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Applied and Environmental Microbiology Nov 2023Persistence of in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face...
Persistence of in the aquatic environment contributes to the fatal diarrheal disease cholera, which remains a global health burden. In the environment, bacteria face predation pressure by heterotrophic protists such as the free-living amoeba . This study explores how a mutant of adapts to acquire essential nutrients and survive predation. Here, we observed that up-regulation of iron acquisition genes and genes regulating resistance to oxidative stress enhances pathogen fitness. Our data show that can defend predation to overcome nutrient limitation and oxidative stress, resulting in an enhanced survival inside the protozoan hosts.
Topics: Animals; Vibrio cholerae; Amoeba; Predatory Behavior; Cholera; Iron
PubMed: 37882527
DOI: 10.1128/aem.01095-23 -
Current Microbiology Oct 2023The obligate biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals, which results in large crop losses. Control of B. graminis in...
The obligate biotrophic fungal pathogen Blumeria graminis causes the powdery mildew disease of cereals, which results in large crop losses. Control of B. graminis in barley is mainly achieved by fungicide treatment and by breeding resistant varieties. Vampyrellid amoebae, just like mycophagous protists, are able to consume a variety of fungi. To reveal the impact of some selected fungus-consuming protists on Blumeria graminis f. sp. hordei (Bgh), and to evaluate the possibility of using these protists as biological agents in the future, their feeding behaviour on B. graminis spores on barley leaves was investigated. An experiment was carried out with five different protist isolates (Leptophrys vorax, Platyreta germanica, Theratromyxa weberi U 11, Theratromyxa weberi G7.2 and Acanthamoeba castellanii) and four matched controls, including the food sources of the cultures and the medium. Ten-day-old leaves of barley (Hordeum vulgare cv. Golden Promise) were first inoculated with Blumeria graminis (f. sp. hordei race A6) spores, then treated with protists and fungal colonies on the leaf surfaces were counted under the microscope after 5 days. The isolates L. vorax, P. germanica, and T. weberi U11 did not show a significant reduction in the number of powdery mildew colonies whereas the isolates T. weberi G7.2 and A. castellanii significantly reduced the number of powdery mildew colonies on the leaf surfaces compared to their respective controls. This indicates that these two isolates are capable of reducing B. graminis colonies on barley leaves and are suitable candidates for further investigation for possible use as biological agents. Nevertheless, the susceptibility to dryness and the cell division rate should be considered during the optimisation of the next steps like application procedure and whole plant treatment.
Topics: Hordeum; Ascomycota; Plant Leaves; Biological Factors; Plant Diseases
PubMed: 37872440
DOI: 10.1007/s00284-023-03497-5 -
European Journal of Protistology Oct 2023Acanthamoeba castellanii is a free-living amoeba and an opportunistic pathogen for humans that can cause encephalitis and, more commonly, Acanthamoeba keratitis. During...
Acanthamoeba castellanii is a free-living amoeba and an opportunistic pathogen for humans that can cause encephalitis and, more commonly, Acanthamoeba keratitis. During its life cycle, A. castellanii may present as proliferative and infective trophozoites or resistant cysts. The adhesion of trophozoites to host cells is a key first step in the pathogenesis of infection. A major virulence protein of Acanthamoeba is a mannose-binding protein (MBP) that mediates the adhesion of amoebae to cell surfaces. Ectophosphatases are ecto-enzymes that can dephosphorylate extracellular substrates and have already been described in several microorganisms. Regarding their physiological roles, there is consistent evidence that ectophosphatase activities play an important role in parasite-host interactions. In the present work, we identified and biochemically characterized the ectophosphatase activity of A. castellanii. The ectophosphatase activity is acidic, stimulated by magnesium, cobalt and nickel, and presents the following apparent kinetic parameters: K = 2.12 ± 0.54 mM p-NPP and V = 26.12 ± 2.53 nmol p-NP × h × 10 cells. We observed that sodium orthovanadate, ammonium molybdate, sodium fluoride, and inorganic phosphate are able to inhibit ectophosphatase activity. Comparing the two stages of the A. castellanii lifecycle, ectophosphatase activity is significantly higher in trophozoites than in cysts. The ectophosphatase activity is stimulated by mannose residues and is significantly increased when trophozoites interact with LLC-MK2 cells. The inhibition of ectophosphatase by pretreatment with sodium orthovanadate also inhibits the adhesion of trophozoites to epithelial cells. These results allow us to conclude that the ectophosphatase activity of A. castellanii is somehow important for the adhesion of trophozoites to their host cells. According to our data, we believe that the activation of MBP by mannose residues triggers the stimulation of ectophosphatase activity to facilitate the adhesion process.
Topics: Humans; Animals; Acanthamoeba castellanii; Mannose; Vanadates; Cell Adhesion; Sodium; Trophozoites; Acanthamoeba Keratitis
PubMed: 37871554
DOI: 10.1016/j.ejop.2023.126026 -
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 -
ACS Infectious Diseases Nov 2023Pathogenic free-living amoebae (pFLA) can cause life-threatening central nervous system (CNS) infections and warrant the investigation of new chemical agents to combat...
Pathogenic free-living amoebae (pFLA) can cause life-threatening central nervous system (CNS) infections and warrant the investigation of new chemical agents to combat the rise of infection from these pathogens. glucokinase (Glck), a key metabolic enzyme involved in generating glucose-6-phosphate, was previously identified as a potential target due to its limited sequence similarity with human Glck (Glck). Herein, we used our previously demonstrated multifragment kinetic target-guided synthesis (KTGS) screening strategy to identify inhibitors against pFLA glucokinases. Unlike the majority of previous KTGS reports, our current study implements a "shotgun" approach, where fragments were not biased by predetermined binding potentials. The study resulted in the identification of 12 inhibitors against 3 pFLA glucokinase enzymes─Glck, Glck (Glck), and Glck (Glck). This work demonstrates the utility of KTGS to identify small-molecule binders for biological targets where resolved X-ray crystal structures are not readily accessible.
Topics: Humans; Amoeba; Glucokinase; Naegleria fowleri; Balamuthia mandrillaris; Acanthamoeba castellanii
PubMed: 37820055
DOI: 10.1021/acsinfecdis.3c00284 -
Heliyon Sep 2023Amoebae of the genus are etiological agents of amoebic keratitis, for which up to now there is no treatment of choice and one of its main risk factors is the use of...
Amoebae of the genus are etiological agents of amoebic keratitis, for which up to now there is no treatment of choice and one of its main risk factors is the use of contact lenses, including cosmetic contact lenses. Recently there has been an increase in amoebic keratitis cases due to the use of cosmetic contact lenses. Therefore, having a solution for the care of lenses with an efficient disinfectant effect that prevents the adhesion of trophozoites to lenses becomes essential. This study was carried out to determine the effect of 8 multipurpose contact lenses care solutions on trophozoites viability, and the efficiency of two of them to prevent the trophozoites adherence onto two cosmetic contact lenses (Acuvue 2, approved by the US Food and Drug Administration, and Magic Eye CCL, not approved). After 3 h of interaction, only AO Sept Plus, OPTI FREE Replenish, Renu Plus, Bio True and Multiplus significantly reduced the number of viable trophozoites with respect to the control; at 6 h Renu Plus, and at 12 h Conta Soft Plus and Multiplus, maintained the inhibitory effect. Only Opti Free Pure Moist did not significantly reduce the number of viable trophozoites. Multiplus and Opti Free Pure Moist (selected for their greater and lesser antiamibic effect) significantly reduced trophozoite adherence to both lenses; however, Opti Free Pure Moist was more efficient, despite the fact that adhered similarly to both lenses. Our results show that in all the multipurpose solutions evaluated, hundreds of viable trophozoites remain after several hours of incubation. Therefore, storage of the lenses in their case with MPS maintains the potential risk of amoebic keratitis in, cosmetic contact lenses wearers. Moreover, the use of CCL, not approved by the FDA, can increase the risk factor for AK since its poor manufacture can favor the permanence of amoebae, in addition to being a risk for corneal integrity.
PubMed: 37809484
DOI: 10.1016/j.heliyon.2023.e19599 -
Parasitology International Feb 2024Acanthamoeba are ubiquitously distributed in the environment and can cause infection of the central nervous system as well a sight-threatening eye infection. Herein, the...
Acanthamoeba are ubiquitously distributed in the environment and can cause infection of the central nervous system as well a sight-threatening eye infection. Herein, the potential anti-amoebic activity of a series of sulfonate/sulfamate derivatives against pathogenic A. castellanii was evaluated. These compounds were tested using several assays namely amoebicidal, adhesion, excystation, cytotoxic, and cytopathogenicity. Amoebicidal assays revealed that the selected compounds reduced amoebae viability significantly (P < 0.05), and exhibited IC values at two-digit micromolar concentrations. Sulfamate derivatives 1j & 1k inhibited 50% of amoebae at 30.65 μM and 27.21 μM, respectively. The tested compounds blocked amoebae binding to host cells as well as inhibited amoebae excystation. Notably, the selected derivatives exhibited minimal human cell cytotoxicity but reduced parasite-mediated host cell damage. Overall, our study showed that sulfamate derivatives 1j & 1k have anti-amoebic potential and offer a promising avenue in the development of potential anti-amoebic drug candidates.
Topics: Humans; Acanthamoeba castellanii; Amebicides; Sulfonic Acids; Alkanesulfonates; Genotype
PubMed: 37806551
DOI: 10.1016/j.parint.2023.102814 -
Acta Tropica Dec 2023Acanthamoeba castellanii is an opportunistic free-living amoeba (FLA) pathogen which can cause fatal central nervous system (CNS) infection, granulomatous amoebic...
Acanthamoeba castellanii is an opportunistic free-living amoeba (FLA) pathogen which can cause fatal central nervous system (CNS) infection, granulomatous amoebic encephalitis (GAE) and potentially blinding ocular infection, Acanthamoeba keratitis (AK). Acanthamoeba species remain a challenging protist to treat due to the unavailability of safe and effective therapeutic drugs and their ability to protect themselves in the cyst stage. Natural products and their secondary metabolites play a pivotal role in drug discovery against various pathogenic microorganisms. In the present study, the ethyl acetate extract of Myristica cinnamomea King fruit was evaluated against A. castellanii (ATCC 50492), showing an IC of 45.102 ± 4.62 µg/mL. Previously, the bio-guided fractionation of the extract resulted in the identification of three active compounds, namely Malabaricones (A-C). The isolated and thoroughly characterized acylphenols were evaluated for their anti-amoebic activity against A. castellanii for the first time. Among tested compounds, Malabaricone B (IC of 101.31 ± 17.41 µM) and Malabaricone C (IC of 49.95 ± 6.33 µM) showed potent anti-amoebic activity against A. castellanii trophozoites and reduced their viability up-to 75 and 80 %, respectively. Moreover, both extract and Malabaricones also significantly (p < 0.05) inhibit the encystation and excystation of A. castellanii, while showed minimal toxicity against human keratinocyte cells (HaCaT cells) at lower tested concentrations. Following that, the explanation of the possible mechanism of action of purified compounds were assessed by detection of the state of chromatin. Hoechst/PI 33342 double staining showed that necrotic cell death occurred in A. castellanii trophozoites after 8 h treatment of Malabaricones (A-C). These findings demonstrate that Malabaricones B and C could serve as promising therapeutic options against A. castellanii infections.
Topics: Animals; Humans; Acanthamoeba castellanii; Amebicides; Myristica; Fruit; Amebiasis; Acanthamoeba Keratitis; Trophozoites
PubMed: 37783284
DOI: 10.1016/j.actatropica.2023.107033 -
Experimental Eye Research Nov 2023Therapeutic management of inflammation in infectious keratitis (IK) requires new strategy and targets for selective immunomodulation. Targeting host cell-type specific...
Therapeutic management of inflammation in infectious keratitis (IK) requires new strategy and targets for selective immunomodulation. Targeting host cell-type specific inflammatory responses might be a viable strategy to curtail unnecessary inflammation and reduce tissue damage without affecting pathogen clearance. This study explores the possibility of pathogen and host cell-type dependent differences in the inflammatory pathways relevant in the pathogenesis of IK. Human corneal epithelial cell line (HCEC) and phorbol 12-myristate-13 acetate (PMA) differentiated THP-1 macrophage line were infected with either Aspergillus flavus conidia or Acanthamoeba castellanii trophozoites and the elicited inflammatory responses were studied in terms of gene expression and secretion of proinflammatory factors interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-α) and an upstream inflammatory regulator and mediator protein-the Macrophage Migration Inhibitory Factor (MIF). Given the pleotropic mode of MIF function in diverse cell types relevant in many human diseases, we tested if MIF driven responses to infection is different in HCECs and THP-1 macrophages by studying its expression, secretion and involvement in inflammation by siRNA mediated knockdown. We also examined IK patient tear samples for MIF levels. Infection with A. flavus or A. castellanii induced IL-8 and TNF-α responses in HCECs and THP-1 macrophages but to different levels. Our preliminary human data showed that the level of secreted MIF protein was elevated in IK patient tear, however, MIF secretion by the two cell types were strikingly different in-vitro, under both normal and infected conditions. We found that HCECs released MIF constitutively, which was significantly inhibited with infection, whereas THP-1 macrophages were stimulated to release MIF during infection. MIF gene expression remained largely unaffected by infection in both the cell lines. Although MIF in HCECs appeared to be intracellularly captured during infection, MIF knockdown in HCECs associated with a partial reduction of the IL-8 and TNF-α expression produced by either of the pathogens, suggesting a pro-inflammatory role for MIF in HCECs, independent of its canonical cytokine like function. In contrast, MIF knockdown in THP-1 macrophages accompanied a dramatic increase in IL-8 and TNF-α expression during A. castellanii infection, while the responses to A. flavus infection remained unchanged. These data imply a host cell-type and pathogen specific distinction in the MIF- related inflammatory signaling and MIF as a potential selective immunomodulatory target in infectious keratitis.
Topics: Humans; Macrophage Migration-Inhibitory Factors; Tumor Necrosis Factor-alpha; Interleukin-8; Keratitis; Inflammation; Immunomodulation; Intramolecular Oxidoreductases
PubMed: 37774962
DOI: 10.1016/j.exer.2023.109669 -
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