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Molecular and Biochemical Parasitology Dec 2023Acanthamoeba are known to cause a vision threatening eye infection typically due to contact lens wear, and an infection of the central nervous system. The ability of...
Acanthamoeba are known to cause a vision threatening eye infection typically due to contact lens wear, and an infection of the central nervous system. The ability of these amoebae to switch phenotypes, from an active trophozoite to a resistant cyst form is not well understood; the cyst stage is often resistant to chemotherapy, which is of concern given the rise of contact lens use and the ineffective disinfectants available, versus the cyst stage. Herein, for the first time, a range of raloxifene sulfonate/sulfamate derivatives which target nucleotide pyrophosphatase/phosphodiesterase enzymes, were assessed using amoebicidal and excystation tests versus the trophozoite and cyst stage of Acanthamoeba. Moreover, the potential for cytopathogenicity inhibition in amoebae was assessed. Each of the derivatives showed considerable anti-amoebic activity as well as the ability to suppress phenotypic switching (except for compound 1a). Selected raloxifene derivatives reduced Acanthamoeba-mediated host cell damage using lactate dehydrogenase assay. These findings suggest that pyrophosphatase/phosphodiesterase enzymes may be valuable targets against Acanthamoeba infections.
Topics: Animals; Acanthamoeba castellanii; Raloxifene Hydrochloride; Sulfonic Acids; Trophozoites; Alkanesulfonates; Phosphoric Diester Hydrolases
PubMed: 37562558
DOI: 10.1016/j.molbiopara.2023.111582 -
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 -
BMC Microbiology Jul 2023Acanthamoeba castellanii is a free-living protist that feeds on diverse bacteria. A. castellanii has frequently been utilized in studies on microbial interactions....
BACKGROUND
Acanthamoeba castellanii is a free-living protist that feeds on diverse bacteria. A. castellanii has frequently been utilized in studies on microbial interactions. Grazing bacteria also exhibit diverse effects on the physiological characteristics of amoebae, such as their growth, encystation, and cytotoxicity. Since the composition of amoebae amino acids is closely related to cellular activities, it can indicate the overall responses of A. castellanii to various stimuli.
METHOD
A. castellanii was exposed to different culture conditions in low-nutrient medium with heat-killed DH5α to clarify their effects. A targeted metabolomic technique was utilized to evaluate the concentration of cellular amino acids. The amino acid composition and pathways were analyzed by two web-based tools: MetaboAnalyst and Pathview. Then, long-term exposure to A. castellanii was investigated through in silico and in vitro methods to elucidate the homeostasis of amino acids and the growth of A. castellanii.
RESULTS
Under short-term exposure, all kinds of amino acids were enriched in all exposed groups. In contrast to the presence of heat-killed bacteria, the medium exhibited obvious effects on the amino acid composition of A. castellanii. After long-term exposure, the amino acid composition was more similar to that of the control group. A. castellanii may achieve amino acid homeostasis through pathways related to alanine, aspartate, citrulline, and serine.
DISCUSSION
Under short-term exposure, compared to the presence of bacteria, the type of medium exerted a more powerful effect on the amino acid composition of the amoeba. Previous studies focused on the interaction of the amoeba and bacteria with effective secretion systems and effectors. This may have caused the effects of low-nutrient environments to be overlooked.
CONCLUSION
When A. castellanii was stimulated in the coculture system through various methods, such as the presence of bacteria and a low-nutrient environment, it accumulated intracellular amino acids within a short period. However, different stimulations correspond to different amino acid compositions. After long-term exposure, A. castellanii achieved an amino acid equilibrium by downregulating the biosynthesis of several amino acids.
Topics: Acanthamoeba castellanii; Escherichia coli; Coculture Techniques; Amino Acids; Acclimatization; Hot Temperature; Culture Media
PubMed: 37495951
DOI: 10.1186/s12866-023-02942-6 -
Biochimica Et Biophysica Acta. General... Oct 2023S1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn-dependent acidic...
BACKGROUND
S1-like nucleases are widespread enzymes commonly used in biotechnology and molecular biology. Although it is commonly believed that they are mainly Zn-dependent acidic enzymes, we have found that numerous members of this family deviate from this rule. Therefore, in this work, we decided to check how broad is the range of non‑zinc-dependent S1-like nucleases and what is the molecular basis of their activities.
METHODS
S1-like nucleases chosen for analysis were achieved through heterologous expression in appropriate eukaryotic hosts. To characterize nucleases' active-site properties, point mutations were introduced in selected positions. The enzymatic activities of wild-type and mutant nucleases were tested by in-gel nuclease activity assay.
RESULTS
We discovered that S1-like nucleases encoded by non-vascular plants and single-celled protozoa, like their higher plant homologues, exhibit a large variety of catalytic properties. We have shown that these individual properties are determined by specific non-conserved active site residues.
CONCLUSIONS
Our findings demonstrate that mutations that occur during evolution can significantly alter the catalytic properties of S1-like nucleases. As a result, different ions can compete for particular S1-type nucleases' active sites. This phenomenon undermines the existing classification of S1-like nucleases.
GENERAL SIGNIFICANCE
Our findings have numerous implications for applications and understanding the S1-like nucleases' biological functions. For example, new biotechnological applications should take into account their unexpected catalytic properties. Moreover, these results demonstrate that the trinuclear zinc-based model commonly used to characterize the catalytic activities of S1-like nucleases is insufficient to explain the actions of non‑zinc-dependent members of this family.
Topics: Catalytic Domain; Endonucleases; Plants; Eukaryotic Cells; Catalysis
PubMed: 37463618
DOI: 10.1016/j.bbagen.2023.130424 -
Infection and Immunity Aug 2023Previously, we showed that Legionella pneumophila secretes rhizoferrin, a polycarboxylate siderophore that promotes bacterial growth in iron-deplete media and the murine...
Previously, we showed that Legionella pneumophila secretes rhizoferrin, a polycarboxylate siderophore that promotes bacterial growth in iron-deplete media and the murine lung. Yet, past studies failed to identify a role for the rhizoferrin biosynthetic gene () in L. pneumophila infection of host cells, suggesting the siderophore's importance was solely linked to extracellular survival. To test the possibility that rhizoferrin's relevance to intracellular infection was missed due to functional redundancy with the ferrous iron transport (FeoB) pathway, we characterized a new mutant lacking both and . This mutant was highly impaired for growth on bacteriological media that were only modestly depleted of iron, confirming that rhizoferrin-mediated ferric iron uptake and FeoB-mediated ferrous iron uptake are critical for iron acquisition. The mutant, but not its -containing complement, was also highly defective for biofilm formation on plastic surfaces, demonstrating a new role for the L. pneumophila siderophore in extracellular survival. Finally, the mutant, but not its complement containing , proved to be greatly impaired for growth in Acanthamoeba castellanii, , and human U937 cell macrophages, revealing that rhizoferrin does promote intracellular infection by L. pneumophila. Moreover, the application of purified rhizoferrin triggered cytokine production from the U937 cells. Rhizoferrin-associated genes were fully conserved across the many sequenced strains of L. pneumophila examined but were variably present among strains from the other species of . Outside of , the closest match to the L. pneumophila rhizoferrin genes was in Aquicella siphonis, another facultative intracellular parasite of amoebae.
Topics: Animals; Mice; Humans; Legionella pneumophila; Siderophores; Amoeba; U937 Cells; Bacterial Proteins; Iron; Macrophages; Biofilms
PubMed: 37428036
DOI: 10.1128/iai.00072-23 -
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 -
Microorganisms May 2023Protozoan grazing is a major cause of bacterial mortality and controls bacterial population size and composition in the natural environment. To enhance their survival,...
Protozoan grazing is a major cause of bacterial mortality and controls bacterial population size and composition in the natural environment. To enhance their survival, bacteria evolved many defense strategies to avoid grazing by protists. Cell wall modification is one of the defense strategies that helps bacteria escape from recognition and/or internalization by its predators. Lipopolysaccharide (LPS) is the major component of Gram-negative bacterial cell wall. LPS is divided into three regions: lipid A, oligosaccharide core and O-specific polysaccharide. O-polysaccharide as the outermost region of LPS provides protection against predation by ; however, the characteristics of O-polysaccharide contribute to this protection remain unknown. Here, we investigate how length, structure and composition of LPS affect recognition and internalization by . We found that length of O-antigen does not play a significant role in regulating bacterial recognition by . However, the composition and structure of O-polysaccharide play important roles in providing resistance to predation.
PubMed: 37374879
DOI: 10.3390/microorganisms11061377 -
Acta Parasitologica Sep 2023This study aimed to examine the ultrastructure, cytotoxicity, phagocytosis, and antioxidant responses of Acanthamoeba castellanii trophozoites exposed to sublethal...
PURPOSE
This study aimed to examine the ultrastructure, cytotoxicity, phagocytosis, and antioxidant responses of Acanthamoeba castellanii trophozoites exposed to sublethal plasma-activated water.
METHODS
Trophozoites were exposed to a sublethal treatment of PAW and compared to untreated viable trophozoites via adhesion assays on macrophage monolayers, osmo- and thermotolerance tests. Bacterial uptake was assessed in treated cells to evaluate their phagocytic characteristics. Oxidative stress biomarkers and antioxidant activities were compared in treated and untreated trophozoites. Finally, the expression of the mannose-binding protein (MBP), cysteine protease 3 (CP3), and serine endopeptidase (SEP) genes was determined in cells.
RESULTS
In PAW-treated trophozoites, cytopathic effects were more extensive and resulted in the detachment of macrophage monolayers. Treated trophozoites could not grow at high temperatures (43 °C). Moreover, they showed osmotolerance to 0.5 M D-mannitol but not to 1 M. Results demonstrated a higher bacterial uptake rate by PAW-treated trophozoites than untreated cells. Activities of superoxide dismutase and catalase and catalase were significantly greater in the treated trophozoites, and the glutathione and glutathione/glutathione disulfide were significantly lower in the PAW-treated cells. Exposure to PAW also significantly increased the malondialdehyde level and total antioxidant capacity. Treatment with PAW led to significantly higher expression of virulent genes like MBP, CP3, and SEP.
CONCLUSION
PAW is a double-edged sword against A. castellanii. PAW is an effective antiamoebic agent in proper usage, whereas its sublethal exposure may reduce its effectiveness and increase amoebas' pathogenicity. An agent's adequate concentration and exposure time are essential to achieve optimum results.
Topics: Acanthamoeba castellanii; Virulence; Catalase; Antioxidants
PubMed: 37338633
DOI: 10.1007/s11686-023-00691-0 -
Microorganisms Apr 2023Amoebae of the genus cause a sight-threatening infection called keratitis. It is considered a rare disease in humans but poses an increasing threat to public health...
BACKGROUND
Amoebae of the genus cause a sight-threatening infection called keratitis. It is considered a rare disease in humans but poses an increasing threat to public health worldwide, including in Poland. We present successive isolates from serious keratitis preliminary examined in terms of the identification and monitoring of, among others, the in vitro dynamics of the detected strains.
METHODS
Clinical and combined laboratory methods were applied; causative agents of the keratitis were identified at the cellular and molecular levels; isolates were cultivated in an axenic liquid medium and regularly monitored.
RESULTS
In a phase-contrast microscope, sp. cysts and live trophozoites from corneal samples and in vitro cultures were assessed on the cellular level. Some isolates that were tested at the molecular level were found to correspond to , , , genotype T4. There was variability in the amoebic strain dynamics; high viability was expressed as trofozoites' long duration ability to intense multiply.
CONCLUSIONS
Some strains from keratitis under diagnosis verification and dynamics assessment showed enough adaptive capability to grow in an axenic medium, allowing them to exhibit significant thermal tolerance. In vitro monitoring that was suitable for verifying in vivo examinations, in particular, was useful to detect the strong viability and pathogenic potential of successive strains with a long duration of high dynamics.
PubMed: 37317148
DOI: 10.3390/microorganisms11051174 -
Photochemical & Photobiological... Sep 2023Despite access to drinking water being a basic human right, the availability of safe drinking water remains a privilege that many do not have and as a result, many lives...
A challenge in washing water with the sun: 24h of SODIS fails to inactivate Acanthamoeba castellanii cysts and internalized Pseudomonas aeruginosa under strong real sun conditions.
Despite access to drinking water being a basic human right, the availability of safe drinking water remains a privilege that many do not have and as a result, many lives are lost each year due to waterborne diseases associated with the consumption of biologically unsafe water. To face this situation, different low-cost household drinking water treatment technologies (HDWT) have been developed, and among them is solar disinfection (SODIS). Despite the effectiveness of SODIS and the epidemiological gains being consistently documented in the literature, there is a lack of evidence of the effectiveness of the batch-SODIS process against protozoan cysts as well as their internalized bacteria under real sun conditions. This work evaluated the effectiveness of the batch-SODIS process on the viability of Acanthamoeba castellanii cysts, and internalized Pseudomonas aeruginosa. Dechlorinated tap water contaminated with 5.6 × 10 cysts/L, contained in PET (polyethylene terephthalate) bottles, was exposed for 8 h a day to strong sunlight (531-1083 W/m of maximum insolation) for 3 consecutive days. The maximum water temperature inside the reactors ranged from 37 to 50 °C. Cyst viability was assessed by inducing excystment on non-nutrient agar, or in water with heat-inactivated Escherichia coli. After sun exposure for 0, 8, 16 and 24 h, the cysts remained viable and without any perceptible impairment in their ability to excyst. 3 and 5.5 log CFU/mL of P. aeruginosa were detected in water containing untreated and treated cysts, respectively, after 3 days of incubation at 30 °C. The batch-SODIS process is unable to inactivate A. castellanii cysts as well as its internalized bacteria. Although the use of batch SODIS by communities should continue to be encouraged, SODIS-disinfected water should be consumed within 3 days.
Topics: Humans; Sunlight; Pseudomonas aeruginosa; Acanthamoeba castellanii; Disinfection; Drinking Water; Water Purification; Bacteria; Water Microbiology
PubMed: 37296325
DOI: 10.1007/s43630-023-00440-2