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Translational Vision Science &... Sep 2023To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human...
PURPOSE
To investigate the combined anti-Acanthamoeba effects of nitric oxide (NO) donors and hypochlorite to maximize amoebicidal outcomes while minimizing damage to human corneal epithelial cells (HCECs).
METHODS
Acanthamoeba castellanii and primary cultured HCECs and keratocytes were treated with sodium hypochlorite (NaOCl), NO donors (sodium nitroprusside [SNP] and sodium nitrite [NaNO2]), or a combination of hypochlorite and NO donors. The viability of A. castellanii, HCECs, and keratocytes was assessed. Minimal inhibitory concentration (MIC) and fractional inhibitory concentration of NaOCl and NO donors were determined. The activation of mammalian targets of rapamycin (mTOR) and ERK and the expression of nitrite reductase and Nrf2 were assessed in HCECs using Western blot analysis. The cysticidal effects of combined NaOCl and NO donors were also evaluated.
RESULTS
A dose-dependent toxicity was observed in A. castellanii, HCECs, and keratocytes when treated with NaOCl and SNP. The range of tested NaNO2 concentrations showed no significant toxicity to HCECs; however, dose-dependent toxicity to A. castellanii was observed. The MIC of NaOCl against HCECs and A. castellanii was 8.0 mg/mL. The MIC of NaNO2 and SNP was 500 mM and 10 mM in both HCECs and A. castellanii, respectively. Weak attenuation of the mTOR and ERK phosphorylation was observed and Nrf2 expression decreased slightly after exposure of HCECs to 2.0 mg/mL NaOCl. For the combination treatment, NaOCl (0.125 mg/mL) was selected based on the safety of HCECs and the toxicity of A. castellanii. A more potent anti-Acanthamoeba effect and HCEC toxicity were observed when NaOCl was combined with SNP rather than NaNO2.
CONCLUSIONS
Combined NaOCl and NO donors had a stronger anti-Acanthamoeba effect compared to either drug alone.
TRANSLATIONAL RELEVANCE
This study demonstrates that the combined use of various drugs for the treatment of Acanthamoeba infection can enhance the anti-Acanthamoeba effect while minimizing the toxicity of the individual drug.
Topics: Humans; Animals; Acanthamoeba castellanii; Nitric Oxide Donors; Hypochlorous Acid; NF-E2-Related Factor 2; TOR Serine-Threonine Kinases; Mammals
PubMed: 37768280
DOI: 10.1167/tvst.12.9.23 -
Cellular and Molecular Life Sciences :... Apr 2021The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative...
The free-living amoeba Acanthamoeba castellanii occurs worldwide in soil and water and feeds on bacteria and other microorganisms. It is, however, also a facultative parasite and can cause serious infections in humans. The annotated genome of A. castellanii (strain Neff) suggests the presence of two different thioredoxin reductases (TrxR), of which one is of the small bacterial type and the other of the large vertebrate type. This combination is highly unusual. Similar to vertebrate TrxRases, the gene coding for the large TrxR in A. castellanii contains a UGA stop codon at the C-terminal active site, suggesting the presence of selenocysteine. We characterized the thioredoxin system in A. castellanii in conjunction with glutathione reductase (GR), to obtain a more complete understanding of the redox system in A. castellanii and the roles of its components in the response to oxidative stress. Both TrxRases localize to the cytoplasm, whereas GR localizes to the cytoplasm and the large organelle fraction. We could only identify one thioredoxin (Trx-1) to be indeed reduced by one of the TrxRases, i.e., by the small TrxR. This thioredoxin, in turn, could reduce one of the two peroxiredoxins tested and also methionine sulfoxide reductase A (MsrA). Upon exposure to hydrogen peroxide and diamide, only the small TrxR was upregulated in expression at the mRNA and protein levels, but not the large TrxR. Our results show that the small TrxR is involved in the A. castellanii's response to oxidative stress. The role of the large TrxR, however, remains elusive.
Topics: Acanthamoeba castellanii; Antioxidants; Glutathione Disulfide; Glutathione Reductase; Humans; Oxidation-Reduction; Oxidative Stress; Thioredoxin-Disulfide Reductase; Thioredoxins
PubMed: 33599799
DOI: 10.1007/s00018-021-03786-x -
Applied and Environmental Microbiology Jul 2017Plague is a flea-borne rodent-associated zoonotic disease caused by The disease is characterized by epizootics with high rodent mortalities, punctuated by...
Plague is a flea-borne rodent-associated zoonotic disease caused by The disease is characterized by epizootics with high rodent mortalities, punctuated by interepizootic periods when the bacterium persists in an unknown reservoir. This study investigates the interaction between and the ubiquitous soil free-living amoeba (FLA) to assess if the bacterium can survive within soil amoebae and whether intracellular mechanisms are conserved between infection of mammalian macrophages and soil amoebae. The results demonstrate that during coculture with amoebae, representative strains of epidemic biovars Medievalis, Orientalis, and Antiqua are phagocytized and able to survive within amoebae for at least 5 days. Key determinants of the intracellular interaction of and phagocytic macrophages, PhoP and the type three secretion system (T3SS), were then tested for their roles in the -amoeba interaction. Consistent with a requirement for the PhoP transcriptional activator in the intracellular survival of in macrophages, a PhoP mutant is unable to survive when cocultured with amoebae. Additionally, induction of the T3SS blocks phagocytic uptake of by amoebae, similar to that which occurs during macrophage infection. Electron microscopy revealed that in , resides intact within spacious vacuoles which were characterized using lysosomal trackers as being separated from the lysosomal compartment. This evidence for prolonged survival and subversion of intracellular digestion of within FLA suggests that protozoa may serve as a protective soil reservoir for is a reemerging flea-borne zoonotic disease. Sylvatic plague cycles are characterized by an epizootic period during which the disease spreads rapidly, causing high rodent mortality, and an interepizootic period when the bacterium quiescently persists in an unknown reservoir. An understanding of the ecology of in the context of its persistence in the environment and its reactivation to initiate a new epizootic cycle is key to implementing novel surveillance strategies to more effectively predict and prevent new disease outbreaks. Here, we demonstrate prolonged survival and subversion of intracellular digestion of within a soil free-living amoeba. This suggests the potential role for protozoa as a protective soil reservoir for , which may help explain the recrudescence of plague epizootics.
Topics: Acanthamoeba castellanii; Bacterial Proteins; Humans; Microbial Viability; Plague; Type III Secretion Systems; Yersinia pestis
PubMed: 28455335
DOI: 10.1128/AEM.00593-17 -
International Journal of Molecular... Aug 2021The bacterial pathogen , which causes enteritis, has a broad host range and extensive environmental longevity. In water and soil, Salmonella interacts with protozoa and...
The bacterial pathogen , which causes enteritis, has a broad host range and extensive environmental longevity. In water and soil, Salmonella interacts with protozoa and multiplies inside their phagosomes. Although this relationship resembles that between and mammalian phagocytes, the interaction mechanisms and bacterial genes involved are unclear. Here, we characterized global gene expression patterns of serovar Typhimurium within at the early stage of infection by Cappable-Seq. Gene expression features of Typhimurium within were presented with downregulation of glycolysis-related, and upregulation of glyoxylate cycle-related genes. Expression of Pathogenicity Island-1 (SPI-1), chemotaxis system, and flagellar apparatus genes was upregulated. Furthermore, expression of genes mediating oxidative stress response and iron uptake was upregulated within as well as within mammalian phagocytes. Hence, global Typhimurium gene expression patterns within help better understand the molecular mechanisms of adaptation to an amoeba cell and intracellular persistence in protozoa inhabiting water and soil ecosystems.
Topics: Acanthamoeba castellanii; Animals; Bacterial Proteins; Ecosystem; Gene Expression Regulation, Bacterial; Genomic Islands; Mammals; Salmonella typhimurium; Virulence
PubMed: 34445780
DOI: 10.3390/ijms22169077 -
Parasites, Hosts and Diseases Nov 2023Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae....
Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.
Topics: Acanthamoeba castellanii; Escherichia coli; Phagocytosis; Legionella pneumophila; Phagosomes
PubMed: 38043535
DOI: 10.3347/PHD.23088 -
Heliyon Sep 2020causes severe diseases such as Granulomatous Amebic Encephalitis (GAE) and keratitis (AK). Improving the culture media classically used for this amoeba could help to...
causes severe diseases such as Granulomatous Amebic Encephalitis (GAE) and keratitis (AK). Improving the culture media classically used for this amoeba could help to identify it quickly and facilitate its study as a biological model. The purpose of this study was to compare the growth of two genotypes (T3 and T4) in several culture media. (T3 genotype) and (T4 genotype) were cultured in PYG, TSY, TYI-S-33, RPMI, and RPMI-FBS medium. The number of amoebas grown in different culture media was counted and compared to each other for 14 days. Findings in this research revealed the highest growth in RPMI-FBS medium. For this reason, we can recommend this culture medium to promote the growth of in its biological studies.
PubMed: 32984575
DOI: 10.1016/j.heliyon.2020.e04805 -
Microbiology Spectrum Jun 2022Traditional cysticidal assays for species revolve around treating cysts with compounds and manually observing the culture for evidence of excystation. This method is...
Traditional cysticidal assays for species revolve around treating cysts with compounds and manually observing the culture for evidence of excystation. This method is time-consuming, labor-intensive, and low throughput. We adapted and trained a YOLOv3 machine learning, object detection neural network to recognize Acanthamoeba castellanii trophozoites and cysts in microscopy images to develop an automated cysticidal assay. This trained neural network was used to count trophozoites in wells treated with compounds of interest to determine if a compound treatment was cysticidal. We validated this new assay with known cysticidal and noncysticidal compounds. In addition, we undertook a large-scale bioluminescence-based screen of 9,286 structurally unique marine microbial metabolite fractions against the trophozoites of A. castellanii and identified 29 trophocidal hits. These hits were then subjected to this machine learning-based automated cysticidal assay. One marine microbial metabolite fraction was identified as both trophocidal and cysticidal. The free-living can exist as a trophozoite or cyst and both stages can cause painful blinding keratitis. Infection recurrence occurs in approximately 10% of cases due to the lack of efficient drugs that can kill both trophozoites and cysts. Therefore, the discovery of therapeutics that are effective against both stages is a critical unmet need to avert blindness. Current efforts to identify new anti- compounds rely primarily upon assays that target the trophozoite stage of the parasite. We adapted and trained a machine learning, object detection neural network to recognize trophozoites and cysts in microscopy images. Our machine learning-based cysticidal assay improved throughput, demonstrated high specificity, and had an exquisite ability to identify noncysticidal compounds. We combined this cysticidal assay with our bioluminescence-based trophocidal assay to screen about 9,000 structurally unique marine microbial metabolites against A. castellanii. Our screen identified a marine metabolite that was both trophocidal and cysticidal.
Topics: Acanthamoeba Keratitis; Acanthamoeba castellanii; Amebicides; Animals; Machine Learning; Trophozoites
PubMed: 35467370
DOI: 10.1128/spectrum.00077-22 -
Acta Tropica Jan 2023We examined the anti-acanthamoebic efficacy of green tea Camellia sinensis solvent extract (SE) or its chemical constituents against Acanthamoeba castellanii by using...
We examined the anti-acanthamoebic efficacy of green tea Camellia sinensis solvent extract (SE) or its chemical constituents against Acanthamoeba castellanii by using anti-trophozoite, anti-encystation, and anti-excystation assays. C. sinensis SE (625-5000 µg/mL) inhibited trophozoite replication within 24-72 h. C. sinensis SE exhibited a dose-dependent inhibition of encystation, with a marked cysticidal activity at 2500-5000 µg/mL. Two constituents of C. sinensis, namely epigallocatechin-3-gallate and caffeine, at 100 μM and 200 μM respectively, significantly inhibited both trophozoite replication and encystation. Cytotoxicity analysis showed that 156.25-2500 µg/mL of SE was not toxic to human corneal epithelial cells, while up to 625 µg/mL was not toxic to Madin-Darby canine kidney cells. This study shows the anti-acanthamoebic potential of C. sinensis SE against A. castellanii trophozoites and cysts. Pre-clinical studies are required to elucidate the in vivo efficacy and safety of C. sinensis SE.
Topics: Animals; Dogs; Humans; Acanthamoeba castellanii; Camellia sinensis; Caffeine; Solvents; Trophozoites
PubMed: 36280206
DOI: 10.1016/j.actatropica.2022.106729 -
Translational Vision Science &... Nov 2020The purpose of this study was to analyze the concentration-dependent effects of biguanides (polyhexamethylene biguanide [PHMB], chlorhexidine [CH]); diamidines...
PURPOSE
The purpose of this study was to analyze the concentration-dependent effects of biguanides (polyhexamethylene biguanide [PHMB], chlorhexidine [CH]); diamidines (hexamidine-diisethionate [HD], propamidine-isethionate [PD], dibromopropamidine-diisethionate [DD]); natamycin (NM); miltefosine (MF); povidone iodine (PVPI), and chlorin e6 PDT on trophozoites and cysts, in vitro.
METHODS
Strain 1BU was cultured in peptone-yeast extract-glucose medium. Trophozoites or cysts were cultured in PYG medium containing each agent at 100%, 50%, and 25% of maximum concentration for 2 hours. The percentage of dead trophozoites was determined using a non-radioactive cytotoxicity assay and trypan blue staining. Treated cysts were also maintained on non-nutrient agar () plates and observed for 3 weeks.
RESULTS
All tested drugs displayed significant cytotoxic effects on 1BU cells based on the biochemical and staining-based viability assays tested. On non-nutrient agar plates, neither trophozoites nor freshly formed cysts were observed after PHMB, PD, NM, and PVPI treatment, respectively, within 3 weeks. However, CH-, HD-, DD-, and MF-treated cysts could excyst, multiply, and encyst again.
CONCLUSIONS
The off-label drugs PHMB, PD, NM, and PVPI are under in vitro conditions more effective against strain 1BU than CH, HD, DD, and MF. Our findings also suggest that the non-nutrient agar plate assay should be considered as method of choice for the in vitro analysis of the treatment efficacy of anti-amoebic agents.
TRANSLATIONAL RELEVANCE
Ophthalmologists may optimize the treatment regime against keratitis by pre-testing the in vitro susceptibilities of the strain against drugs of interest with the non-nutrient agar plate assay.
Topics: Acanthamoeba castellanii; Amebicides; Animals; Escherichia coli; Triazenes; Trophozoites
PubMed: 33262903
DOI: 10.1167/tvst.9.12.29 -
International Journal For Parasitology.... Dec 2021Free-living amoebae of Acanthamoeba spp. are causative agents of human infections such as granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK). The...
Free-living amoebae of Acanthamoeba spp. are causative agents of human infections such as granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK). The exploration of innovative chemical entities from natural sources that induce intrinsic apoptotic pathway or a Programmed Cell Death (PCD) in Acanthamoeba protozoa is essential to develop new therapeutic strategies. In this work, the antiamoeboid activity of squamins C-F (1-4), four cyclooctapeptides isolated from Annona globiflora was tested in vitro against Acanthamoeba castellanii Neff, A. polyphaga, A. quina, and A. griffini, and a structure-activity relationship was also established. The most sensitive strain against all tested cyclooctapeptides was A. castellanii Neff being the R conformers of the S-oxo-methionine residue, squamins D (2) and F (4), the most active against the trophozoite stage. It is remarkable that all four peptides showed no cytotoxic effects against murine macrophages cell line J774A.1. The analysis of the mode of action of squamins C-F against A. castellanii indicate that these cyclopeptides induced the mechanisms of programmed cell death (PCD). All peptides trigger mitochondrial damages, significant inhibition of ATP production compared to the negative control, chromatin condensation and slight damages in membrane that affects its permeability despite it conserves integrity at the IC for 24 h. An increase in reactive oxygen species (ROS) was observed in all cases.
Topics: Acanthamoeba Keratitis; Acanthamoeba castellanii; Amebiasis; Animals; Annona; Humans; Mice; Trophozoites
PubMed: 34411895
DOI: 10.1016/j.ijpddr.2021.08.003