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Fulltext Amoebicidal activity of Cassia angustifolia extract and its effect on Acanthamoeba triangularis autophagy-related gene expression at the transcriptional level

Boonhok R, Sangkanu S, Norouzi R, Siyadatpanah A, Mirzaei F, Mitsuwan W, et al.

Parasitology, 2021 Aug;148(9):1074-1082.
PMID: 33966667 DOI: 10.1017/S0031182021000718

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.
Fulltext Preparation and evaluation of a niosomal delivery system containing G. mangostana extract and study of its anti-Acanthamoeba activity

Sangkana S, Eawsakul K, Ongtanasup T, Boonhok R, Mitsuwan W, Chimplee S, et al.

Nanoscale Adv, 2024 Feb 27;6(5):1467-1479.
PMID: 38419876 DOI: 10.1039/d3na01016c

Garcinia mangostana extract (GME) has severe pharmacokinetic deficiencies and is made up of a variety of bioactive components. GME has proven its anti-Acanthamoeba effectiveness. In this investigation, a GME-loaded niosome was developed to increase its potential therapeutic efficacy. A GME-loaded niosome was prepared by encapsulation in a mixture of span60, cholesterol, and chloroform by the thin film hydration method. The vesicle size, zeta potential, percentage of entrapment efficiency, and stability of GME-loaded niosomes were investigated. The values for GME-loaded niosome size and zeta potential were 404.23 ± 4.59 and -32.03 ± 0.95, respectively. The delivery system enhanced the anti-Acanthamoeba activity, which possessed MIC values of 0.25-4 mg mL-1. In addition, the niosomal formulation decreased the toxicity of GME by 16 times. GME-loaded niosome must be stored at 4 °C, as the quantity of remaining GME encapsulated is greater at this temperature than at room temperature. SEM revealed the damage to the cell membrane caused by trophozoites and cysts, which led to dead cells. In light of the above, it was found that GME-loaded niosomes had better anti-Acanthamoeba activity. The study suggested that GME-loaded niosomes could be used as an alternative to Acanthamoeba's therapeutic effects.
Fulltext Development of a novel encystment medium: Enhancing diagnostic potential of Acanthamoeba spp

Chuprom J, Sangkanu S, Mitsuwan W, Boonhok R, Paul AK, Rodrigues Oliveira SM, et al.

Vet World, 2025 Jan;18(1):110-121.
PMID: 40041505 DOI: 10.14202/vetworld.2025.110-121

BACKGROUND AND AIM: Acanthamoeba spp. are pathogenic microorganisms linked to severe infections in humans and animals, requiring a deeper understanding of their encystation process for effective diagnostics and research. This study focused on developing a novel encystment medium to induce synchronized encystation of Acanthamoeba spp. efficiently and rapidly.
MATERIALS AND METHODS: The study employed response surface methodology with a central composite design to optimize the encystment medium formulation. The key components included Tris-HCl, NaCl, glucose, and MgCl2. The optimized liquid medium was spray-dried to produce a dehydrated powder for practical application. The encystation efficiency of different Acanthamoeba strains was assessed using hemocytometry and fluorescence microscopy.
RESULTS: The optimized medium, comprising 3.152 g/L Tris-HCl, 5.55 g/L NaCl, 8% (w/v) glucose, and 5.0 mM MgCl2 at pH 9.0, demonstrated exceptional encystation efficiency with rates ranging from 99% to 100%. A spray-dried powdered version of this medium was equally effective, achieving a 98.77% encystation rate for A. castellanii American Type Culture Collection 50739 in glucose-free conditions. Notably, optimal glucose concentrations varied among Acanthamoeba strains, with certain strains reaching maximum encystation at 6-8% glucose.
CONCLUSION: This study successfully developed an innovative encystment medium that promotes rapid and efficient cyst production in Acanthamoeba spp. The medium enhances laboratory research and diagnostic capabilities, paving the way for future advancements in understanding and managing Acanthamoeba infections.
Fulltext Acanthamoeba castellanii-Mediated Reduction of Interleukin-1β Secretion and Its Association With Macrophage Autophagy

Boonhok R, Senghoi W, Sangkanu S, Lim CL, Pudla M, Pereira ML, et al.

Scientifica (Cairo), 2025;2025:3430892.
PMID: 40109888 DOI: 10.1155/sci5/3430892

Noncanonical autophagy including unconventional protein secretion has been extensively studied. Our work focused on a leaderless IL-1β protein secretion from human macrophage in response to Acanthamoeba castellanii components, Acanthamoeba culture supernatant (CS) and cell lysate (CL), as well as its association with macrophage autophagy. Phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages were treated with Acanthamoeba components of pathogenic (ATCC50739) and nonpathogenic (ATCC30010) strains in vitro. The data showed that Acanthamoeba treatment resulted in low IL-1β secretion from macrophages. In addition, Acanthamoeba CL of both strains was able to upregulate autophagy-related (Atg) protein 8, an autophagy marker, whereas Acanthamoeba CS downregulated Atg8 expression. We further manipulated autophagy and found that autophagy induction by starvation diminished IL-1β secretion while autophagy inhibition by 3-methyladenine (3MA) increased IL-1β secretion. Interestingly, in the presence of Acanthamoeba components either under starvation or 3MA treatment, IL-1β secretion was significantly reduced. Transcriptional expression of other ATG genes, i.e., ATG6, ATG7, and ATG5, were investigated and showed that their mRNA expression was maintained at the basal level under A. castellanii CS or CL treatment. Inflammasome-related genes, NLRP3 and CASPASE1, were upregulated following A. castellanii 50739 CS treatment but not in A. castellanii 50739 CL-treated condition. However, both conditions were able to increase IL-1β mRNA expression. TEM micrographs revealed that 3MA treatment induced the formation of large vacuoles and accumulation of autophagosome at the edge of THP-1 macrophages. However, the number and size of their structures were declined in the presence of A. castellanii 50739 CS with 3MA. Furthermore, immunofluorescence staining demonstrated the association between Atg8/LC3 and IL-1β expression, where downregulation of Atg8 by A. castellanii 50739 CS led to the upregulation of IL-1β. Altogether, the data indicate that Acanthamoeba can manipulate macrophage autophagy, thereby controlling low IL-1β secretion. The expression of autophagy- and inflammasome-related genes also indicates multiple mechanisms in IL-1β secretion in response to Acanthamoeba components. However, further characterization of Atg proteins and investigations into other intracellular pathways or defense mechanisms are needed to fully understand the unconventional secretion of IL-1β in macrophages. This knowledge could eventually lead to the development of innovative therapeutic strategies against Acanthamoeba infection by modulating autophagy or macrophage responses.
Fulltext Anti-Acanthamoeba activity of a semi-synthetic mangostin derivative and its ability in removal of Acanthamoeba triangularis WU19001 on contact lens

Chuprom J, Sangkanu S, Mitsuwan W, Boonhok R, Mahabusarakam W, Singh LR, et al.

PeerJ, 2022;10:e14468.
PMID: 36523474 DOI: 10.7717/peerj.14468

Garcinia mangostana L., also known as the mangosteen tree, is a native medicinal plant in Southeast Asia having a wide variety of pharmacologically active compounds, including xanthonoid mangostin. In this study, we examined the pharmacological activities of the selected semi-synthetic mangostin derivative, namely, amoebicidal activity, encystation inhibition, excystation activity, and removal capacity of adhesive Acanthamoeba from the surface of contact lens (CL). Among the three derivatives, C1 exhibited promising anti-Acanthamoeba activity against Acanthamoeba triangularis WU19001 trophozoites and cysts. SEM images displayed morphological changes in Acanthamoeba trophozoites, including the loss of acanthopodia, pore formation in the cell membrane, and membrane damage. In addition, the treated cyst was shrunken and adopted an irregular flat cyst shape. Under a fluorescence microscope, acridine orange and propidium iodide (AO/PI) staining revealed C1 induced condensation of cytoplasm and chromatin with the loss of cell volume in the treated trophozoites, while calcofluor white staining demonstrated the leakage of cell wall in treated cysts, leading to cell death. Interestingly, at the concentration ranges in which C1 showed the anti-Acanthamoeba effects (IC50 values ranging from 0.035-0.056 mg/mL), they were not toxic to Vero cells. C1 displayed the highest inhibitory effect on A. triangularis encystation at 1/16×MIC value (0.004 mg/mL). While C1 demonstrated the excystation activity at 1/128×MIC value with a high rate of 89.47%. Furthermore, C1 exhibited the removal capacity of adhesive Acanthamoeba from the surface of CL comparable with commercial multipurpose solutions (MPSs). Based on the results obtained, C1 may be a promising lead agent to develop a therapeutic for the treatment of Acanthamoeba infections and disinfectant solutions for CL.
Fulltext Curcumin effect on Acanthamoeba triangularis encystation under nutrient starvation

Boonhok R, Sangkanu S, Phumjan S, Jongboonjua R, Sangnopparat N, Kwankaew P, et al.

PeerJ, 2022;10:e13657.
PMID: 35811814 DOI: 10.7717/peerj.13657

BACKGROUND: Curcumin is an active compound derived from turmeric, Curcuma longa, and is known for its benefits to human health. The amoebicidal activity of curcumin against Acanthamoeba triangularis was recently discovered. However, a physiological change of intracellular pathways related to A. triangularis encystation mechanism, including autophagy in the surviving amoeba after curcumin treatment, has never been reported. This study aims to investigate the effect of curcumin on the survival of A. triangularis under nutrient starvation and nutrient-rich condition, as well as to evaluate the A. triangularis encystation and a physiological change of Acanthamoeba autophagy at the mRNA level.
METHODS: In this study, A. triangularis amoebas were treated with a sublethal dose of curcumin under nutrient starvation and nutrient-rich condition and the surviving amoebas was investigated. Cysts formation and vacuolization were examined by microscopy and transcriptional expression of autophagy-related genes and other encystation-related genes were evaluated by real-time PCR.
RESULTS: A. triangularis cysts were formed under nutrient starvation. However, in the presence of the autophagy inhibitor, 3-methyladenine (3-MA), the percentage of cysts was significantly reduced. Interestingly, in the presence of curcumin, most of the parasites remained in the trophozoite stage in both the starvation and nutrient-rich condition. In vacuolization analysis, the percentage of amoebas with enlarged vacuole was increased upon starvation. However, the percentage was significantly declined in the presence of curcumin and 3-MA. Molecular analysis of A. triangularis autophagy-related (ATG) genes showed that the mRNA expression of the ATG genes, ATG3, ATG8b, ATG12, ATG16, under the starvation with curcumin was at a basal level along the treatment. The results were similar to those of the curcumin-treated amoebas under a nutrient-rich condition, except AcATG16 which increased later. On the other hand, mRNA expression of encystation-related genes, cellulose synthase and serine proteinase, remained unchanged during the first 18 h, but significantly increased at 24 h post treatment.
CONCLUSION: Curcumin inhibits cyst formation in surviving trophozoites, which may result from its effect on mRNA expression of key Acanthamoeba ATG-related genes. However, further investigation into the mechanism of curcumin in A. triangularis trophozoites arrest and its association with autophagy or other encystation-related pathways is needed to support the future use of curcumin.
Targeting Acanthamoeba proteins interaction with flavonoids of Propolis extract by in vitro and in silico studies for promising therapeutic effects

Sama-Ae I, Sangkanu S, Siyadatpanah A, Norouzi R, Chuprom J, Mitsuwan W, et al.

F1000Res, 2022;11:1274.
PMID: 36936052 DOI: 10.12688/f1000research.126227.1

Background : Propolis is a natural resinous mixture produced by bees. It provides beneficial effects on human health in the treatment/management of many diseases. The present study was performed to demonstrate the anti- Acanthamoeba activity of ethanolic extracts of Propolis samples from Iran. The interactions of the compounds and essential proteins of Acanthamoeba were also visualized through docking simulation. Methods: The minimal inhibitory concentrations (MICs) of Propolis extract against Acanthamoeba trophozoites and cysts was determined in vitro. In addition, two-fold dilutions of each of the agents were tested for encystment, excystment and adhesion inhibitions. Three major compounds of Propolis extract such as chrysin, tectochrysin and pinocembrin have been selected in molecular docking approach to predict the compounds that might be responsible for encystment, excystment and adhesion inhibitions of A. castellanii. Furthermore, to confirm the docking results, molecular dynamics (MD) simulations were also carried out for the most promising two ligand-pocket complexes from docking studies. Results : The minimal inhibitory concentrations (MICs) 62.5 and 125 µg/mL of the most active Propolis extract were assessed in trophozoites stage of Acanthamoeba castellanii ATCC30010 and ATCC50739, respectively. At concentrations lower than their MICs values (1/16 MIC), Propolis extract revealed inhibition of encystation. However, at 1/2 MIC, it showed a potential inhibition of excystation and anti-adhesion. The molecular docking and dynamic simulation revealed the potential capability of Pinocembrin to form hydrogen bonds with A. castellanii Sir2 family protein (AcSir2), an encystation protein of high relevance for this process in Acanthamoeba. Conclusions : The results obtained provided a candidate for the development of therapeutic drugs against Acanthamoeba infection. In vivo experiments and clinical trials are necessary to support this claim.
Fulltext Peganum harmala Extract Has Antiamoebic Activity to Acanthamoeba triangularis Trophozoites and Changes Expression of Autophagy-Related Genes

Boonhok R, Sangkanu S, Chuprom J, Srisuphanunt M, Norouzi R, Siyadatpanah A, et al.

Pathogens, 2021 Jul 04;10(7).
PMID: 34357992 DOI: 10.3390/pathogens10070842

Peganum harmala, a well-known medicinal plant, has been used for several therapeutic purposes as it contains numerous pharmacological active compounds. Our study reported an anti-parasitic activity of P. harmala seed extract against Acanthamoeba triangularis. The stress induced by the extract on the surviving trophozoites for Acanthamoeba encystation and vacuolization was examined by microscopy, and transcriptional expression of Acanthamoeba autophagy-related genes was investigated by quantitative PCR. Our results showed that the surviving trophozoites were not transformed into cysts, and the number of trophozoites with enlarged vacuoles were not significantly different from that of untreated control. Molecular analysis data demonstrated that the mRNA expression of tested AcATG genes, i.e., ATG3, ATG8b, and ATG16, was at a basal level along the treatment. However, upregulation of AcATG16 at 24 h post treatment was observed, which may indicate an autophagic activity of this protein in response to the stress. Altogether, these data revealed the anti-Acanthamoeba activity of P. harmala extract and indicated the association of autophagy mRNA expression and cyst formation under the extract stress, representing a promising plant for future drug development. However, further identification of an active compound and a study of autophagy at the protein level are needed.