Members of the genus Acanthamoeba, amphizoic protozoan parasites, are causative agents of granulomatous amoebic encephalitis and amoebic keratitis. Proteinases play a role in various biologic actions in Acanthamoeba, including host tissue destruction, pathogenesis, and digestion of phagocytosed food. Interestingly, we found that encystation of Acanthamoeba was inhibited by the serine proteinase inhibitor phenylmethanesulfonyl fluoride. In this study, we characterize a serine proteinase that is involved in mediating the encystation of Acanthamoeba. This encystation-mediating serine proteinase (EMSP) is shown to be highly expressed during encystation by real-time PCR and Western blot analysis. Chemically synthesized small interfering RNA against EMSP inhibited the expression of EMSP mRNA and significantly reduced the encystation efficiency of Acanthamoeba. An EMSPenhanced green fluorescent protein fusion protein localized to vesicle-like structures within the amoeba. Using LysoTracker analysis, these vesicular structures were confirmed to be lysosomes. After incubation of the transfected amoeba in encystment media, small fluorescent vesicle-like structures gathered and formed ball-like structures, which were identified as colocalizing with the autophagosome. Taken together, these results indicate that EMSP plays an important role in the differentiation of Acanthamoeba by promoting autolysis.
BackgroundDespite the extensive endeavours, developing an effective malaria vaccine remains as a great challenge. Apical membrane antigen 1 (AMA-1) located on the merozoite surface of parasites belonging to the genus Plasmodium is involved in red blood cell invasion.MethodsInfluenza virus-like particle (VLP) vaccines containing codon-optimized or native (non-codon optimized) AMA-1 from Plasmodium berghei were generated. VLP-induced protective immunity was evaluated in a mouse model.ResultsMice immunized with VLP vaccine containing the codon-optimized AMA-1 elicited higher levels of P. berghei-specific IgG and IgG2a antibody responses compared to VLPs containing non-codon optimized AMA-1 before and after challenge infection. Codon-optimized AMA-1 VLP vaccination induced higher levels of CD4+ T cells, CD8+ T cells, B cells, and germinal centre cell responses compared to non-codon optimized AMA-1 VLPs. Importantly, the codon-optimized AMA-1 VLP vaccination showed lower body weight loss, longer survival and a significant decrease in parasitaemia compared to non-codon optimized VLP vaccination.ConclusionOverall, VLP vaccine expressing codon-optimized AMA-1 induced better protective efficacy than VLPs expressing the non-codon optimized AMA-1. Current findings highlight the importance of codon-optimization for vaccine use and its potential involvement in future malaria vaccine design strategies.
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 Acanthamoeba castellanii (AcAtg3). AcAtg3 encoding a 304 amino acid protein showed high similarity with the catalytic cysteine site of other E2 like enzymes of ubiquitin system. Predicted 3D structure of AcAtg3 revealed a hammer-like shape, which is the characteristic structure of E2-like enzymes. The expression level of AcAtg3 did not increase during encystation. However, the formation of mature cysts was significantly reduced in Atg3-siRNA transfected cells in which the production of Atg8-phosphatidylethanolamine conjugate was inhibited. Fluorescent microscopic analysis revealed that dispersed AcAtg3-EGFP fusion protein gathered around autophagosomal membranes during encystation. These results provide important information for understanding autophagic machinery through the lipidation reaction mediated by Atg3 in Acanthamoeba.
Abstract:The genus Acanthamoeba can cause severe infections such as granulomatous amebic encephalitis and amebic keratitis in humans. However, little genomic information of Acanthamoeba has been reported. Here, we constructed Acanthamoeba expressed sequence tags (EST) database (Acanthamoeba EST DB) derived from our 4 kinds of Acanthamoeba cDNA library. The Acanthamoeba EST DB contains 3,897 EST generated from amebae under various conditions of long term in vitro culture, mouse brain passage, or encystation, and downloaded data of Acanthamoeba from National Center for Biotechnology Information (NCBI) and Taxonomically Broad EST Database (TBestDB). The almost reported cDNA/genomic sequences of Acanthamoeba provide stand alone BLAST system with nucleotide (BLAST NT) and amino acid (BLAST AA) sequence database. In BLAST results, each gene links for the significant information including sequence data, gene orthology annotations, relevant references, and a BlastX result. This is the first attempt for construction of Acanthamoeba database with genes expressed in diverse conditions. These data were integrated into a database (http://www. amoeba.or.kr).
Encystation of Acanthamoeba leads to the formation of resilient cysts from vegetative trophozoites. This process is essential for parasite survival under unfavorable conditions such as starvation, low temperatures, and exposure to biocides. During encystation, a massive turnover of intracellular components occurs, and a large number of organelles and proteins are degraded by proteases. Previous studies with specific protease inhibitors have shown that cysteine and serine proteases are involved in encystation of Acanthamoeba, but little is known about the role of metalloproteases in this process. Here, we have biochemically characterized an M17 leucine aminopeptidase of Acanthamoeba castellanii (AcLAP) and analyzed its functional involvement in encystation of the parasite. Recombinant AcLAP shared biochemical properties such as optimal pH, requirement of divalent metal ions for activity, substrate specificity for Leu, and inhibition profile by aminopeptidase inhibitors and metal chelators with other characterized M17 family LAPs. AcLAP was highly expressed at a late stage of encystation and mainly localized in the cytoplasm of A. castellanii. Knockdown of AcLAP using small interfering RNA induced a decrease of LAP activity during encystation, a reduction of mature cyst formation, and the formation of abnormal cyst walls. In summary, these results indicate that AcLAP is a typical M17 family enzyme that plays an essential role during encystation of Acanthamoeba.
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