| Eun-Kyung Moon | 27 Articles |
Acanthamoeba is an opportunistic pathogen that causes Acanthamoeba keratitis, granulomatous amoebic encephalitis, and other cutaneous diseases. The life cycle of Acanthamoeba consists of 2 stages of trophozoites and cysts. Under adverse environmental conditions, Acanthamoeba encysts, while the conditions become favorable for growth, it reverts to the trophozoite form. Acanthamoeba excystation is crucial for its proliferation and can lead to recurrent infections after incomplete treatment. To identify the factors involved in excystation, A. castellanii was subjected to either encystation- or excystation-inducing conditions, and gene expression profiles were compared using mRNA sequencing. A. castellanii samples were collected at 8 h intervals for analysis under both conditions. Differentially expressed gene analysis revealed that 1,214 and 1,163 genes were upregulated and downregulated, respectively, by more than 2-fold during early excystation. Five genes markedly upregulated in early excystation (ACA1_031140, ACA1_032330, ACA1_374400, ACA1_275740, and ACA1_112650) were selected, and their expression levels were confirmed via real-time PCR. Small interfering RNA (siRNA) targeting these 5 genes was transfected into Acanthamoeba and gene knockdown was validated through real-time PCR. The silencing of ACA1_031140, ACA1_032330, ACA1_374400, and ACA1_112650 inhibited excystation and suggested that these genes might be essential for excystation. Our findings provide valuable insights for suppressing Acanthamoeba proliferation and recurrence.
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Malaria is a global disease affecting a large portion of the world’s population. Although vaccines have recently become available, their efficacies are suboptimal. We generated virus-like particles (VLPs) that expressed either apical membrane antigen 1 (AMA1) or microneme-associated antigen (MIC) of Plasmodium berghei and compared their efficacy in BALB/c mice. We found that immune sera acquired from AMA1 VLP- or MIC VLP-immunized mice specifically interacted with the antigen of choice and the whole P. berghei lysate antigen, indicating that the antibodies were highly parasite-specific. Both VLP vaccines significantly enhanced germinal center B cell frequencies in the inguinal lymph nodes of mice compared with the control, but only the mice that received MIC VLPs showed significantly enhanced CD4+ T cell responses in the blood following P. berghei challenge infection. AMA1 and MIC VLPs significantly suppressed TNF-α and interleukin-10 production but had a negligible effect on interferon-γ. Both VLPs prevented excessive parasitemia buildup in immunized mice, although parasite burden reduction induced by MIC VLPs was slightly more effective than that induced by AMA1. Both VLPs were equally effective at preventing body weight loss. Our findings demonstrated that the MIC VLP was an effective inducer of protection against murine experimental malaria and should be the focus of further development.
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Acanthamoeba species are free-living amoebae those are widely distributed in the environment. They feed on various microorganisms, including bacteria, fungi, and algae. Although majority of the microbes phagocytosed by Acanthamoeba spp. are digested, some pathogenic bacteria thrive within them. Here, we identified the roles of 3 phagocytosis-associated genes (ACA1_077100, ACA1_175060, and AFD36229.1) in A. castellanii. These 3 genes were upregulated after the ingestion of Escherichia coli. However, after the ingestion of Legionella pneumophila, the expression of these 3 genes was not altered after the consumption of L. pneumophila. Furthermore, A. castellanii transfected with small interfering RNS (siRNA) targeting the 3 phagocytosis-associated genes failed to digest phagocytized E. coli. Silencing of ACA1_077100 disabled phagosome formation in the E. coli-ingesting A. castellanii. Alternatively, silencing of ACA1_175060 enabled phagosome formation; however, phagolysosome formation was inhibited. Moreover, suppression of AFD36229.1 expression prevented E. coli digestion and consequently led to the rupturing of A. castellanii. Our results demonstrated that the ACA1_077100, ACA1_175060, and AFD36229.1 genes of Acanthamoeba played crucial roles not only in the formation of phagosome and phagolysosome but also in the digestion of E. coli.
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Toxoplasma gondii infections are primarily diagnosed by serological assays, whereas molecular and fluorescence-based techniques are garnering attention for their high sensitivity in detecting these infections. Nevertheless, each detection method has its limitations. The toxoplasmosis detection capabilities of most of the currently available methods have not been evaluated under identical experimental conditions. This study aimed to assess the diagnostic potential of enzyme-linked immunosorbent assay (ELISA), real-time polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and immunofluorescence (IF) in BALB/c mice experimentally infected with various doses of T. gondii ME49. The detection of toxoplasmosis from sera and brain tissues was markedly enhanced in mice subjected to high infection doses (200 and 300 cysts) compared to those subjected to lower doses (10 and 50 cysts) for all the detection methods. Additionally, increased B1 gene expression levels and cyst sizes were observed in the brain tissues of the mice. Importantly, IHC, IF, and ELISA, but not RT-PCR, successfully detected T. gondii infections at the lowest infection dose (10 cysts) in the brain. These findings may prove beneficial while designing experimental methodologies for detecting T. gondii infections in mice.
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Acanthamoeba keratitis (AK) is a rare ocular disease, but it is a painful and sight-threatening infectious disease. Early diagnosis and adequate treatment are necessary to prevent serious complications. While AK is frequently diagnosis via several PCR assays or Acanthamoeba-specific antibodies, a more specific and effective diagnostic method is required. This study described the production of a polyclonal peptide antibody against the periplasmic binding protein (PBP) of A. castellanii and investigated its diagnostic potential. Western blot analysis showed that the PBP antibody specifically reacted with the cell lysates of A. castellanii. However, the PBP antibody did not interact with human corneal epithelial (HCE) cells and the other 3 major causative agents of keratitis. Immunocytochemistry (ICC) results revealed the specific detection of A. castellanii trophozoites and cysts by PBP antibodies when A. castellanii were co-cultured with HCE cells. PBP antibody specificity was further confirmed by co-culture of A. castellanii trophozoites with F. solani, S. aureus, and P. aeruginosa via ICC. The PBP antibody specifically reacted with the trophozoites and cysts of A. polyphaga, A. hatchetti, A. culbertsoni, A. royreba, and A. healyi, thus demonstrated its genus-specific nature. These results showed that the PBP polyclonal peptide antibody of A. castellanii could specifically detect several species of Acanthamoeba, contributing to the development of an effective antibody-based AK diagnostics.
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The encystation of Acanthamoeba leads to the development of metabolically inactive and dormant cysts from vegetative trophozoites under unfavorable conditions. These cysts are highly resistant to anti-Acanthamoeba drugs and biocides. Therefore, the inhibition of encystation would be more effective in treating Acanthamoeba infection. In our previous study, a sirtuin family protein—Acanthamoeba silent-information regulator 2-like protein (AcSir2)—was identified, and its expression was discovered to be critical for Acanthamoeba castellanii proliferation and encystation. In this study, to develop Acanthamoeba sirtuin inhibitors, we examine the effects of sirtinol, a sirtuin inhibitor, on trophozoite growth and encystation. Sirtinol inhibited A. castellanii trophozoites proliferation (IC50=61.24 μM). The encystation rate of cells treated with sirtinol significantly decreased to 39.8% (200 μM sirtinol) after 24 hr of incubation compared to controls. In AcSir2-overexpressing cells, the transcriptional level of cyst-specific cysteine protease (CSCP), an Acanthamoeba cysteine protease involved in the encysting process, was 11.6- and 88.6-fold higher at 48 and 72 hr after induction of encystation compared to control. However, sirtinol suppresses CSCP transcription, resulting that the undegraded organelles and large molecules remained in sirtinol-treated cells during encystation. These results indicated that sirtinol sufficiently inhibited trophozoite proliferation and encystation, and can be used to treat Acanthamoeba infections.
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Acanthamoeba keratitis (AK) is a rare infectious disease and accurate diagnosis has remained arduous as clinical manifestations of AK were similar to keratitis of viral, bacterial, or fungal origins. In this study, we described the production of a polyclonal peptide antibody against the adenylyl cyclase-associated protein (ACAP) of A. castellanii, and evaluated its differential diagnostic potential. Enzyme-linked immunosorbent assay revealed high titers of A. castellanii-specific IgG and IgA antibodies being present in low dilutions of immunized rabbit serum. Western blot analysis revealed that the ACAP antibody specifically interacted with A. castellanii, while not interacting with human corneal epithelial (HCE) cells and other causes of keratitis such as Fusarium solani, Pseudomonas aeruginosa, and Staphylococcus aureus. Immunocytochemistry (ICC) results confirmed the specific detection of trophozoites and cysts of A. castellanii co-cultured with HCE cells. The ACAP antibody also specifically interacted with the trophozoites and cysts of 5 other Acanthamoeba species. These results indicate that the ACAP antibody of A. castellanii can specifically detect multiple AK-causing members belonging to the genus Acanthamoeba and may be useful for differentially diagnosing Acanthamoeba infections.
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Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term ‘integral component of membrane’ were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.
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Pathogenic Acanthamoeba spp. cause granulomatous amoebic encephalitis and keratitis. Acanthamoeba keratitis (AK) is a rare but serious ocular infection that can result in permanent visual impairment or blindness. However, pathogenic factors of AK remain unclear and treatment for AK is arduous. Expression levels of proteins secreted into extracellular space were compared between A. castellanii pathogenic (ACP) and non-pathogenic strains. Two-dimensional polyacrylamide gel electrophoresis revealed 123 differentially expressed proteins, including 34 increased proteins , 7 qualitative increased proteins, 65 decreased proteins, and 17 qualitative decreased proteins in ACP strain. Twenty protein spots with greater than 5-fold increase in ACP strain were analyzed by liquid chromatography triple quadrupole mass spectrometry. These proteins showed similarity each to inosine-uridine preferring nucleoside hydrolase, carboxylesterase, oxygen-dependent choline dehydrogenase, periplasmic-binding protein proteinases and hypothetical proteins. These proteins expressed higher in ACP may provide some information to understand pathogenicity of Acanthamoeba.
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Multipurpose contact lens disinfecting solutions (MPDS) are widely used to cleanse and disinfect microorganisms. However, disinfection efficacy of these MPDS against Acanthamoeba cyst remain insufficient. 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, is capable of increasing the amoebical effect against Acanthamoeba by inhibiting its encystation. In this study, we investigated the possibility of DCB as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba cyst. Eight commercial MPDS (from a to h) were assessed, all of which displayed insufficient amoebicidal activity against the mature cysts. Solution e, f, and h showed strong amoebicidal effect on the immature cysts. Amoebicidal efficacy against mature cysts remained inadequate even when the 8 MPDS were combined with 100 μM DCB. However, 4 kinds of MPDS (solution d, e, f, and h) including 100 μM DCB demonstrated strong amoebicidal activity against the immature cysts. The amoebicidal activity of solution d was increased by addition of DCB. Cytotoxicity was absent in human corneal epithelial cells treated with either DCB or mixture of DCB with MPDS. These results suggested that DCB can enhance the amoebicical activity of MPDS against Acanthamoeba immature cyst in vitro.
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Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFPAcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.
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Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1-3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.
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Toxoplasma gondii infections occur throughout the world, and efforts are needed to develop various vaccine candidates expressing recombinant protein antigens. In this study, influenza matrix protein (M1) virus-like particles (VLPs) consisting of T. gondii rhoptry antigen 4 (ROP4 protein) were generated using baculovirus (rBV) expression system. Recombinant ROP4 protein with influenza M1 were cloned and expressed in rBV. SF9 insect cells were coinfected with recombinant rBVs expressing T. gondii ROP4 and influenza M1. As the results, influenza M1 VLPs showed spherical shapes, and T. gondii ROP4 protein exhibited as spikes on VLP surface under transmission electron microscopy (TEM). The M1 VLPs resemble virions in morphology and size. We found that M1 VLPs reacted with antibody from T. gondii-infected mice by western blot and ELISA. This study demonstrated that T. gondii ROP4 protein can be expressed on the surface of influenza M1 VLPs and the M1 VLPs containing T. gondii ROP4 reacted with T. gondii-infected sera, indicating the possibility that M1 VLPs could be used as a coating antigen for diagnostic and/or vaccine candidate against T. gondii infection.
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Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis.
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Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.
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Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Citations Citations to this article as recorded by
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Autophagy is a catabolic process involved in the degradation of a cell's own components for cell growth, development, homeostasis, and the recycling of cellular products. Autophagosome is an essential component in the protozoan parasite during differentiation and encystation. The present study identified and characterized autophagy-related protein (Atg) 3, a member of Atg8 conjugation system, in Citations Citations to this article as recorded by
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Actin binding proteins play key roles in cell structure and movement particularly as regulators of the assembly, stability and localization of actin filaments in the cytoplasm. In the present study, a cDNA clone encoding an actin bundling protein named as AhABP was isolated from Citations Citations to this article as recorded by
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Naegleria fowleri is a free-living amoeba that can cause primary amebic meningoencephalitis (PAM), a very serious infection of the central nervous system. Early diagnosis of PAM is challenging, and the condition is almost always fatal. In this study, we conducted 2-dimensional gel electrophoresis (2-DE) analysis using N. fowleri trophozoite lysates and conditioned media to identify preferentially secreted proteins. As a result of the 2-dimensional gel electrophoresis analysis, 1 protein was found to increase, 5 proteins were found to decrease, 3 proteins showed a qualitative increase, and 15 proteins showed a qualitative decrease in the conditioned media compared to the proteins in the trophozoite lysates. Using cDNA from N. fowleri, Acanthamoeba castellanii, and Balamuthia mandrillaris, all of which can cause encephalitis, real-time PCR was performed on 5 genes corresponding to the p23-like domain-containing protein, cystatin-like domain-containing protein, fowlerpain-2, hemerythrin family non-heme iron protein, and an uncharacterized protein. The results showed that all 5 genes were highly expressed in N. fowleri. In animal models infected with N. fowleri resulting in PAM, real-time PCR analysis of brain tissue revealed significant overexpression of the p23-like domain-containing protein and fowlerpain-2. These results suggest that the 2 secreted proteins could provide valuable insights for developing antibody-based or molecular diagnostic methods to detect N. fowleri in patients with PAM.
Acanthamoeba is a genus of free-living amoebae commonly found in soil, water, and other habitats. This organism undergoes 2 distinct stages in its life cycle, the trophozoite and the cyst. Under adverse conditions, trophozoites transform into cysts, which are notably resistant to harsh physical and chemical conditions. Infected by Legionella pneumophila has been shown to decrease the number of cysts in its host Acanthamoeba species, although the mechanisms responsible for this effect remain poorly understood. In this study, A. castellanii was co-cultured with either L. pneumophila or Escherichia coli to assess the impact on encystation and to explore the genes involved in this process. Following a 72-h encystation induction period, it was observed that Acanthamoeba infected with Legionella exhibited a 45.8% reduction in cyst formation compared to the control group. In contrast, Acanthamoeba that phagocytosed E. coli showed a 21.7% decrease. To identify the genes involved in this phenomenon, real-time PCR analysis was conducted on 20 genes known to be upregulated during encystation. This analysis was performed to verify their expression patterns at 24, 48, and 72 h. Notably, ten genes, including cyst-specific protein 21, glycosyltransferase, RSNARE, and cellulose synthase, did not exhibit increased expression in Legionella-infected Acanthamoeba. However, these genes showed elevated expression levels in both the control group and the bacteria-phagocytosed Acanthamoeba. This suggests that several cellular processes, including cell wall formation, are inhibited in Acanthamoeba infected with Legionella, resulting in reduced encystation.
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