Several studies have shown the mechanisms and importance of immune responses against Toxoplasma gondii infection and the notable role of cholinesterases in inflammatory reactions. However, the association between those factors has not yet been investigated. Therefore, the aim of this study was to evaluate the acetylcholinesterase (AChE) activity in blood and lymphocytes and the activity of butyrylcholinesterase (BChE) in serum of rats experimentally infected with T. gondii during the acute phase of infection. For that, an in vivo study was performed with evaluations of AChE and BChE activities on days 5 and 10 post-infection (PI). The activity of AChE in blood was increased on day 5 PI, while in lymphocytes its activity was enhanced on days 5 and 10 PI (P<0.05). No significant difference was observed between groups regarding to the activity of BChE in serum. A positive (P<0.01) correlation was observed between AChE activity and number of lymphocytes. The role of AChE as an inflammatory marker is well known in different pathologies; thus, our results lead to the hypothesis that AChE has an important role in modulation of early immune responses against T. gondii infection.
Acetylcholinesterase/blood/*metabolism ; Animals ; Butyrylcholinesterase/blood/*metabolism ; Humans ; Lymphocytes/enzymology/parasitology ; Male ; Rats ; Rats, Wistar ; Toxoplasma/*physiology ; Toxoplasmosis/*enzymology/genetics/parasitology

Monoclonal antibody (mAb) Tg786 against Toxoplasma gondii has been found to detect a 42-kDa rhoptry protein (ROP6) which showed protease activity and host cell binding characteristics after secretion. Using the mAb, a colony containing a 3'-UTR was probed in a T. gondii cDNA expression library. A full length cDNA sequence of the rhoptry protein was completed after 5'-RACE, which consisted of 1,908 bp with a 1,443 bp ORF. The deduced amino acid sequence of ROP6 consisted of a polypeptide of 480 amino acids without significant homology to any other known proteins. This sequence contains an amino terminal stop transfer sequence downstream of a short neutral sequence, hydrophilic middle sequence, and hydrophobic carboxy terminus. It is suggested that the ROP6 is inserted into the rhoptry membrane with both N- and C-termini.
Toxoplasma/enzymology/*genetics ; Protozoan Proteins/*genetics/metabolism ; Peptide Hydrolases/genetics/metabolism ; Molecular Sequence Data ; Gene Library ; Cloning, Molecular ; Animals ; Amino Acid Sequence ; 3' Untranslated Regions

OBJECTIVEThe protozoan Toxoplasma gondii expresses large amounts of a 37 kDa Type 2C serine-threonine phosphatase, the so-called TgPP2C which has been suggested to contribute to parasite growth regulation. Ectopic expression in mammalian cells also indicated that the enzyme could regulate growth and survival. In this study, we aimed to investigate the interaction of TgPP2C with human SSRP1 (structure-specific recognition protein 1) and the effects of TgPP2C on cell viability.

METHODSThe yeast two hybrid system, His-tag pull-down and co-immunoprecipitation assays were used to confirm the interaction of TgPP2C with SSRP1 and determine the binding domain on SSRP1. The evaluation of cell apoptosis was performed using cleaved caspase-3 antibody and Annexin-V/PI kit combined with flow cytometry.

RESULTSWe identified human SSRP1 as an interacting partner of TgPP2C. The C-terminal region of SSRP1 including the amino acids 471 to 538 was specifically mapped as the region responsible for interaction with TgPP2C. The overexpression of TgPP2C down-regulated cell apoptosis and negatively regulated apoptosis induced by DRB, casein kinase II (CKII) inhibitor, through enhanced interaction with SSRP1.

CONCLUSIONTgPP2C may be a parasitic factor capable of promoting cell survival through interaction with the host protein SSRP1, thereby creating a favorable environment for parasite growth.

Apoptosis ; Blotting, Western ; DNA-Binding Proteins ; genetics ; metabolism ; Flow Cytometry ; HeLa Cells ; High Mobility Group Proteins ; genetics ; metabolism ; Humans ; Immunoprecipitation ; Phosphoprotein Phosphatases ; genetics ; metabolism ; Protein Phosphatase 2C ; Toxoplasma ; enzymology ; Transcriptional Elongation Factors ; genetics ; metabolism ; Two-Hybrid System Techniques

Toxoplasma gondii, an obligate intracellular protozoan parasite of the phylum Apicomplexa, can infect all warm-blooded vertebrates, including humans, livestock, and marine mammals. The aim of this study was to investigate whether superoxide dismutase (SOD) of T. gondii can be used as a new marker for genetic study or a potential vaccine candidate. The partial genome region of the SOD gene was amplified and sequenced from 10 different T. gondii isolates from different parts of the world, and all the sequences were examined by PCR-RFLP, sequence analysis, and phylogenetic reconstruction. The results showed that partial SOD gene sequences ranged from 1,702 bp to 1,712 bp and A + T contents varied from 50.1% to 51.1% among all examined isolates. Sequence alignment analysis identified total 43 variable nucleotide positions, and these results showed that 97.5% sequence similarity of SOD gene among all examined isolates. Phylogenetic analysis revealed that these SOD sequences were not an effective molecular marker for differential identification of T. gondii strains. The research demonstrated existence of low sequence variation in the SOD gene among T. gondii strains of different genotypes from different hosts and geographical regions.
Amino Acid Sequence ; Animals ; Base Sequence ; Cats ; *Genetic Variation ; Goats ; Humans ; Molecular Sequence Data ; Phylogeny ; Protozoan Proteins/chemistry/*genetics/metabolism ; Sequence Alignment ; Sheep ; Superoxide Dismutase/chemistry/*genetics/metabolism ; Toxoplasma/classification/*enzymology/genetics/isolation & purification ; Toxoplasmosis/*parasitology ; Toxoplasmosis, Animal/*parasitology

A cDNA of 1.1 kb comprising the gene encoding the peroxiredoxin of Toxoplasma gondii (TgPrx) has been cloned. The open reading frame of 591 bp was translated into a protein of 196 amino acids with a molecular mass of 25 kDa. Conserved 2 cysteine domains of Phe-Val-Cys-Pro and Glu-Val-Cys-Pro indicated TgPrx belonged to 2-Cys Prx families. TgPrx showed the highest homology with that of Arabidopsis thaliana by 53.9% followed by Entamoeba histolytica with 39.5% by the amino acid sequence alignment. Polyclonal antibody against recombinant TgPrx detected 25 kDa band in T. gondii without binding to host cell proteins. TgPrx was located in the cytoplasm of T. gondii extracellularly or intracellularly by immunofluorescence assay. The expression of TgPrx was increased as early as 30 min after the treatment with artemisinin in the intracellular stage, while no changes in those of host Prx I and TgSOD. This result implies that TgPrx may function as an antioxidant protecting the cell from the attack of reactive oxygen intermediates. It is also suggested that TgPrx is a possible target of chemotherapy.
Amino Acid Sequence ; Animals ; Antioxidants ; *Artemisinins ; Base Sequence ; *Cloning, Molecular ; Cysteine/metabolism ; Molecular Sequence Data ; Nucleic Acid Amplification Techniques ; Peroxidases/chemistry/*genetics/physiology ; Sesquiterpenes/pharmacology ; Support, Non-U.S. Gov't ; Toxoplasma/*enzymology