| Hae-Ahm Lee | 5 Articles |
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.
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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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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