Acanthamoeba Castellanii Trophozoites Research Articles

Acanthamoeba castellanii trophozoites need oxygen for normal functioning and lipids are their preferred substrate, offering new possibilities for treatment

Acanthamoebae, pathogenic free-living amoebae, can cause Granulomatous Amoebic Encephalitis (GAE) and keratitis, and for both types of infection, no adequate treatment options are available. As the metabolism of pathogens is an attractive treatment target, we set out to examine the energy metabolism of Acanthamoeba castellanii and studied the aerobic and anaerobic capacities of the trophozoites. Under anaerobic conditions, or in the presence of inhibitors of the electron-transport chain, A. castellanii trophozoites became rounded, moved sluggishly and stopped multiplying. This demonstrates that oxygen and the respiratory chain are essential for movement and replication. Furthermore, the simultaneous activities of both terminal oxidases, cytochrome c oxidase and the plant-like alternative oxidase, are essential for normal functioning and replication. The inhibition of normal function caused by the inactivity of the respiratory chain was reversible. Once respiration was made possible again, the rounded, rather inactive amoebae formed acanthopodia within 4 h and resumed moving, feeding and multiplying. Experiments with radiolabelled nutrients revealed a preference for lipids over glucose and amino acids as food. Subsequent experiments showed that adding lipids to a standard culture medium of trophozoites strongly increased the growth rate. Acanthamoeba castellanii trophozoites have a strictly aerobic energy metabolism and β-oxidation of fatty acids, the Krebs cycle, and an aerobic electron-transport chain coupled to the ATP synthase, producing most of the used ATP. The preference for lipids can be exploited, as we show that three known inhibitors of lipid oxidation strongly inhibited the growth of A. castellanii. In particular, thioridazine and perhexiline showed potent effects in low micromolar concentrations. Therefore, this study revealed a new drug target with possibly new options to treat Acanthamoeba infections.

Sodium Metabisulfite Inhibits Acanthamoeba Trophozoite Growth through Thiamine Depletion.

Acanthamoeba keratitis (AK) is a severe infection of the cornea. Prevention and treatment are difficult due to the inefficacy of currently available compounds. The impact of many commonly used compounds for routine examinations of Acanthamoeba is unexplored but might offer insight useful in combatting AK. In this study, we demonstrate that sodium metabisulfite, a common preservation constituent of eye care solutions, was found to be active against Acanthamoeba trophozoites at concentrations lower than that commonly found in eye drops (IC50 0.03 mg/mL). We demonstrate that sodium metabisulfite depletes thiamine from growth medium and that Acanthamoeba is a thiamine auxotroph, requiring thiamine salvage for growth. The inhibitory effects of sodium metabisulfite can be overcome by thiamine supplementation. These results are consistent with the lack of key enzymes for thiamine biosynthesis in the genome of Acanthamoeba, an area which might prove exploitable using new or existing compounds. Indeed, this study highlights sodium metabisulfite as a useful inhibitor of Acanthamoeba castellanii trophozoites in vitro and that it acts, at least in part, by limiting available thiamine.

The ecto-3′-nucleotidase activity of Acanthamoeba castellanii trophozoites increases their adhesion to host cells through the generation of extracellular adenosine

Acanthamoeba castellanii, a free-living amoeba, can be pathogenic to humans causing a corneal infection named Acanthamoeba keratitis (AK). The mannose-binding protein (MBP) is well established as the major factor related to Acanthamoeba pathogenesis. However, additional factors that participate in the adhesion process and protect trophozoites from cytolytic effects caused by host immune responses remain unknown. Ectonucleotidases, including 3′-nucleotidase/nuclease (3′-NT/NU), a bifunctional enzyme that was recently reported in A. castellanii, are frequently related to the establishment of parasitic infections. We verified that trophozoites can hydrolyze 3′-AMP, and this activity is similar to that observed in other protists. The addition of 3′-AMP increases the adhesion of trophozoites to LLC-MK2 epithelial cells, and this stimulation is completely reversed by DTT, an inhibitor of ecto-3′-nucleotidase activity. Lesions in corneal cells caused by AK infection may elevate the extracellular level of 3′-AMP. We believe that ecto-3′-nucleotidase activity can modulate the host immune response, thus facilitating the establishment of parasitic infection. This activity results from the generation of extracellular adenosine, which can bind to purinergic receptors present in host immune cells. Positive feedback may occur in this cascade of events once the ecto-3′-nucleotidase activity of trophozoites is increased by the adhesion of trophozoites to LLC-MK2 cells.

Anti-Acanthamoeba metallopharmaceuticals: Amoebicidal activity and synergistic effect of copper(II)coordination compound.

Acanthamoeba spp. emerged as a clinically important pathogen related to amoebic keratitis. It is among the main causes of corneal transplantation and vision loss in ophthalmology. The treatment protocols have a low cure rate, high toxicity, and need for drug combination. Transition metal compounds have shown promising antiprotozoal effects. This study evaluates the amoebicidal activity of copper(II) coordination compounds in combination with chlorhexidine and the cytotoxicity to topical ocular application. These copper(II) coordination compounds were screened against Acanthamoeba castellanii trophozoites (ATCC 50492). The cytotoxicity on rabbit corneal cell line (ATCC-CCL 60) was performed. The compounds showed high amoebicidal potential, with inhibition of trophozoite viability above 80%. The Cp12 and Cp13 compounds showed Minimal Inhibitory Amoebicidal Concentration (MIAC) at 200µM and mean inhibitory concentration (IC50) values lower than 10µM. Against the cysts, Cp12 showed a reduction in viability (48%) in the longest incubation period. A synergistic effect for Cp12 with chlorhexidine was observed. The compounds have a dose-dependent effect against rabbit corneal cells. Compound Cp12 haspotential for future application in developing ophthalmic formulations against Acanthamoeba keratitis and its use in multipurpose solutions is highlighted.

In vitro, amoebicidal activities of submerged plant Ceratophyllum demersum L. extract against Acanthamoeba castellanii trophozoites

Ceratophyllum demersum L. is a hydrophyte with potential for use as an analgesic, antipyretic and anti-inflammatory drug. It has also been stated that it is a hepatoprotective and anti-diarrheal agent with potential use in stomach disorders and respiratory diseases. Our study investigated the anti-amoebic activity of C. demersum, which became a hydrophyte underwater collected from Samsun River Miliç. Different concentrations of the pathogenic strain of Acanthamoeba castellanii (ATCC 30010) and the ethanolic extract of C. demersum were used to determine anti-Acanthamoeba activity. A. castellanii trophozoites were treated with C. demersum extract at different concentrations ranging from 1.9, 3.8, 7.6, 15.2, 30.4, 60.8 µg/mL and incubated at 26°C for 72 h. The 50% inhibitory concentration (IC50) of C. demersum extract was approximately 42.5 µg/mL at 72 hours. Trophozoite cell viability decreased to 42% and 58.33% in the presence of 30.4 and 60.8 µg/mL C. demersum extract at 72 hours, respectively. These results indicate that the ethanolic extract of C. demersum has anti-Acanthamoeba activity against A. castellanii trophozoites. The study highlights that this extract can be a potential protective drug source against Acanthamoeba trophozoites.

Acanthamoeba castellanii trophozoites escape killing by neutrophil extracellular traps using their 3′-nucleotidase/nuclease activity

Acanthamoeba castellanii is a free-living amoeba that acts as an opportunistic pathogen for humans and is the pathogenic agent of Acanthamoeba keratitis (AK). A. castellanii may present as proliferative and infective trophozoites or as resistant cysts during their life cycle. The immune response against AK is still poorly explored; however, it is well established that macrophages and neutrophils play essential roles in controlling corneal infection during the disease outcome. The release of NETs is one of the innate immune strategies to prevent parasite infection, especially when neutrophils interact with microorganisms that are too large to be phagocytosed, which is the case for amoeba species. The present work demonstrated that A. castellanii trophozoites can trigger NET formation upon in vitro interaction with neutrophils. Using DNase as a control, we observed increased parasite survival after coinciding with neutrophils, which may be correlated with NET degradation. Indeed, A. castellanii trophozoites degrade the NET DNA scaffold. Molecular analysis confirmed the occurrence of a 3′-nucleotidase/nuclease (3′-NT/NU) in the A. castellanii genome. We also demonstrated that trophozoites exhibit significantly higher 3′-NT/NU activity than cysts, which cannot trigger NET release. Considering that previous studies indicated the pathological role of 3′-NT-/NU in parasite infection, we suggest that this enzyme may act as the mechanism of escape of A. castellanii trophozoites from NETs.

Host cell-type and pathogen-specific immunomodulatory functions of macrophage migration inhibitory factor (MIF) in infectious keratitis

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.

Acanthamoeba castellanii trophozoites that survive multipurpose solutions are able to adhere to cosmetic contact lenses, increasing the risk of infection

Amoebae of the genus Acanthamoeba 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 Acanthamoeba castellanii 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 A. castellanii adhered similarly to both lenses. Our results show that in all the multipurpose solutions evaluated, hundreds of viable A. castellanii 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.

Taurine, a Component of the Tear Film, Exacerbates the Pathogenic Mechanisms of Acanthamoeba castellanii in the Ex Vivo Amoebic Keratitis Model.

Acanthamoeba spp. is the etiological agent of amoebic keratitis. In this study, the effect of taurine in physiological concentrations in tears (195 μM) on trophozoites of Acanthamoeba castellanii through the ex vivo amoebic keratitis model was evaluated. Trophozoites were coincubated with the Syrian golden hamster cornea (Mesocricetus auratus) for 3 and 6 h. Group 1: Control (-). Corneas coincubated with amoebic culture medium and taurine. Group 2: Control (+). Corneas coincubated with trophozoites without taurine. Group 3: Corneas coincubated with taurine 15 min before adding trophozoites. Group 4: Trophozoites coincubated 15 min with taurine before placing them on the cornea. Group 5: Corneas coincubated for 15 min with trophozoites; subsequently, taurine was added. Results are similar for both times, as evaluated by scanning electron microscopy. As expected, in the corneas of Group 1, no alterations were observed in the corneal epithelium. In the corneas of Group 2, few adhered trophozoites were observed on the corneal surface initiating migrations through cell junctions as previously described; however, in corneas of Groups 3, 4 and 5, abundant trophozoites were observed, penetrating through different corneal cell areas, emitting food cups and destabilizing corneal surface in areas far from cell junctions. Significant differences were confirmed in trophozoites adherence coincubated with taurine (p < 0.05). Taurine does not prevent the adhesion and invasion of the amoebae, nor does it favor its detachment once these have adhered to the cornea, suggesting that taurine in the physiological concentrations found in tears stimulates pathogenic mechanisms of A. castellanii.

Sublethal Exposure to Plasma-Activated Water Influences the Morphological Characteristics, Phagocytic Ability, and Virulence of Acanthamoeba castellanii.

This study aimed to examine the ultrastructure, cytotoxicity, phagocytosis, and antioxidant responses of Acanthamoeba castellanii trophozoites exposed to sublethal plasma-activated water. 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. 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 0.5M D-mannitol but not to 1M. 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. 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.

Amoebicidal and Cytotoxic Activity of Propolis collected from Different Regions in Turkey on Acanthamoeba castellanii Trophozoites

Objective: The present study aimed to investigate the amoebicidal effects of propolis collected from different regions in Turkey on Acanthamoeba trophozoites. Method: The propolis was collected from different geographic sites (Van, Erzurum, Gümüşhane, Ordu, Rize, and Muğla) in Turkey. Different concentrations of propolis ethanolic extract (in quantities from 1, 2, 3, 4, 5, 6, 7, and 8 mg/mL) and the same volume of trophozoites in 100 μl culture were blended for the identification of the amoebicidal efficiency of propolis. Results: The growth of trophozoites stopped in Turkish propolis extracts with 50% inhibitory concentrations (IC50)/48h for 5 mg/mL extract solution. Propolis showed more potent inhibitory effects on Acanthamoeba trophozoites at concentrations of 7, 6, 5, 4, and 3 mg/mL for 72 h. Propolis extract substantially inhibited human bronchial epithelial cells, especially at higher concentrations (7, 8, and 16 mg/mL). Propolis can kill Acanthamoeba trophozoites at a concentration (of 3-6 mg/mL) but is safe for human bronchial epithelial cells at the same concentrations after 72 h treatment, this paves the way for propolis to be an alternative source of therapeutic drugs in the treatment of Acanthamoeba spp. Conclusion: Acanthamoeba infection still cannot be treated with drugs. In this study, propolis collected from different regions of Turkey showed amoebic and cytotoxic activities. Propolis extract, which is a natural product that can be used against Acanthamoeba trophozoites can be an alternative source of therapeutic drugs in the treatment of Acanthamoeba, supported by in vivo studies.

In vitro Evaluation the Efficacy of Some New Plant Extracts and Biocides on the Viability of Acanthamoeba castellanii

The purpose of this study was to assess the efficacy of certain plant extracts and to compare them with current biocides on the viability of Acanthamoeba castellanii cysts and trophozoites in vitro. Amoebicidal and cysticidal assays were performed against both trophozoites and cysts of Acanthamoeba castellanii (ATCC 50370). Ten plant extracts were evaluated alongside the current agents included polyhexamethylene biguanide (PHMB), octenidine and chlorhexidine digluconate. A. castellanii (ATCC 50370) was treated to serial two-fold dilutions of the test compounds and extracts in microtitre plate wells to investigate the effect on trophozoites and cysts of A. castellanii (ATCC 50370). Furthermore, the toxicity of each of the test compounds and extracts were assessed towards a mammalian cell line. Minimum trophozoite inhibitory concentration (MTIC), minimum trophozoite amoebicidal concentration (MTAC), and minimum cysticidal concentration (MCC) were used to establish A. castellanii (ATCC 50370) in vitro sensitivity. The findings of this research revealed that the biguanides PHMB, chlorhexidine, and octenidine all had excellent effectiveness against trophozoites and cysts of A. castellanii (ATCC 50370). The plant extracts testing results showed that, great activity against trophozoites and cysts ofA. castellanii (ATCC 50370) at lower concentrations. This is the first study to demonstrate that the Proskia plant extract had the lowest MCC value, which was 3.9 µg/mL. The time kill experiment confirmed this finding, as this extract reduced cysts of A. castellanii (ATCC 50370) by more than 3-log at 6 hour and by 4-log after 24 hour. The anti-amoebic efficacy of new plant extracts on the viability of A. castellanii (ATCC 50370) cysts and trophozoites was comparable to existing biocide treatments and was not toxic when tested on a mammalian cell line. This could be a promising novel Acanthamoeba treatment by using the tested plant extracts as a monotherapy against trophozoites and cysts.

Exposure to sublethal concentrations of chlorine enhances the cytotoxicity of Acanthamoeba castellanii.

Free-living amoebae belonging to the genus Acanthamoeba are the causative agents of infections in humans and animals. Many studies are being conducted to find effective compounds against amoebae, but their sublethal concentration effects on surviving amoebae seem to have been overlooked. Chlorine is a common disinfection agent commonly added to public water facilities and supplies. In this study, the cytopathic and phagocytic properties of Acanthamoeba castellanii trophozoites following exposure to sublethal concentrations of chlorine were examined. Two hours of exposure to 5 ppm hypochlorite calcium was considered the sublethal concentration for A. castellanii trophozoites. To compare the pathogenic potential of treated and untreated Acanthamoeba trophozoites, cytotoxicity, adhesion assays in RAW 264.7 macrophages, osmo, and thermotolerance tests were carried out. 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 mRNA expression of the mannose-binding protein (MBP), cysteine protease 3 (CP3), and serine endopeptidase (SEP) genes was determined in cells. In all the experiments, untreated trophozoites were considered the control. In comparison to untreated trophozoites, in chlorine-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). Besides, they showed osmotolerance to 0.5 M D-mannitol but not to 1 M. Results demonstrated a higher bacterial uptake rate by chlorine-treated trophozoites than untreated cells. The treated and untreated cells had significantly different glutathione and glutathione/glutathione disulfide ratios. Antioxidant enzyme activities, total antioxidant capacity, and malondialdehyde levels were increased significantly in chlorine-treated cells. Quantifying mRNA expression in chlorine-treated trophozoites revealed that virulence genes were upregulated. Chlorine can form resistance and virulent amoebae if it is not used at a proper concentration and exposure time. Identification of stress responses, their mechanisms in Acanthamoeba, and their relation to amoeba virulence would give us a better perception of their pathophysiology.

Acanthamoeba castellanii Genotype T4: Inhibition of Proteases Activity and Cytopathic Effect by Bovine Apo-Lactoferrin.

Acanthamoeba castellanii genotype T4 is a clinically significant free-living amoeba that causes granulomatous amoebic encephalitis and amoebic keratitis in human beings. During the initial stages of infection, trophozoites interact with various host immune responses, such as lactoferrin (Lf), in the corneal epithelium, nasal mucosa, and blood. Lf plays an important role in the elimination of pathogenic microorganisms, and evasion of the innate immune response is crucial in the colonization process. In this study, we describe the resistance of A. castellanii to the microbicidal effect of bovine apo-lactoferrin (apo-bLf) at different concentrations (25, 50, 100, and 500 µM). Acanthamoeba castellanii trophozoites incubated with apo-bLf at 500 µM for 12 h maintained 98% viability. Interestingly, despite this lack of effect on viability, our results showed that the apo-bLf inhibited the cytopathic effect of A. castellanii in MDCK cells culture, and analysis of amoebic proteases by zymography showed significant inhibition of cysteine and serine proteases by interaction with the apo-bLf. From these results, we conclude that bovine apo-Lf influences the activity of A. castellanii secretion proteases, which in turn decreases amoebic cytopathic activity.

A Promising Antifungal and Antiamoebic Effect of Silver Nanorings, a Novel Type of AgNP.

Silver nanoparticles (AgNPs) play an important role in the medical field due to their potent antimicrobial activity. This, together with the constant emergence of resistance to antimicrobial drugs, means AgNPs are often investigated as an alternative to solve this problem. In this article, we analyzed the antifungal and antiamoebic effects of a recently described type of AgNP, silver nanorings (AgNRs), and compared them with other types of AgNPs. Tests of the activity of AgNPs against various fungal and amoebic species were carried out. In all cases, AgNPs showed a high biocidal effect, although with fungi this depended on the species involved. Antifungal activity was detected by the conditioning of culture media or water but this effect was not dependent on the release of Ag ions. On the other hand, the proliferation of Acanthamoeba castellanii trophozoites was reduced by silver nanorings (AgNRs) and silver nanowires (AgNWs), with AgNWs being capable of totally inhibiting the germination of A. castellanii cysts. AgNRs constitute a new type of AgNP with an antifungal and antiacanthamoebic activity. These results open the door to new and effective antimicrobial therapies as an alternative to the use of antifungals or antiamoebic drugs, thus avoiding the constant appearance of resistance and the difficulty of eradicating infections.

Recognition of Cell Wall Mannosylated Components as a Conserved Feature for Fungal Entrance, Adaptation and Survival Within Trophozoites of Acanthamoeba castellanii and Murine Macrophages.

Acanthamoeba castellanii (Ac) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms.

5-Nitroindazole derivatives as potential therapeutic alternatives against Acanthamoeba castellanii

Amoebas of the genus Acanthamoeba are distributed worldwide, including species with a high pathogenic capacity for humans. In a similar way to what occurs with other parasitic protozoa, the available treatments show variable effectiveness in addition to high toxicity, which demands the development of new treatments. Positive results of 5-nitroindazole derivatives against several protozoa parasites suggest that these compounds may be a promising tool for the development of efficient antiparasitic drugs. In the present work we have evaluated the in vitro activity of ten 5-nitroindazole derivatives against Acanthamoeba castellanii trophozoites and cysts. To that end, AlamarBlue Assay Reagent® was used to determine the activity against trophozoites compared to the reference drug chlorhexidine digluconate. Cytotoxicity of the compounds was evaluated using Vero cells. The activity on cysts was evaluated by light microscopy and using a Neubauer chamber to quantifying cysts and presence of trophozoites, as an indication of cyst. Our results showed the effectiveness of the 5-nitroindazole derivatives tested against both trophozoites and cysts of A. castellani highlighting 5-nitroindazole derivative 8 which showed a 80% activity on cysts, which is higher than that of the reference drug. Moreover, 5-nitroindazole derivatives 8, 9 and 10 were more effective on trophozoites than the reference drug showing IC50 values lower than 5 µM. Taking together these results, these 5-nitroindazole derivatives specially compound 8, might be a promising alternative for the development of more efficient treatments against A. castellani infection.

Development of a Machine Learning-Based Cysticidal Assay and Identification of an Amebicidal and Cysticidal Marine Microbial Metabolite against Acanthamoeba.

ABSTRACTTraditional cysticidal assays for Acanthamoeba 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.IMPORTANCE The free-living Acanthamoeba 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-Acanthamoeba 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 Acanthamoeba 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.

Acanthamoeba castellanii: Effect of neuroactive substances on trophozoite migration

Acanthamoeba castellanii is the etiological agent of granulomatous amebic encephalitis, amebic keratitis, and skin lesions. In vitro and in vivo studies have demonstrated that Acanthamoeba trophozoites induce contact-dependent, and contact-independent pathogenic mechanisms. We have explored the potential role neuroactive substances may have in the migration of Acanthamoeba castellanii trophozoites using Transwell permeable supports in the presence of physiological concentrations of dopamine, glutamate, serotonin, or taurine diluted in PBS. Quantitation of migrated amoebae was carried out in scanning electron micrographs of the upper and under compartments sides of the chamber membranes. Our results showed that at 2 h of interaction, a statistically significant larger proportion of A. castellanii trophozoites migrated through the chamber membranes when neurotransmitters were placed in the lower compartments of the chambers compared to control. This migration effect was more evident under the presence of glutamate and taurine on the three surfaces (upper/lower membrane and bottom compartment) when the percentage of migrated trophozoites was analyzed. Scanning electron microscopy of trophozoites revealed that glutamate and taurine induced the formation of large adhesion lamellas and phagocytic stomas. These observations suggest that certain neuroactive substances could stimulate the migration of A. castellanii trophozoites in the central nervous system.

Nisin Induces Cell-Cycle Arrest in Free-Living Amoebae Acanthamoeba castellanii.

Acanthamoeba spp. are free-living amoebas with worldwide distribution and play an important role as disease-causing agents in humans. Drug inability to completely eradicate these parasites along with their toxic effects suggest urgent need for new antimicrobials. Nisin is a natural antimicrobial peptide produced by Lactococcus lactis. Nisin is also the only bacteriocin approved for use in food preservation. In this work, we analyzed the effect of nisin on the growth of Acanthamoeba castellanii trophozoites. A total of 8 × 104 trophozoites were exposed to increasing concentrations of nisin to determine its activity. Changes in cell membrane and cellular cycle of trophozoites were investigated by flow cytometry, and nisin cytotoxicity in mammalian cells was evaluated in L929 cells by MTT method. After 24h exposure to increasing nisin concentrations, an IC50 of 4493.2IUmL-1 was obtained for A. castellanii trophozoites. However, after 72h a recovery in amoebic growth was observed, and it was no longer possible to determine IC50. Flow cytometry analysis showed that nisin has no effect on the membrane integrity. Treatment with nisin induced cell-cycle arrest during G1 and S phases in A. castellanii trophozoites, which recovered their growth after 72h. This is one of the first studies showing the effect of internationally approved nisin against A. castellanii trophozoites. Nisin caused cell-cycle arrest in trophozoites, momentarily interfering with the DNA replication process. The data highlight the amoebostatic activity of nisin, and suggest its use as an adjuvant for the treatment of infections caused by Acanthamoeba spp.