The mature cyst of Acanthamoeba is highly resistant to various antibiotics and therapeutic agents. Cyst wall of Acanthamoeba are composed of cellulose, acid-resistant proteins, lipids, and unidentified materials. Because cellulose is one of the primary components of the inner cyst wall, cellulose synthesis is essential to the process of cyst formation in Acanthamoeba. In this study, we hypothesized the key and short-step process in synthesis of cellulose from glycogen in encysting Acanthamoeba castellanii, and confirmed it by comparing the expression pattern of enzymes involving glycogenolysis and cellulose synthesis. The genes of 3 enzymes, glycogen phosphorylase, UDP-glucose pyrophosphorylase, and cellulose synthase, which are involved in the cellulose synthesis, were expressed high at the 1st and 2nd day of encystation. However, the phosphoglucomutase that facilitates the interconversion of glucose 1-phosphate and glucose 6-phosphate expressed low during encystation. This report identified the short-cut pathway of cellulose synthesis required for construction of the cyst wall during the encystation process in Acanthamoeba. This study provides important information to understand cyst wall formation in encysting Acanthamoeba.
Acanthamoeba castellanii/*enzymology/genetics/growth & development ; Amebiasis/*parasitology ; Cell Wall/*metabolism ; Cellulose/*biosynthesis/genetics ; Glucosyltransferases/genetics/metabolism ; Glycogen Phosphorylase/genetics/metabolism ; Protozoan Proteins/genetics/*metabolism ; UTP-Glucose-1-Phosphate Uridylyltransferase/genetics/metabolism

Toxoplasmic encephalitis is caused by reactivation of bradyzoites to rapidly dividing tachyzoites of the apicomplexan parasite Toxoplasma gondii in immunocompromised hosts. Diagnosis of this life-threatening disease is problematic, because it is difficult to discriminate between these 2 stages. Toxoplasma PCR assays using gDNA as a template have been unable to discriminate between an increase or decrease in SAG1 and BAG1 expression between the active tachyzoite stage and the latent bradyzoite stage. In the present study, real-time RT-PCR assay was used to detect the expression of bradyzoite (BAG1)- and tachyzoite-specific genes (SAG1) during bradyzoite/tachyzoite stage conversion in mice infected with T. gondii Tehran strain after dexamethasone sodium phosphate (DXM) administration. The conversion reaction was observed in the lungs and brain tissues of experimental mice, indicated by SAG1 expression at day 6 after DXM administration, and continued until day 14. Bradyzoites were also detected in both organs throughout the study; however, it decreased at day 14 significantly. It is suggested that during the reactivation period, bradyzoites not only escape from the cysts and reinvade neighboring cells as tachyzoites, but also converted to new bradyzoites. In summary, the real-time RT-PCR assay provided a reliable, fast, and quantitative way of detecting T. gondii reactivation in an animal model. Thus, this method may be useful for diagnosing stage conversion in clinical specimens of immunocompromised patients (HIV or transplant patients) for early identification of tachyzoite-bradyzoite stage conversion.
Animals ; Antigens, Protozoan/*biosynthesis ; Brain/parasitology ; Female ; *Gene Expression ; Heat-Shock Proteins/*biosynthesis ; Immunocompromised Host ; Life Cycle Stages ; Lung/parasitology ; Mice ; Protozoan Proteins/*biosynthesis ; Real-Time Polymerase Chain Reaction ; Toxoplasma/*genetics/physiology ; Toxoplasmosis, Animal

OBJECTIVETo establish an tachyzoite-brachyzoite interconversion system for Toxoplasma gondii RH strain in vitro.

METHODSCOS-7 cells were inoculated with purified tachyzoites of T.gondii RH strain and cultured in vitro. The morphology of the cultured cells and parasites was observed and the total cellular RNA extracted on days 1 to 6 following the inoculation for detecting the expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) using RT-PCR.

RESULTSWith the passage of time, the number of parasites in COS-7 cells increased but the proliferation rate was lowered gradually. The intracellular tachyzoites proliferated by means of budding and binary fission, which led to the changes in the alignment of the parasites in the cells from curved pairs, rosette or clustered, and semi-circular patterns to spherical encapsulation-like structures. These changes indicated the gradual transformation of the tachyzoites into bradyzoites. The expressions of the tachyzoite-specific SAG1 gene were detected throughout the 6 days of in vitro culture. The expression of the bradyzoite-specific BAG1 gene had been detected since the second day after the inoculation and SAG2C gene since the fifth day. Alteration of the culture condition resulted in gradual transformation of the bradyzoites into tachyzoites.

CONCLUSIONAn in vitro tachzoites-bradyzoite interconversion system for T.gondii has been successfully established, which provides the basis for further study of the mechanism of interconversion.

Animals ; COS Cells ; Cell Culture Techniques ; Cercopithecus aethiops ; Cysts ; Female ; Genes, Protozoan ; genetics ; Host-Parasite Interactions ; Mice ; Protozoan Proteins ; biosynthesis ; genetics ; Toxoplasma ; growth & development ; physiology

Protozoan ciliates are a group of unicellular eukaryotes. The special characteristics of stop codons usage in termination of protein biosynthesis in ciliates cells makes them an ideal model to study the mechanism of stop codon recognition of polypeptides release factors. To localize the functional positions of biomolecules in ciliates cell, we constructed a macronuclear artificial chromosome containing a gene encoding red fluorescence protein (EoMAC_R) based on the structural characteristics of ciliates chromosome. Three factors, L11, eRF1a, and eRF3 that are involved in termination process of protein synthesis were colocalized in Euplotes octocarinatus cells by using novel EoMAC_R and the previously constructed EoMAC_G. The results indicated that protein synthesis mainly occurred inside the "C" shape macronucleus, suggesting that EoMAC could be a useful tool for localizing biomolecules in ciliates cell.
Chromosomes, Artificial ; Codon, Terminator ; metabolism ; Euplotes ; chemistry ; Peptide Termination Factors ; analysis ; genetics ; metabolism ; Peptides ; metabolism ; Protein Biosynthesis ; genetics ; Protozoan Proteins ; analysis ; genetics ; Ribosomal Proteins ; analysis ; genetics

Phenylalany--tRNA synthetase is a key enzyme for protein synthesis in Trypanosoma. Its validation as an inhibition. target will enable the development of a new generation of anti-Trypanosoma drugs. However, little is known about the isolation of the Trypanosoma Phenylalanyl-tRNA synthetase. Here we report the cloning, expression, purification, and activity assay of Phenylalanyl-tRNA synthetase from Trypanosoma brucei in Escherichia coli host. We co-cloned the alpha-subunit and beta-subunit of Phenylalanyl-tRNA synthetase from Trypanosoma brucei genomic DNA into the co-expression vector pCOLADuet. We successfully expressed the Trypanosoma brucei Phenylalanyl-tRNA synthetase in E. coli host, purified the whole enzyme by Ni-Hind affinity column and verified it by Western blotting. In addition, we tested its enzymatic activity by isotope labeling. The whole work laid a solid foundation for in vitro the screening and optimization of Trypanosoma brucei phenylalanyl-tRNA synthetase inhibitors.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Phenylalanine-tRNA Ligase ; biosynthesis ; genetics ; Protozoan Proteins ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Trypanosoma brucei brucei ; enzymology ; genetics

Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis.
Animals ; Biosynthetic Pathways ; Glycosylphosphatidylinositols/*biosynthesis/chemistry ; Humans ; Protozoan Proteins/genetics/metabolism ; Trypanosoma brucei brucei/chemistry/genetics/*metabolism ; Trypanosomiasis, African/*parasitology

Trypanosoma brucei, a protozoan parasite, causes sleeping sickness in humans and Nagana disease in domestic animals in central Africa. The trypanosome surface is extensively covered by glycosylphosphatidylinositol (GPI)-anchored proteins known as variant surface glycoproteins and procyclins. GPI anchoring is suggested to be important for trypanosome survival and establishment of infection. Trypanosomes are not only pathogenically important, but also constitute a useful model for elucidating the GPI biosynthesis pathway. This review focuses on the trypanosome GPI biosynthesis pathway. Studies on GPI that will be described indicate the potential for the design of drugs that specifically inhibit trypanosome GPI biosynthesis.
Animals ; Biosynthetic Pathways ; Glycosylphosphatidylinositols/*biosynthesis/chemistry ; Humans ; Protozoan Proteins/genetics/metabolism ; Trypanosoma brucei brucei/chemistry/genetics/*metabolism ; Trypanosomiasis, African/*parasitology

The objective of this study was to construct and express recombinant prokaryotic plasmid pET32a (+)- ast1 in E. coli BL21(DE3). Amastin gene was amplified from genomic DNA of Leishmania Donovani and its transmembran region was predicted by the methods of SOSUI and Tmpred; astl located in N-terminus of amastin gene was amplified and cloned into prokaryotic plasmid pET32a(+), which was named pET32a(+)-ast1, and then rAST1 was expressed in E. coli BL21(DE3). The results of SDS-PAGE and immunobloting assay showed that a fusion protein rAST1 (relative molecular mass about 27 kDa) was able to express in BL21. The recombinant prokaryotic plasmid pET32a(+)- ast1 was successfully constructed, and noted to be efficiently expressed in E. coli BL21(DE3).
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; Genes, Protozoan ; Leishmania donovani ; genetics ; Plasmids ; genetics ; Protozoan Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

OBJECTIVETo develop a technology for production of recombinant SAG1 of Toxoplasma gondii (T.g) in batches.

METHODSThe rSAG1 of T.g was expressed in E.coli by high-density fermentation and purified by Sephadex G-75 column chromatography after Ni-NTA agarose at native condition. The activity of rSAG1 and its efficacy in T.g diagnosis were identified by Western blotting and ELISA, respectively.

RESULTSThe optical density (OD) of the bacteria reached 20.21 after induction, and 300 g bacteria were harvested from 11.5 L broth. The rSAG1 was highly expressed in E.coli as a fusion protein, accounting for about 25.82% of the total bacterial protein. The purity of rSAG1 reached 98.54% after purification by Ni-NTA combined with Sephadex G-75 column chromatography. Western blotting revealed a distinct band reacting with the sera of rabbits vaccinated by T.g. Twenty-four of the 25 sera of mice infected with T.g and 36 of the 38 sera of human subjects with IgG antibody against T.g were detected by rSAG1-ELISA.

CONCLUSIONA large-scale production of immunoreactive SAG1 of T.g is developed by high-density fermentation and purification with Ni-NTA combined with Sephadex G-75 column chromatography.

Animals ; Antigens, Protozoan ; biosynthesis ; genetics ; immunology ; Antigens, Surface ; immunology ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification ; Toxoplasma ; immunology

Toxoplasma gondii GRA10 expressed as a GFP-GRA10 fusion protein in HeLa cells moved to the nucleoli within the nucleus rapidly and entirely. GRA10 was concentrated specifically in the dense fibrillar component of the nucleolus morphologically by the overlap of GFP-GRA10 transfection image with IFA images by monoclonal antibodies against GRA10 (Tg378), B23 (nucleophosmin) and C23 (nucleolin). The nucleolar translocalization of GRA10 was caused by a putative nucleolar localizing sequence (NoLS) of GRA10. Interaction of GRA10 with TATA-binding protein associated factor 1B (TAF1B) in the yeast two-hybrid technique was confirmed by GST pull-down assay and immunoprecipitation assay. GRA10 and TAF1B were also co-localized in the nucleolus after co-transfection. The nucleolar condensation of GRA10 was affected by actinomycin D. Expressed GFP-GRA10 was evenly distributed over the nucleoplasm and the nucleolar locations remained as hollows in the nucleoplasm under a low dose of actinomycin D. Nucleolar localizing and interacting of GRA10 with TAF1B suggested the participation of GRA10 in rRNA synthesis of host cells to favor the parasitism of T. gondii.
Alpha-Amanitin/pharmacology ; Animals ; Antibodies, Monoclonal/analysis/metabolism ; Antibodies, Protozoan/analysis/metabolism ; Dactinomycin/pharmacology ; Fluorescent Antibody Technique, Direct ; Gene Expression/*physiology ; Green Fluorescent Proteins/genetics ; Hela Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleic Acid Synthesis Inhibitors/pharmacology ; Nucleolus Organizer Region/drug effects/*metabolism ; Pol1 Transcription Initiation Complex Proteins/metabolism ; Protein Sorting Signals/physiology ; Protozoan Proteins/*biosynthesis/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Toxoplasma/*physiology ; Transfection