RESUMO
BACKGROUND: Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms. OBJECTIVES: To synthesize and evaluate the anti-Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development. METHODS: A panel of 12 S-MGBs was synthesized and anti-Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei, Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. RESULTS AND DISCUSSION: S-MGB-241 has an IC50 of 6.6 µM against A. castellanii, comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50â>â100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a ΔTmâ=â1â±â0.1°C. CONCLUSIONS: Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii, providing a starting point from which to begin optimization of DNA binding and potency.
Assuntos
Acanthamoeba castellanii , Acanthamoeba castellanii/efeitos dos fármacos , Humanos , Células HEK293 , Escherichia coli/efeitos dos fármacos , Antiprotozoários/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Testes de Sensibilidade Parasitária , Sobrevivência Celular/efeitos dos fármacosRESUMO
Soluble factors in the secretome of Acanthamoeba castellanii play crucial roles in the pathogenesis of Acanthamoeba keratitis (AK). Investigating the pathological effects of A. castellanii-derived conditioned medium (ACCM) on ocular cells can provide insights into the damage inflicted during AK. This study examined ACCM-induced cytotoxicity in primary human corneal stromal cells (CSCs) and a human SV40 immortalized corneal epithelial cell line (ihCECs) at varying ACCM concentrations (25 %, 50 %, 75 %, and 100 %). MTT, AlamarBlue, Sulforhodamine B, lactate dehydrogenase, and Caspase-3/7 activation assays were used to assess the impact of ACCM on the cell viability, proliferation and apoptosis. Additionally, fluorescent staining was used to reveal actin cytoskeleton changes. ACCM exposure significantly decreased cell viability, increased apoptosis, and disrupted the actin cytoskeleton, particularly at higher concentrations and longer exposures. Proteases were found to mediate these cytopathogenic effects, highlighting the need for characterization of A. castellanii proteases as key virulence factors in AK pathogenesis.
Assuntos
Acanthamoeba castellanii , Sobrevivência Celular , Células Epiteliais , Humanos , Acanthamoeba castellanii/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Células Epiteliais/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Linhagem Celular , L-Lactato Desidrogenase/metabolismo , Células Cultivadas , Proliferação de Células/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Caspase 3/metabolismo , Tiazóis/metabolismo , Sais de Tetrazólio , OxazinasRESUMO
Natural products play a significant role in providing the current demand as antiparasitic agents, which offer an attractive approach for the discovery of novel drugs. The present study aimed to evaluate in vitro the potential impact of seaweed Padina pavonica (P. pavonica) extract in combating Acanthamoeba castellanii (A. castellanii). The phytochemical constituents of the extract were characterized by Gas chromatography-mass spectrometry. Six concentrations of the algal extract were used to evaluate its antiprotozoal activity at various incubation periods. Our results showed that the extract has significant inhibition against trophozoites and cysts viability, with complete inhibition at the high concentrations. The IC50 of P. pavonica extract was 4.56 and 4.89 µg/mL for trophozoites and cysts, respectively, at 24 h. Morphological alterations of A. castellanii trophozoites/cysts treated with the extract were assessed using inverted and scanning electron microscopes and showed severe damage features upon treatment with the extract at different concentrations. Molecular Docking of extracted compounds against Acanthamoeba cytochrome P450 monooxygenase (AcCYP51) was performed using Autodock vina1.5.6. A pharmacokinetic study using SwissADME was also conducted to investigate the potentiality of the identified bioactive compounds from Padina extract to be orally active drug candidates. In conclusion, this study highlights the in vitro amoebicidal activity of P. pavonica extract against A. castellanii adults and cysts and suggests potential AcCYP51 inhibition.
Assuntos
Ceratite por Acanthamoeba , Acanthamoeba castellanii , Simulação de Acoplamento Molecular , Extratos Vegetais , Acanthamoeba castellanii/efeitos dos fármacos , Ceratite por Acanthamoeba/tratamento farmacológico , Ceratite por Acanthamoeba/parasitologia , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Antiprotozoários/farmacologia , Antiprotozoários/química , Trofozoítos/efeitos dos fármacos , Animais , HumanosRESUMO
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 MgCl2 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.
Assuntos
Acanthamoeba castellanii , Biguanidas , Ouabaína , Biguanidas/farmacologia , Acanthamoeba castellanii/efeitos dos fármacos , Ouabaína/farmacologia , Ceratite por Acanthamoeba/parasitologia , Ceratite por Acanthamoeba/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Humanos , Sinergismo Farmacológico , Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/efeitos dos fármacos , Desinfetantes/farmacologia , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidoresRESUMO
Acanthamoeba 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. Acanthamoeba 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 Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. 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 IC50 0.45 µM), VT1598 (0.25 µM), and VT1161 (0.20 µM). The crystal structures of A. castellanii 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.
Assuntos
Inibidores de 14-alfa Desmetilase , Esterol 14-Desmetilase , Esterol 14-Desmetilase/metabolismo , Esterol 14-Desmetilase/química , Inibidores de 14-alfa Desmetilase/farmacologia , Inibidores de 14-alfa Desmetilase/química , Inibidores de 14-alfa Desmetilase/síntese química , Relação Estrutura-Atividade , Acanthamoeba/enzimologia , Acanthamoeba/efeitos dos fármacos , Acanthamoeba castellanii/enzimologia , Acanthamoeba castellanii/efeitos dos fármacos , Cristalografia por Raios X , Antiprotozoários/farmacologia , Antiprotozoários/química , Antiprotozoários/síntese química , Modelos Moleculares , Estrutura MolecularRESUMO
Purpose: To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pÉK+) against Acanthamoeba castellanii. Methods: A. castellanii trophozoites and cysts were grown in the presence of pÉK solution (0-2.17 mM), pÉK or pÉK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pÉK, pÉK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration. Results: The toxicity of pÉK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pÉK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pÉK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pÉK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pÉK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL. Conclusions: pÉK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pÉK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.
Assuntos
Ceratite por Acanthamoeba/tratamento farmacológico , Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/farmacologia , Córnea/parasitologia , Infecções Oculares Parasitárias/tratamento farmacológico , Hidrogéis/farmacologia , Polilisina/farmacologia , Ceratite por Acanthamoeba/parasitologia , Amebicidas/toxicidade , Animais , Células Cultivadas , Soluções para Lentes de Contato/farmacologia , Modelos Animais de Doenças , Epitélio Corneano/efeitos dos fármacos , Infecções Oculares Parasitárias/parasitologia , Humanos , Hidrogéis/toxicidade , Microscopia de Fluorescência , Polilisina/toxicidade , Suínos , Trofozoítos/efeitos dos fármacosRESUMO
PURPOSE: To describe a small case series of infectious keratitis with poor visual outcomes after amniotic membrane (AM) placement and to prospectively evaluate whether AM demonstrates antibacterial activity in vitro against pathogens commonly isolated from infectious corneal ulcers. METHODS: A retrospective case series and in vitro study of antibacterial activity of dehydrated AM using disk diffusion and measurement of inhibitory zones for bacterial assessment and inverted microscopy analysis for Acanthamoeba sp. growth. RESULTS: Three cases of known etiology infectious keratitis are described where the clinical presentation worsened after treatment with AM. In vitro analysis of dehydrated AM, with and without a soft contact lens, demonstrated no inhibition of growth against Pseudomonas aeruginosa or Streptococcus pneumoniae. There was minimal growth inhibition of Staphylococcus aureus, although these zones of inhibition were much smaller than that surrounding the positive control. For Acanthamoeba sp., solubilized, dehydrated AM did not alter cyst density. CONCLUSIONS: In an in vitro analysis, dehydrated AM did not provide evidence for a potentially clinically meaningful antibacterial effect against organisms commonly isolated from corneal ulcers.
Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Âmnio/microbiologia , Âmnio/parasitologia , Moxifloxacina/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Ceratite por Acanthamoeba/parasitologia , Ceratite por Acanthamoeba/cirurgia , Adolescente , Adulto , Âmnio/transplante , Antibacterianos/farmacologia , Infecções Oculares Bacterianas/microbiologia , Infecções Oculares Bacterianas/cirurgia , Humanos , Ceratite/microbiologia , Ceratite/cirurgia , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Infecções por Pseudomonas/cirurgia , Estudos Retrospectivos , Infecções Estafilocócicas/cirurgia , Infecções Estreptocócicas/cirurgiaRESUMO
This work aimed to investigate, for the first time, the chemical composition, antioxidant, antiparasitic, cytotoxicity, and antimicrobial activities of the aromatic plant Limonium oleifolium Mill. essential oil (EO) and organic extracts. L.â oleifolium aerial parts essential oil was analyzed by GC-FID and GC-MS, and 46 constituents representing 98.25±1.12 % of the oil were identified. γ-Muurolene (10.81±0.07 %), cis-caryophyllene (7.71±0.06 %), o-cymene (7.07±0.01 %) and α-copaene (5.02±0.05 %) were the essential oil main compounds. The antioxidant activity of L.â oleifolium EO and organic extracts (MeOH, CHCl3 , AcOEt, BuOH) was explored using 2,2-diphenyl-1-picrylhydrazyl (DPPH), ABTS, ß-carotene/linoleic acid, cupric reducing antioxidant capacity (CUPRAC), and ferric reducing power assays. The results showed that L.â oleifolium EO exhibit antioxidant capacity (IC50 =17.40±1.32â µg/mL for DPPH assay, IC50 =29.82±1.08â µg/mL for ß-carotene assay, IC50 =25.23±1.01â µg/mL for ABTS assay, IC50 =9.11±0.08â µg/mL for CUPRAC assay and IC50 =19.41±2.06â mg/mL for reducing power assay). Additionally, the EO showed significant activity against trophozoite form of Acanthamoeba castellanii (IC50 =7.48±0.41â µg/mL) and promastigote form of Leishmania amazonensis (IC50 =19.36±1.06â µg/mL) and low cytotoxicity on murine macrophages (LC50 â 90.23±1.09â µg/mL), as well as good antimicrobial activity against Staphylococcus aureus, Escherichia coli, Klebsiella oxytoca, and Pseudomonas aeruginosa. These results suggest that L.â oleifolium essential oil is a valuable source of bioactive compounds presenting antioxidant, antiparasitic, and antimicrobial activities. Furthermore, it is considered nontoxic.
Assuntos
Antibacterianos/farmacologia , Antioxidantes/farmacologia , Antiparasitários/farmacologia , Extratos Vegetais/farmacologia , Plumbaginaceae/química , Acanthamoeba castellanii/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antiparasitários/química , Antiparasitários/isolamento & purificação , Bactérias/efeitos dos fármacos , Benzotiazóis/antagonistas & inibidores , Compostos de Bifenilo/antagonistas & inibidores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Testes de Sensibilidade Parasitária , Picratos/antagonistas & inibidores , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Ácidos Sulfônicos/antagonistas & inibidoresRESUMO
Cassia angustifolia Vahl. plant is used for many therapeutic purposes, for example, in people with constipation, skin diseases, including helminthic and parasitic infections. In our study, we demonstrated an amoebicidal activity of C. angustifolia extract against Acanthamoeba triangularis trophozoite at a micromolar level. Scanning electron microscopy (SEM) images displayed morphological changes in the Acanthamoeba trophozoite, which included the formation of pores in cell membrane and the membrane rupture. In addition to the amoebicidal activity, effects of the extract on surviving trophozoites were observed, which included cyst formation and vacuolization by a microscope and transcriptional expression of Acanthamoeba autophagy in response to the stress by quantitative polymerase chain reaction. Our data showed that the surviving trophozoites were not transformed into cysts and the trophozoite number with enlarged vacuole was not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of AcATG genes was slightly changed. Interestingly, AcATG16 decreased significantly at 12 h post treatment, which may indicate a transcriptional regulation by the extract or a balance of intracellular signalling pathways in response to the stress, whereas AcATG3 and AcATG8b remained unchanged. Altogether, these data reveal the anti-Acanthamoeba activity of C. angustifolia extract and the autophagic response in the surviving trophozoites under the plant extract pressure, along with data on the formation of cysts. These represent a promising plant for future drug development. However, further isolation and purification of an active compound and cytotoxicity against human cells are needed, including a study on the autophagic response at the protein level.
Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/farmacologia , Genes de Protozoários/efeitos dos fármacos , Extratos Vegetais/farmacologia , Senna/química , Transcrição Gênica/efeitos dos fármacos , Acanthamoeba castellanii/genética , Extratos Vegetais/químicaRESUMO
Acanthamoeba species are free-living amoebae isolated from many ecological areas such as swimming pools, dams, lakes, soil, and air filters. These amoebae are usually causing granulomatous amebic encephalitis and amebic keratitis in immunosuppressive individuals. In this study, the reproductive potential and morphological changes determined of Acanthamoeba castellanii trophozoite and cyst forms exposed to three different active substances derived from benzothiazole. Furthermore, the cytotoxic potential of these active substances determined by XTT analysis. In the study, axenic cultures prepared for Acanthamoeba castellanii cyst and trophozoite forms and parasite exposed to different concentrations of active substances. Cell counts of parasite cultures were performed at the 30 minutes, 1st, 6th, 12th, 24th, and 48th hour periods. As a result of the study, the reproductive potential suppressive effects of all three substances on Acanthamoeba castellanii trophozoites and cysts were determined. The most effective of these substances was 2-Amino-6(trifluoromethoxy)-benzothiazole. In the first three concentrations of this substance (0.1%, 0.05%, 0.025%), no determined trophozoite and cysts at the end of twenty four. Due to its strong ameobicidal effect, it is thought that 2-Amino-6(trifluoromethoxy)-benzothiazole may be a new therapeutic agent in diseases caused by acanthamoeba parasites by supporting this study with animal experiments.
Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Acanthamoeba castellanii/crescimento & desenvolvimento , Amebíase/tratamento farmacológico , Benzotiazóis/farmacologia , Amebicidas/farmacologia , Trofozoítos/efeitos dos fármacosRESUMO
Acanthamoeba keratitis is a sight-endangering eye infection, and causative organism Acanthamoeba presents a significant concern to public health, given escalation of contact lens wearers. Contemporary therapy is burdensome, necessitating prompt diagnosis and aggressive treatment. None of the contact lens disinfectants (local and international) can eradicate Acanthamoeba effectively. Using a range of compounds targeting cellulose, ion channels, and biochemical pathways, we employed bioassay-guided testing to determine their anti-amoebic effects. The results indicated that acarbose, indaziflam, terbuthylazine, glimepiride, inositol, vildagliptin and repaglinide showed anti-amoebic effects. Compounds showed minimal toxicity on human cells. Therefore, effects of the evaluated compounds after conjugation with nanoparticles should certainly be the subject of future studies and will likely lead to promising leads for potential applications.
Assuntos
Ceratite por Acanthamoeba/tratamento farmacológico , Ceratite por Acanthamoeba/parasitologia , Acanthamoeba castellanii/efeitos dos fármacos , Antiprotozoários/farmacologia , Lentes de Contato/parasitologia , Acarbose/farmacologia , Carbamatos/farmacologia , Linhagem Celular , Soluções para Lentes de Contato/farmacologia , Lentes de Contato/efeitos adversos , Células HaCaT , Humanos , Indenos/farmacologia , Inositol/farmacologia , Nanopartículas , Piperidinas/farmacologia , Compostos de Sulfonilureia/farmacologia , Triazinas/farmacologia , Vildagliptina/farmacologiaRESUMO
Acanthamoeba spp. cause a corneal infection, Acanthamoeba keratitis (AK), and a cerebral infection, granulomatous amoebic encephalitis (GAE). Though aggressive chemotherapy has been able to kill the active trophozoite form of Acanthamoeba, the encysted form of this parasite has remained problematic to resist physiological concentrations of drugs. The emergence of encysted amoeba into active trophozoite form poses a challenge to eradicate this parasite. Acanthamoeba trophozoites have active metabolic machinery that furnishes energy in the form of ATPs by subjecting carbohydrates and lipids to undergo pathways including glycolysis and beta-oxidation of free fatty acids, respectively. However, very little is known about the metabolic preferences and dependencies of an encysted trophozoite on minerals or potential nutrients that it consumes to live in an encysted state. Here, we investigate the metabolic and nutrient preferences of the encysted trophozoite of Acanthamoeba castellanii and the possibility to target them by drugs that act on calcium ion dependencies of the encysted amoeba. The experimental assays, immunostaining coupled with bioinformatics tools show that the encysted Acanthamoeba uses diverse nutrient pathways to obtain energy in the quiescent encysted state. These findings highlight potential pathways that can be targeted in eradicating amoebae cysts successfully.
Assuntos
Acanthamoeba castellanii/metabolismo , Antiprotozoários/química , Acanthamoeba castellanii/efeitos dos fármacos , Acanthamoeba castellanii/crescimento & desenvolvimento , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Sítios de Ligação , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Bases de Dados Factuais , Humanos , Ceratite/tratamento farmacológico , Ceratite/parasitologia , Ceratite/patologia , Simulação de Acoplamento Molecular , Nutrientes/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trofozoítos/efeitos dos fármacos , Trofozoítos/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/metabolismoRESUMO
BACKGROUND: Free-living amoebae of the genus Acanthamoeba are cosmopolitan, widely distributed protozoans that cause a severe, vision-threatening corneal infection known as Acanthamoeba keratitis (AK). The majority of the increasing number of AK cases are associated with contact lens use. Appropriate eye hygiene and effective contact lens disinfection are crucial in the prevention of AK because of the lack of effective therapies against it. Currently available multipurpose contact lens disinfection systems are not fully effective against Acanthamoeba trophozoites and cysts. There is an urgent need to increase the disinfecting activity of these systems to prevent AK infections. Synthesized nanoparticles (NPs) have been recently studied and proposed as a new generation of anti-microbial agents. It is also known that some plant metabolites, including tannins, have anti-parasitic activity. The aim of this study was to evaluate the anti-amoebic activity and cytotoxicity of tannic acid-modified silver NPs (AgTANPs) conjugated with selected multipurpose contact lens solutions. METHODS: The anti-amoebic activities of pure contact lens care solutions, and NPs conjugated with contact lens care solutions, were examined in vitro by a colorimetric assay based on the oxido-reduction of alamarBlue. The cytotoxicity assays were performed using a fibroblast HS-5 (ATCC CRL-11882) cell line. The results were statistically analysed by ANOVA and Student-Newman-Keuls test using P < 0.05 as the level of statistical significance. RESULTS: We show that the NPs enhance the anti-Acanthamoeba activities of the tested contact lens solutions without increasing their cytotoxicity profiles. The activities are enhanced within the minimal disinfection time recommended by the manufacturers. CONCLUSIONS: The conjugation of the selected contact lens solutions with AgTANPs might be a novel and promising approach for the prevention of AK infections among contact lens users.
Assuntos
Ceratite por Acanthamoeba/prevenção & controle , Acanthamoeba castellanii/efeitos dos fármacos , Soluções para Lentes de Contato/farmacologia , Nanopartículas Metálicas/química , Prata/farmacologia , Taninos/farmacologia , Animais , HumanosRESUMO
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 Acanthamoeba 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 Escherichia coli (E.coli) 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 E. coli 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 E.coli 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 Acanthamoeba keratitis by pre-testing the in vitro susceptibilities of the Acanthamoeba strain against drugs of interest with the non-nutrient E.coli agar plate assay.
Assuntos
Acanthamoeba castellanii , Amebicidas , Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/farmacologia , Animais , Escherichia coli , Triazenos , TrofozoítosRESUMO
Cytochromes P450 (P450, CYP) metabolize a wide variety of endogenous and exogenous lipophilic molecules, including most drugs. Sterol 14α-demethylase (CYP51) is a target for antifungal drugs known as conazoles. Using X-ray crystallography, we have discovered a domain-swap homodimerization mode in CYP51 from a human pathogen, Acanthamoeba castellanii CYP51 (AcCYP51). Recombinant AcCYP51 with a truncated transmembrane helix was purified as a heterogeneous mixture corresponding to the dimer and monomer units. Spectral analyses of these two populations have shown that the CO-bound ferrous form of the dimeric protein absorbed at 448 nm (catalytically competent form), whereas the monomeric form absorbed at 420 nm (catalytically incompetent form). AcCYP51 dimerized head-to-head via N-termini swapping, resulting in formation of a nonplanar protein-protein interface exceeding 2000 Å2 with a total solvation energy gain of -35.4 kcal/mol. In the dimer, the protomers faced each other through the F and G α-helices, thus blocking the substrate access channel. In the presence of the drugs clotrimazole and isavuconazole, the AcCYP51 drug complexes crystallized as monomers. Although clotrimazole-bound AcCYP51 adopted a typical CYP monomer structure, isavuconazole-bound AcCYP51 failed to refold 74 N-terminal residues. The failure of AcCYP51 to fully refold upon inhibitor binding in vivo would cause an irreversible loss of a structurally aberrant enzyme through proteolytic degradation. This assumption explains the superior potency of isavuconazole against A. castellanii The dimerization mode observed in this work is compatible with membrane association and may be relevant to other members of the CYP family of biologic, medical, and pharmacological importance. SIGNIFICANCE STATEMENT: We investigated the mechanism of action of antifungal drugs in the human pathogen Acanthamoeba castellanii. We discovered that the enzyme target [Acanthamoeba castellanii sterol 14α-demethylase (AcCYP51)] formed a dimer via an N-termini swap, whereas drug-bound AcCYP51 was monomeric. In the AcCYP51-isavuconazole complex, the protein target failed to refold 74 N-terminal residues, suggesting a fundamentally different mechanism of AcCYP51 inactivation than only blocking the active site. Proteolytic degradation of a structurally aberrant enzyme would explain the superior potency of isavuconazole against A. castellanii.
Assuntos
Inibidores de 14-alfa Desmetilase/farmacologia , Acanthamoeba castellanii/efeitos dos fármacos , Amebíase/tratamento farmacológico , Proteínas de Protozoários/antagonistas & inibidores , Esterol 14-Desmetilase/metabolismo , Inibidores de 14-alfa Desmetilase/uso terapêutico , Acanthamoeba castellanii/metabolismo , Amebíase/parasitologia , Cristalografia por Raios X , Humanos , Simulação de Dinâmica Molecular , Nitrilas/farmacologia , Nitrilas/uso terapêutico , Ligação Proteica , Domínios Proteicos/fisiologia , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/fisiologia , Proteólise/efeitos dos fármacos , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/ultraestrutura , Piridinas/farmacologia , Piridinas/uso terapêutico , Proteínas Recombinantes , Esterol 14-Desmetilase/ultraestrutura , Triazóis/farmacologia , Triazóis/uso terapêuticoRESUMO
The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1-R) play key roles in growth, regulation of nutrient metabolism and carbohydrate homeostasis. Insulin-like molecules in prokaryotes and other early life have been reported. However, an account of metabolic effects of insulin, transcriptomic evidence of expression of glucose transporting channels (GLUT) and homology modelling of IR and IGF1-R like proteins in unicellular life-forms have yet to be established. Acanthamoeba spp. has existed for about 2 billion years and is one of the earliest mitochondriate unicellular eukaryotic cells on Earth. Despite Acanthamoeba spp. being grown in a medium called peptone-yeast-glucose (PYG) for over 50 years, the mechanism and regulation of glucose uptake by IR or IGF1-R molecules in this microbe has not yet been reported. Several methods were utilized to validate the effects of insulin on trophozoites of A. castellanii, including: growth assays with insulin, estimation of glucose and potassium (K+) entry into the cell, and histology showing anabolic effects on proteins. Bioinformatic computational tools and homology modeling demonstrated the involvement of IR like proteins, GLUT, and adapter proteins in mediating the IR cascade. Growth assays showed proliferative effects in a dose range of 2.98-5.97 µmol/mL of insulin. After insulin exposure, A. castellanii trophozoites displayed enhanced Periodic acid-Sciff (PAS) staining. Amino acid sequence similarities and homology modelling revealed ACA1_163470 in Acanthamoeba spp. to be a homolog of human-IR. Acanthamoeba protein ACA1_336150 shares similarities with IGF1-R. Additionally, some proteins like ACA1_060920 have attributes of GLUT like channels on homology modelling and show similarity with human GLUT. Knowledge of IR and insulin effects in Acanthamoeba spp. contributes to its biology and advances current understanding behind the evolution of IR and IGF1-R signalling cascade.
Assuntos
Acanthamoeba castellanii/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Insulina/metabolismo , Receptor de Insulina/metabolismo , Acanthamoeba castellanii/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Sequência de Aminoácidos , Evolução Biológica , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica , Glucose/metabolismo , Imuno-Histoquímica , Insulina/farmacologia , Metformina/farmacologia , Modelos Moleculares , Conformação Proteica , Receptor de Insulina/química , Receptor de Insulina/genética , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Acanthamoeba is one of the organisms that cause corneal infection. In this study, attention was focused on potassium isostearate (iso-C18K, a branched chain fatty acid salt) for use in a multipurpose solution (MPS) against Acanthamoeba. An anti-amoebic test against Acanthamoeba castellanii ATCC 30010 (trophozoites type) was conducted. As a result, a growth reduction effect of 4 log units (99.99% suppression) was observed after incubation with 150 mM (5.0 w/v%) iso-C18K for 10 minutes. Furthermore, after the amoeba suspension was mixed with iso-C18K, disruption of cell membranes were observed, and the minimum amoebacidal concentration (MAC) at that time was 9.6 mM (0.31 w/v%). To evaluate the effectiveness as an MPS, assessment by verification tests was conducted using contact lenses. Reducing the concentration of iso-C18K caused a decrease in the number of viable cells, which was confirmed at a MAC of 1.2 mM (0.039 w/v%).
Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/farmacologia , Potássio/farmacologia , Estearatos/farmacologia , Trofozoítos/efeitos dos fármacos , Acanthamoeba castellanii/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Membrana Celular/efeitos dos fármacos , Córnea , Fusarium/efeitos dos fármacos , Fusarium/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Serratia marcescens/efeitos dos fármacos , Serratia marcescens/crescimento & desenvolvimento , Soluções , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Trofozoítos/crescimento & desenvolvimentoRESUMO
Acanthamoeba castellanii is a free-living amoeba which can cause a blinding keratitis and fatal granulomatous amoebic encephalitis. The treatment of Acanthamoeba infections is challenging due to formation of cyst. Quinazolinones are medicinally important scaffold against parasitic diseases. A library of nineteen new 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives was synthesized to evaluate their antiamoebic activity against Acanthamoeba castellanii. One-pot synthesis of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-ones (1-19) was achieved by reaction of 2-amino-4,5-dimethoxybenzoic acid, trimethoxymethane, and different substituted anilines. These compounds were purified and characterized by standard chromatographic and spectroscopic techniques. Antiacanthamoebic activity of these compounds was determined by amoebicidal, encystation, excystation and host cell cytopathogenicity in vitro assays at concentrations of 50 and 100 µg/mL. The IC50 was found to be between 100 and 50 µg/mL for all the compounds except compound 5 which did not exhibit amoebicidal effects at these concentrations. Furthermore, lactate dehydrogenase assay was also performed to evaluate the in vitro cytotoxicity of these compounds against human keratinocyte (HaCaT) cells. The results revealed that eighteen out of nineteen derivatives of quinazolinones significantly decreased the viability of A. castellanii. Furthermore, eighteen out of nineteen tested compounds inhibited the encystation and excystation, as well as significantly reduced the A. castellanii-mediated cytopathogenicity against human cells. Interestingly, while tested against human normal cell line HaCaT keratinocytes, all compounds did not exhibit any overt cytotoxicity. Furthermore, a detailed structure-activity relationship is also studied to optimize the most potent hit from these synthetic compounds. This report presents several potential lead compounds belonging to 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives for drug discovery against infections caused by Acanthamoeba castellanii.
Assuntos
Acanthamoeba castellanii/efeitos dos fármacos , Amebicidas/química , Amebicidas/farmacologia , Quinazolinonas/química , Quinazolinonas/farmacologia , Acanthamoeba castellanii/crescimento & desenvolvimento , Amebíase/tratamento farmacológico , Amebíase/parasitologia , Amebicidas/síntese química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Encistamento de Parasitas/efeitos dos fármacos , Quinazolinonas/síntese química , Relação Estrutura-AtividadeRESUMO
The effect of Camellia sinensis (green tea) on the growth of Acanthamoeba castellanii trophozoites was examined using a microplate based-Sulforhodamine B (SRB) assay. C. sinensis hot and cold brews at 75% and 100% concentrations significantly inhibited the growth of trophozoites. We also examined the structural alterations in C. sinensis-treated trophozoites using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). This analysis showed that C. sinensis compromised the cell membrane integrity and caused progressive destruction of trophozoites. C. sinensis also significantly inhibited the parasite's ability to form cysts in a dose-dependent manner and reduced the rate of excystation from cysts to trophozoites. C. sinensis exhibited low cytotoxic effects on primary corneal stromal cells. However, cytotoxicity was more pronounced in SV40-immortalized corneal epithelial cells. Chromatographic analysis showed that both hot and cold C. sinensis brews contained the same number and type of chemical compounds. This work demonstrated that C. sinensis has anti-acanthamoebic activity against trophozoite and cystic forms of A. castellanii. Further studies are warranted to identify the exact substances in C. sinensis that have the most potent anti-acanthamoebic effect.
Assuntos
Acanthamoeba castellanii , Antiprotozoários/farmacologia , Camellia sinensis , Extratos Vegetais/farmacologia , Acanthamoeba castellanii/efeitos dos fármacos , Acanthamoeba castellanii/ultraestrutura , Animais , Técnicas In Vitro , Trofozoítos/efeitos dos fármacos , Trofozoítos/ultraestruturaRESUMO
Nowadays, infections caused by fungi and protists constitute a serious problem for public health services. The limited number of treatment options coupled with the increasing number of resistant microorganisms makes necessary the development of new non-toxic antifungal and antiprotozoal agents. Cationic amino acid-based rhamnolipids have been recently prepared by our group and exhibited good antibacterial activity. In this work, the antifungal, antibiofilm and antiprotozoal activity of these new rhamnolipids was investigated against a collection of fluconazole-resistant strains of different Candida species and Acanthamoeba castellanii, respectively. The arginine-RLs exhibited good antifungal activity against all fluconazole-resistant Candida spp. strains tested at MICs ranging from 6.5 to 20.7â¯mg/L. Their mechanism of action involves alterations in the permeability of the cell membranes that provoke death by apoptosis. The Arginine based-RLs also disperse Candida biofilms at low concentrations, similar to the MICs. All RLs tested (anionic and cationic) showed antiprotozoal activity, the arginine derivatives had the best activity killing the Acanthamoeba castellanii at concentrations of 4â¯mg/L. Interestingly, these surfactants have a wide range of action against yeast and A. castellanii in which they do not show toxicity against keratinocytes and fibroblasts. These results indicate that these new rhamnolipids have a sufficiently wide safety margin to be considered good candidates for several pharmaceutical applications such as combating fungal resistance and microbial biofilms and the formulation of antiprotozoal drugs.