blindness by affecting the cornea. AK commonly affects contact lens wearers who neglect proper cleaning habits. The symptoms of AK include epithelial and stromal destruction, corneal infiltrate, and intense ocular pain, occasionally necessitating surgical removal of the entire eyeball. Current AK treatment involves the hourly application of eye drops containing polyhexamethylene biocide (PHMB). However, studies have revealed their ineffectiveness against drug-resistant strains. Acanthamoeba can form cysts as a survival mechanism in adverse environments, though the exact mechanism remains unknown. Our experiments revealed that sodium P-type ATPase (ACA1_065450) is closely linked to encystation. In addition, various encystation buffers, such as MgCl or NaCl, induced the expression of P-type ATPase. Furthermore, we used ouabain, an ATPase inhibitor, to inhibit the Na/K ion pump, consequently decreasing the encystation rate of Acanthamoeba. Our primary objective is to develop an advanced treatment for AK. We anticipate that the combination of ouabain and PHMB may serve as an effective therapeutic approach against AK in the future.
PubMed: 38821038
DOI: 10.1016/j.ijpddr.2024.100550 -
Parasites & Vectors Jun 2023Legionella spp. can survive and replicate inside host cells such as protozoa and macrophages. After enough growth, Legionella is released from the host cells as free...
BACKGROUND
Legionella spp. can survive and replicate inside host cells such as protozoa and macrophages. After enough growth, Legionella is released from the host cells as free legionellae or Legionella-filled vesicles. The vesicles support Legionella to survive for a long time in the environment and transmit to a new host. In this study, we identified the differentially expressed genes of Acanthamoeba infected by Legionella (ACA1_114460, ACA1_091500, and ACA1_362260) and examined their roles in the formation of the excreted vesicles and escape of Legionella from the Acanthamoeba.
METHODS
After ingestion of Escherichia coli and Legionella pneumophila, expression levels of target genes in Acanthamoeba were measured by real-time polymerase chain reaction (PCR) analysis. The roles of target genes were investigated by transfection of small interfering RNA (siRNA). The formation of Legionella-containing excreted vesicles and the vesicular co-localization with the lysosomes were examined by Giemsa stain and LysoTracker stain.
RESULTS
ACA1_114460, ACA1_091500, and ACA1_362260 were upregulated after ingestion of Legionella in Acanthamoeba. ACA1_114460- and ACA1_091500-silenced Acanthamoeba failed to form the Legionella-containing excreted vesicles. Legionella was released as free legionellae from the Acanthamoeba. When the ACA1_362260 of Acanthamoeba was silenced, Legionella-containing excreted vesicles were fused with the lysosome.
CONCLUSIONS
These results indicated that ACA1_114460, ACA1_091500, and ACA1_362260 of Acanthamoeba played important roles in the formation of Legionella-containing excreted vesicles and inhibition of the lysosomal co-localization with the phagosome.
Topics: Legionella pneumophila; Acanthamoeba castellanii; Azure Stains; Coloring Agents; Endocytosis; Escherichia coli; RNA, Small Interfering
PubMed: 37380986
DOI: 10.1186/s13071-023-05824-y -
Applied and Environmental Microbiology Feb 2024In this study, we conducted an in-depth analysis to characterize potential () proteins capable of recognizing fungal β-1,3-glucans. specifically anchors curdlan or...
In this study, we conducted an in-depth analysis to characterize potential () proteins capable of recognizing fungal β-1,3-glucans. specifically anchors curdlan or laminarin, indicating the presence of surface β-1,3-glucan-binding molecules. Using optical tweezers, strong adhesion of laminarin- or curdlan-coated beads to was observed, highlighting their adhesive properties compared to controls (characteristic time τ of 46.9 and 43.9 s, respectively). Furthermore, () G217B, possessing a β-1,3-glucan outer layer, showed significant adhesion to compared to a G186 strain with an α-1,3-glucan outer layer (τ of 5.3 s vs τ 83.6 s). The addition of soluble β-1,3-glucan substantially inhibited this adhesion, indicating the involvement of β-1,3-glucan recognition. Biotinylated β-1,3-glucan-binding proteins from exhibited higher binding to G217B, suggesting distinct recognition mechanisms for laminarin and curdlan, akin to macrophages. These observations hinted at the β-1,3-glucan recognition pathway's role in fungal entrance and survival within phagocytes, supported by decreased fungal viability upon laminarin or curdlan addition in both phagocytes. Proteomic analysis identified several proteins capable of binding β-1,3-glucans, including those with lectin/glucanase superfamily domains, carbohydrate-binding domains, and glycosyl transferase and glycosyl hydrolase domains. Notably, some identified proteins were overexpressed upon curdlan/laminarin challenge and also demonstrated high affinity to β-1,3-glucans. These findings underscore the complexity of binding via β-1,3-glucan and suggest the existence of alternative fungal recognition pathways in .IMPORTANCE () and macrophages both exhibit the remarkable ability to phagocytose various extracellular microorganisms in their respective environments. While substantial knowledge exists on this phenomenon for macrophages, the understanding of 's phagocytic mechanisms remains elusive. Recently, our group identified mannose-binding receptors on the surface of that exhibit the capacity to bind/recognize fungi. However, the process was not entirely inhibited by soluble mannose, suggesting the possibility of other interactions. Herein, we describe the mechanism of β-1,3-glucan binding by and its role in fungal phagocytosis and survival within trophozoites, also using macrophages as a model for comparison, as they possess a well-established mechanism involving the Dectin-1 receptor for β-1,3-glucan recognition. These shed light on a potential parallel evolution of pathways involved in the recognition of fungal surface polysaccharides.
Topics: Acanthamoeba castellanii; Amoeba; Mannose; Proteomics; beta-Glucans; Glucans; Histoplasma
PubMed: 38259076
DOI: 10.1128/aem.01736-23 -
Journal of Medicinal Chemistry Jan 2024is an amoeba that inhabits soil and water in every part of the world. Acanthamoeba infection of the eye causes keratitis and can lead to a loss of vision. Current...
is an amoeba that inhabits soil and water in every part of the world. Acanthamoeba infection of the eye causes keratitis and can lead to a loss of vision. Current treatment options are only moderately effective, have multiple harmful side effects, and are tedious. In our study, we developed a novel drug screening method to define the inhibitory properties of potential new drugs against in vitro. We found that the clinically used carbonic anhydrase inhibitors, acetazolamide, ethoxzolamide, and dorzolamide, have promising antiamoebic properties.
Topics: Acanthamoeba castellanii; Carbonic Anhydrase Inhibitors; Amoeba; Drug Evaluation, Preclinical
PubMed: 38150360
DOI: 10.1021/acs.jmedchem.3c01020 -
Heliyon Jan 2024A rare but lethal central nervous system disease known as granulomatous amoebic encephalitis (GAE) and potentially blinding keratitis are diseases caused by free-living...
A rare but lethal central nervous system disease known as granulomatous amoebic encephalitis (GAE) and potentially blinding keratitis are diseases caused by free-living . Currently, no therapeutic agent can completely eradicate or prevent GAE. Synthetic compounds are a likely source of bioactive compounds for developing new drugs. This study synthesized seventeen 1,4-benzothiazine derivatives (I -XVII) by a base-catalyzed one-pot reaction of 2-amino thiophenol with substituted bromo acetophenones. Different spectroscopic techniques, such as EI-MS, H-, and C NMR (only for the new compounds), were used for the structural characterization and conformation of compounds. These compounds were assessed for the first time against All compounds showed anti-amoebic potential against , reducing its ability to encyst and excyst at 100 μM. Compounds , , and showed the most potent activities among all derivatives and significantly reduced the viability to 5.3 × 10 ( < 0.0003), 2 × 10 ( < 0.006), and 2.4 × 10 ( < 0.002) cells/mL, respectively. The cytotoxicity profile revealed that these molecules showed lower to moderate cytotoxicity, i.e., 36 %, 2 %, and 21 %, respectively, against human keratinocytes . These results indicate that 1,4-benzothiazines showed potent activity against trophozoites and cysts of . Hence, these 1,4-benzothiazine derivatives should be considered to develop new potential therapeutic agents against infections.
PubMed: 38205285
DOI: 10.1016/j.heliyon.2023.e23258 -
PloS One 2024Exploration of interspecies interactions between microorganisms can have taxonomic, ecological, evolutionary, or medical applications. To better explore interactions...
Exploration of interspecies interactions between microorganisms can have taxonomic, ecological, evolutionary, or medical applications. To better explore interactions between microorganisms it is important to establish the ideal conditions that ensure survival of all species involved. In this study, we sought to identify the ideal biotic and abiotic factors that would result in high co-culture viability of two interkingdom species, Pseudomonas aeruginosa and Acanthamoeba castellanii, two soil dwelling microbes. There have been limited studies showing long-term interactions between these two organisms as co-culture can result in high mortality for one or both organisms suggesting a predator-predator interaction may exist between them. In this study, we identified biotic and abiotic conditions that resulted in a high viability for both organisms in long-term co-culture, including optimizing temperature, nutrient concentration, choice of bacterial strains, and the initial ratio of interacting partners. These two species represent ideal partners for studying microbial interactions because amoebae act similarly to mammalian immune cells in many respects, and this can allow researchers to study host-pathogen interactions in vitro. Therefore, long-term interaction studies between these microbes might reveal the evolutionary steps that occur in bacteria when subjected to intense predation, like what occurs when pathogens enter the human body. The culture conditions characterized here resulted in high viability for both organisms for at least 14-days in co-culture suggesting that long-term experimental studies between these species can be achieved using these culture conditions.
Topics: Acanthamoeba castellanii; Pseudomonas aeruginosa; Coculture Techniques; Microbial Interactions; Temperature; Soil Microbiology
PubMed: 38913685
DOI: 10.1371/journal.pone.0305973 -
Antioxidants (Basel, Switzerland) Dec 2023is a ubiquitous genus of amoebae that can act as opportunistic parasites in both humans and animals, causing a variety of ocular, nervous and dermal pathologies....
is a ubiquitous genus of amoebae that can act as opportunistic parasites in both humans and animals, causing a variety of ocular, nervous and dermal pathologies. Despite advances in therapy, the management of patients with infections remains a challenge for health services. Therefore, there is a need to search for new active substances against Acanthamoebae. In the present study, we evaluated the amoebicidal activity of nitroxoline against the trophozoite and cyst stages of six different strains of . The strain showed the lowest IC value in the trophozoite stage (0.69 ± 0.01 µM), while the strain L-10 showed the lowest IC value in the cyst stage (0.11 ± 0.03 µM). In addition, nitroxoline induced in treated trophozoites of features compatibles with apoptosis and autophagy pathways, including chromatin condensation, mitochondrial malfunction, oxidative stress, changes in cell permeability and the formation of autophagic vacuoles. Furthermore, proteomic analysis of the effect of nitroxoline on trophozoites revealed that this antibiotic induced the overexpression and the downregulation of proteins involved in the apoptotic process and in metabolic and biosynthesis pathways.
PubMed: 38136200
DOI: 10.3390/antiox12122081 -
Microbiology Spectrum Mar 2024species are clinically relevant free-living amoebae (FLA) ubiquitously found in soil and water bodies. Metabolically active trophozoites graze on diverse microbes via...
species are clinically relevant free-living amoebae (FLA) ubiquitously found in soil and water bodies. Metabolically active trophozoites graze on diverse microbes via phagocytosis. However, functional studies on Rab GTPases (Rabs), which are critical for controlling vesicle trafficking and maturation, are scarce for this FLA. This knowledge gap can be partly explained by the limited genetic tools available for cell biology. Here, we developed plasmids to generate fusions of strain Neff proteins to the N- or C-termini of mEGFP and mCherry2. Phylogenomic and structural analyses of the 11 Neff Rab7 paralogs found in the RefSeq assembly revealed that eight of them had non-canonical sequences. After correcting the gene annotation for the Rab7A ortholog, we generated a line stably expressing an mEGFP-Rab7A fusion, demonstrating its correct localization to acidified macropinocytic and phagocytic vacuoles using fluorescence microscopy live cell imaging (LCI). Direct labeling of live ESTM1D_MKCAZ16_6a (Sm18) cells with pHrodo Red, a pH-sensitive dye, demonstrated that they reside within acidified, Rab7A-positive vacuoles. We constructed new mini-Tn7 delivery plasmids and tagged Sm18 with constitutively expressed mScarlet-I. Co-culture experiments of Neff trophozoites with Sm18::mTn7TC1_Pc_mScarlet-I, coupled with LCI and microplate reader assays, demonstrated that Sm18 underwent multiple replication rounds before reaching the extracellular medium via non-lytic exocytosis. We conclude that belongs to the class of bacteria that can use amoeba as an intracellular replication niche within a -containing vacuole that interacts extensively with the endocytic pathway.IMPORTANCEDiverse lineages (genotypes) are of increasing clinical concern, mainly causing amoebic keratitis and granulomatous amebic encephalitis among other infections. ranks among the top 10 most prevalent multidrug-resistant opportunistic nosocomial pathogens and is a recurrent member of the microbiome hosted by and other free-living amoebae. However, little is known about the molecular strategies deployed by for an intracellular lifestyle in amoebae and other professional phagocytes such as macrophages, which allow the bacterium to evade the immune system and the action of antibiotics. Our plasmids and easy-to-use microtiter plate co-culture assays should facilitate investigations into the cellular microbiology of interactions with and other opportunistic pathogens, which may ultimately lead to the discovery of new molecular targets and antimicrobial therapies to combat difficult-to-treat infections caused by these ubiquitous microbes.
Topics: Acanthamoeba castellanii; Stenotrophomonas maltophilia; Vacuoles; Phylogeny; Bacteria
PubMed: 38319117
DOI: 10.1128/spectrum.02988-23 -
Nature Communications May 2024Motion of a passive deformable object in an active environment serves as a representative of both in-vivo systems such as intracellular particle motion in Acanthamoeba...
Motion of a passive deformable object in an active environment serves as a representative of both in-vivo systems such as intracellular particle motion in Acanthamoeba castellanii, or in-vitro systems such as suspension of beads inside dense swarms of Escherichia coli. Theoretical modeling of such systems is challenging due to the requirement of well resolved hydrodynamics which can explore the spatiotemporal correlations around the suspended passive object in the active fluid. We address this critical lack of understanding using coupled hydrodynamic equations for nematic liquid crystals with finite active stress to model the active bath, and a suspended nematic droplet with zero activity. The droplet undergoes deformation fluctuations and its movement shows periods of "runs" and "stays". At relatively low interfacial tension, the droplet begins to break and mix with the outer active bath. We establish that the motion of the droplet is influenced by the interplay of spatial correlations of the flow and the size of the droplet. The mean square displacement shows a transition from ballistic to normal diffusion which depends on the droplet size. We discuss this transition in relation to spatiotemporal scales associated with velocity correlations of the active bath and the droplet.
PubMed: 38697961
DOI: 10.1038/s41467-024-47727-1 -
Frontiers in Microbiology 2024Giant viruses, categorized under are believed to exist ubiquitously in natural environments. However, comprehensive reports on isolated giant viruses remain scarce,...
Giant viruses, categorized under are believed to exist ubiquitously in natural environments. However, comprehensive reports on isolated giant viruses remain scarce, with limited information available on unrecoverable strains, viral proliferation sites, and natural hosts. Previously, the author highlighted , , and sp. styx, isolated from brackish water soil, as potential hotspots for giant virus multiplication. This study presents findings from nearly a year of monthly sampling within the same brackish water region after isolating the three aforementioned strains. This report details the recurrent isolation of a wide range of giant viruses. Each month, four soil samples were randomly collected from an approximately 5 × 10 m plot, comprising three soil samples and one water sample containing sediment from the riverbed. was used as a host for virus isolation. These efforts consistently yielded at least one viral species per month, culminating in a total of 55 giant virus isolates. The most frequently isolated species was (24 isolates), followed by (23 isolates), (6 isolates), and singular isolates of and . Notably, viruses were not consistently isolated from any of the four samples every month, with certain sites yielding no viruses. Cluster analysis based on isolate numbers revealed that soil samples from May and water and sediment samples from January produced the highest number of viral strains. These findings underscore brackish coastal soil as a significant site for isolating numerous giant viruses, highlighting the non-uniform distribution along coastlines.
PubMed: 38756730
DOI: 10.3389/fmicb.2024.1402690