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Parasites & Vectors May 2024Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing...
Characterization of novel extracellular proteases produced by Acanthamoeba castellanii after contact with human corneal epithelial cells and their relevance to pathogenesis.
BACKGROUND
Proteases produced by Acanthamoeba spp. play an important role in their virulence and may be the key to understanding Acanthamoeba pathogenesis; thus, increasing attention has been directed towards these proteins. The present study aimed to investigate the lytic factors produced by Acanthamoeba castellanii during the first hours of in vitro co-culture with human corneal epithelial cells (HCECs).
METHODS
We used one old and one recent Acanthamoeba isolate, both from patients with severe keratitis, and subsets of these strains with enhanced pathogenic potential induced by sequential passaging over HCEC monolayers. The proteolytic profiles of all strains and substrains were examined using 1D in-gel zymography.
RESULTS
We observed the activity of additional proteases (ranging from 33 to 50 kDa) during the early interaction phase between amoebae and HCECs, which were only expressed for a short time. Based on their susceptibilities to protease inhibitors, these proteases were characterized as serine proteases. Protease activities showed a sharp decline after 4 h of co-incubation. Interestingly, the expression of Acanthamoeba mannose-binding protein did not differ between amoebae in monoculture and those in co-culture. Moreover, we observed the activation of matrix metalloproteinases in HCECs after contact with Acanthamoeba.
CONCLUSIONS
This study revealed the involvement of two novel serine proteases in Acanthamoeba pathogenesis and suggests a pivotal role of serine proteases during Acanthamoeba-host cell interaction, contributing to cell adhesion and lysis.
Topics: Humans; Acanthamoeba castellanii; Epithelial Cells; Coculture Techniques; Epithelium, Corneal; Peptide Hydrolases; Acanthamoeba Keratitis; Serine Proteases; Protozoan Proteins; Virulence
PubMed: 38812022
DOI: 10.1186/s13071-024-06304-7 -
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 -
MSystems May 2024We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two strains, Neff (environmental) and T4 (clinical). Morphological analysis via...
We conducted a comprehensive comparative analysis of extracellular vesicles (EVs) from two strains, Neff (environmental) and T4 (clinical). Morphological analysis via transmission electron microscopy revealed slightly larger Neff EVs (average = 194.5 nm) compared to more polydisperse T4 EVs (average = 168.4 nm). Nanoparticle tracking analysis (NTA) and dynamic light scattering validated these differences. Proteomic analysis of the EVs identified 1,352 proteins, with 1,107 common, 161 exclusive in Neff, and 84 exclusively in T4 EVs. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping revealed distinct molecular functions and biological processes and notably, the T4 EVs enrichment in serine proteases, aligned with its pathogenicity. Lipidomic analysis revealed a prevalence of unsaturated lipid species in Neff EVs, particularly triacylglycerols, phosphatidylethanolamines (PEs), and phosphatidylserine, while T4 EVs were enriched in diacylglycerols and diacylglyceryl trimethylhomoserine, phosphatidylcholine and less unsaturated PEs, suggesting differences in lipid metabolism and membrane permeability. Metabolomic analysis indicated Neff EVs enrichment in glycerolipid metabolism, glycolysis, and nucleotide synthesis, while T4 EVs, methionine metabolism. Furthermore, RNA-seq of EVs revealed differential transcript between the strains, with Neff EVs enriched in transcripts related to gluconeogenesis and translation, suggesting gene regulation and metabolic shift, while in the T4 EVs transcripts were associated with signal transduction and protein kinase activity, indicating rapid responses to environmental changes. In this novel study, data integration highlighted the differences in enzyme profiles, metabolic processes, and potential origins of EVs in the two strains shedding light on the diversity and complexity of EVs and having implications for understanding host-pathogen interactions and developing targeted interventions for -related diseases.IMPORTANCEA comprehensive and fully comparative analysis of extracellular vesicles (EVs) from two strains of distinct virulence, a Neff (environmental) and T4 (clinical), revealed striking differences in their morphology and protein, lipid, metabolites, and transcripts levels. Data integration highlighted the differences in enzyme profiles, metabolic processes, and potential distinct origin of EVs from both strains, shedding light on the diversity and complexity of EVs, with direct implications for understanding host-pathogen interactions, disease mechanisms, and developing new therapies for the clinical intervention of -related diseases.
PubMed: 38717186
DOI: 10.1128/msystems.01226-23 -
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 -
Journal of Medicinal Chemistry May 2024are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The...
are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from . The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC 0.45 μM), VT1598 (0.25 μM), and VT1161 (0.20 μM). The crystal structures of CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.
Topics: Sterol 14-Demethylase; 14-alpha Demethylase Inhibitors; Structure-Activity Relationship; Acanthamoeba; Acanthamoeba castellanii; Crystallography, X-Ray; Antiprotozoal Agents; Models, Molecular; Molecular Structure
PubMed: 38683753
DOI: 10.1021/acs.jmedchem.4c00303 -
Frontiers in Cellular and Infection... 2024Amoebae are micropredators that play an important role in controlling fungal populations in ecosystems. However, the interaction between fungi and their amoebic...
Amoebae are micropredators that play an important role in controlling fungal populations in ecosystems. However, the interaction between fungi and their amoebic predators suggests that the pressure from predatory selection can significantly influence the development of fungal virulence and evolutionary processes. Thus, the purpose of this study was to investigate the adaptation of saprotrophic strains during their interactions with . We conducted a comprehensive analysis of survival after co-culture by colony counting of the yeast cells and examining yeast cell phenotypic and genetic characteristics. Our results indicated that exposure to amoebae enhanced the survival capacity of environmental and induced visible morphological alterations in , particularly by an increase in filamentation. These observed phenotypic changes were closely related to concurrent genetic variations. Notably, mutations in genes encoding transcriptional repressors ( and ), recognized for their negative regulation of filamentous growth, were exclusively identified in amoeba-passaged isolates, and absent in unexposed isolates. Furthermore, these adaptations increased the exposed isolates' fitness against various stressors, simultaneously enhancing virulence factors and demonstrating an increased ability to invade A549 lung human epithelial cells. These observations indicate that the sustained survival of under ongoing amoebic predation involved a key role of mutation events in microevolution to modulate the ability of these isolates to change phenotype and increase their virulence factors, demonstrating an enhanced potential to survive in diverse environmental niches.
Topics: Humans; Candida albicans; Virulence; Amoeba; Ecosystem; Virulence Factors; Mutation; Phenotype
PubMed: 38550616
DOI: 10.3389/fcimb.2024.1367656 -
PloS One 2024Acanthamoeba castellanii is infected with diverse nucleocytoplasmic large DNA viruses. Here, we report the co-isolation of 12 viral strains from marine sediments in...
Acanthamoeba castellanii is infected with diverse nucleocytoplasmic large DNA viruses. Here, we report the co-isolation of 12 viral strains from marine sediments in Uranouchi Inlet, Kochi, Japan. Based on the morphological features revealed by electron microscopy, these isolates were classified into four viral groups including Megamimiviridae, Molliviridae, Pandoraviridae, and Pithoviridae. Genomic analyses indicated that these isolates showed high similarities to the known viral genomes with which they are taxonomically clustered, and their phylogenetic relationships were also supported by core gene similarities. It is noteworthy that Molliviridae was isolated from the marine sediments in the Japanese warm temperate zone because other strains have only been found in the subarctic region. Furthermore, this strain has 19 and 4 strain-specific genes found in Mollivirus sibericum and Mollivirus kamchatka, respectively. This study extends our knowledge about the habitat and genomic diversity of Molliviridae.
Topics: Japan; Phylogeny; Virion; Viruses; Acanthamoeba castellanii; Genome, Viral
PubMed: 38547190
DOI: 10.1371/journal.pone.0301185 -
Microorganisms Feb 2024Free-living amoebae (FLA) are widely distributed protozoa in both natural and artificial environments such as drinking water. In addition to the ability of all FLA to...
Free-living amoebae (FLA) are widely distributed protozoa in both natural and artificial environments such as drinking water. In addition to the ability of all FLA to transport various pathogenic microorganisms, certain species, such as spp. or , have intrinsic pathogenic abilities and cause severe cerebral infections. Previous work has shown an enrichment of FLA cysts in biofilm developed in upper levels of Drinking Water Storage Towers (DWSTs), suggesting that differences in densities of FLA cysts may play a role in their unequal distribution in the water column. To evaluate this hypothesis, a model of a water column was created for this study and used to analyze the vertical distribution of cysts of the FLA , , and from 0 to 23 weeks. Interestingly, our data showed that the cysts of both and were enriched in upper water levels during their aging. However, cysts were equally distributed in the water column during the entire study. These results show that, in addition to the role of water level variation in the DWST, some FLA cysts can become less dense during their aging, which contributes to their enrichment in upper water and therefore biofilm levels.
PubMed: 38543525
DOI: 10.3390/microorganisms12030474 -
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 -
Mikrobiyoloji Bulteni Jan 2024Free-living amoebae belonging to the genus Acanthamoeba are microorganisms that live in air, soil and aquatic environments. In humans, they cause infections such as...
Free-living amoebae belonging to the genus Acanthamoeba are microorganisms that live in air, soil and aquatic environments. In humans, they cause infections such as amoebic keratitis, graulamotous amoebic encephalitis that are difficult to treat and can be fatal. In addition, it is known that they contribute to the replication of bacteria and increase their pathogenicity by being a host for various bacteria. However, information on its inhibitory properties against bacteria and its production of antimicrobial agents is very limited. In this context, in this study, it was aimed to investigate whether cell-free supernatants of Acanthamoeba strains have antibacterial effects against Pseudomonas aeruginosa isolates. Four different Acanthamoeba strains (A10, A13, A14, U.GÖL) isolated from aquatic environments in our country were selected and used in the study, P.aeruginosa isolates (PA2, PA3, PA4, PA5) were selected from clinical strains belonging to patients in our country. Acanthamoeba castellanii ATCC 50373 and P.aeruginosa ATCC 27853 were used as standard strains. P.aeruginosa isolates were grown on nutrient agar at 37 °C and Acanthamoeba strains were grown on E.coli spread non-nutrient agar at 30 °C under aerobic conditions. Pepton yeast extract glucose (PYG) medium supplemented with penicillin and streptomycin was used to obtain axenic cultures of Acanthamoeba strains. After the centrifugation of axenic cultures at 3000 rpm for five minutes, Acanthamoeba-cell-free supernatants were obtained by filtering the supernatant part through a sterile filter with a pore diameter of 0.22 µm. The antibacterial activities of these supernatants against P.aeruginosa isolates were determined using the colony counting method. Analysis of each Acanthamoeba-cell-free supernatants was performed according to the GC-MS method. Acanthamoeba-cell-free supernatants were found to have varying degrees of inhibitory effects (3.9-91.5%) against tested P.aeruginosa isolates. It was determined that the cell-free supernatant of A.castellanii ATCC 50373 strain showed the highest antibacterial effect (91.5%) against PA5 isolate. A14 strain showed similar inhibitory effects (89.4%) against the same Pseudomonas isolate. The average inhibitory effect of most of the Acanthamoeba strains of our country was found to be higher than that of the reference strain A.castellanii ATCC 50492. It is thought that the compounds responsible for the anti-Pseudomonas activity of the tested Acanthamoeba strains may be fructose, phosphoric acid, galactose, N-Acetylphenylalanine and glucopyranose determined as major compounds. This is the first study showing the anti-Pseudomonas activity of microorganisms of the genus Acanthamoeba living in the waters of our country. Acanthamoeba, which is widely found in nature, appears to be a good source for new antimicrobial agents.
Topics: Humans; Pseudomonas aeruginosa; Agar; Pseudomonas; Acanthamoeba castellanii; Anti-Bacterial Agents; Escherichia coli
PubMed: 38263942
DOI: 10.5578/mb.20249950