Toxoplasma gondii is an obligatory intracellular parasite that causes severe disease after congenital infection and in immunocompromized patients such as AIDS. During infection, immune effector cells are able to kill or inhibit its intracellular growth. This antiprotozoan activity produces a number of toxic products such as reactive oxygen intermediates. And within the host cell, T. gondii itself produces oxidants as by-products of normal metabolism.

Reactive oxygen species (ROS) are potentially destructive, capable of oxidizing proteins or lipids and causing chemical modification to nucleic acids (Halliwell and Gutteridge, 1991), even though some of these species also function as second messenger molecules (Dalton, 1999). Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase are the three major enzymes to be involved in the cellular anti-ROS system. Hydrogen peroxide, which is produced by SOD at the initial step of ROS detoxification, is subsequently eliminated by catalase and/or Gpx (Halliwell and Gutteridge, 1991). The presence of endogeneous SOD, GPx, and catalase have been demonstrated in T. gondii (Hughes et al., 1989; Ding et al., 2000; Odberg-Ferragut et al., 2000; Kaasch and Joiner, 2000), which suggests to be related with protection from ROS attacks.

Peroxiredoxin (Prx) is a recently described family of antioxidants that are highly conserved in eukaryotes and prokaryotes (McGonigle et al., 1998), which has been called as thioredoxin peroxidase (Tpx,) or thiolspecific antioxidant (TSA). Prx acts as an antioxidant enzyme by eliminating hydrogen peroxide and hydroxyl radicals. The catalytic mechanism of the enzyme involves the redoxactive cysteine, which is highly conserved in the vicinity of the 47th position of its amino acid sequence (Chae et al., 1994). We report here the cDNA cloning, expression, and functional characterization of a Prx from T. gondii (TgPrx).

The DNA fragments of 634 bp (Fig. 1, lane 1) and 744 bp (Fig. 1, lane 3) were amplified by 5'-RACE and 3'-RACE on the cDNA of tachyzoites, respectively. Both the DNA fragments were confirmed to be specific to T. gondii by nested PCR. Nested PCR with 5-NEST and GSP1 primers produced a 424 bp DNA fragment (Fig. 1, lane 2) on the DNA fragment of lane 1 as the template, which made a shorter fragment of 210 bp as expected. And nested PCR with GSP2 and 3-NEST primers amplified a 675 bp DNA fragment (Fig. 1, lane 4) with the DNA fragment of lane 3 as the template which resulted in 70 bp difference. A full-length cDNA sequence of TgPrx was obtained by combining the sequences of the DNA fragments of lane 2 and 4 of Fig. 1. It was 1,101 bp long including a predicted open reading frame of 591 bp. Conserved 2 cysteine domains were present as Phe-Val-Cys-Pro and Glu-Val-Cys-Pro in deduced 196 amino acid sequence (Fig. 2), which indicated that TgPrx belonged to 2-Cys Prx families. The cysteine residue of the anterior domain localized at 51 amino acid (Cys-51) and that of posterior at 171 amino acid (Cys-171). A search of the Toxoplasma database of expressed sequence tags (EST) identified a sequence of 197 bp (Ctoxqual2_665) with perfect match. This EST sequence is overlapped with the anterior cysteine (Cys-51) domain and used in the analysis of peroxidoxins by McGonigle et al. (1998). Deduced amino acid sequence of TgPrx revealed significant sequence similarities with other 2-Cys Prx (Fig. 3).

TgPrx and TgSOD were expressed as GST fusion proteins in HB101 bacteria by the IPTG induction with molecular mass of 51 kDa and 52 kDa, respectively (data not shown). Polyclonal antibodies against recombinant TgPrx and TgSOD detected those proteins of T. gondii each other specifically without binding to host cells (Fig. 4). TgPrx was detected as 25 kDa in extracellular and intracellular stages as traced by anti-SAG1 monoclonal antibody and TgSOD as 26 kDa with the same pattern. TgPrx was localized in the cytoplasm of tachyzoites as revealed by IFA images (Fig. 5A) comparing with spot distribution of dense granular protein (Tg378). Its distribution was limited in the tachyzoites and was not secreted into PV or PVM whereas Tg378 was secreted into PVM during the intracellular stages (Fig. 5B).

Artemisinin, a sesquiterpene endoperoxide, was treated to trace the changes in TgPrx after being exposed to the peroxide during the intracellular growing stage of tachyzoites. The concentration of LD₅₀ was 1 µg/ml in our culture system (data not shown). Artemisinin itself evoked little changes in host cell Prx as detected by anti-mammalian Prx I antibody (Fig. 6A). However, the expression of TgPrx increased as early as 30 min and reached to the plateau in 1 hr by the treatment with artemisinin in the intracellular stage (Fig. 6B), while there were little changes in those of host Prx I and TgSOD. Host cell Prx had approximately equal response to artemisinin regardless of infection by T. gondii.

A full-length cDNA sequence of TgPrx was composed of 1,101 bp including a predicted open reading frame of 591 bp, which coded 196 amino acid sequence of 25 kDa TgPrx. It was preceded by a T-rich region in the 5' untranslated region which was seen in other T. gondii genes (Cesbron-Delauw et al., 1989; Ossorio et al., 1992; Mercier et al., 1998; Lecordier et al., 1999; Carey et al., 2000). The first ATG did conform to the T. gondii consensus translation initiation sequence of NAAA except for C at position +4 (Seeber, 1997).

Two cysteine residues were positioned at 51 amino acid (Cys-51) and 171 amino acid (Cys-171) as compare to 47 (Cys 47) and 170 (Cys 170) in other Prx (Chae et al., 1994a; 1994b). These site variations were also found in tryparedoxin peroxidase (Alphey, 2000). All the sequences of anterior cysteine domains of Prx compared were the same as Phe-Val-Cys-Pro, and the posterior also as Glu-Val-Cys-Pro except for Entamoeba histolytica of Ala at the first position. The amino acid sequence of TgPrx showed the highest homology with that of Arabidopsis thaliana by 53.9%. Among Prx of parasitic organisms, Prx of E. histolytica had the homologous sequence of 39.5%, which made this organism belong to a cluster in upper hierarchy with AhpC of Escherichia coli (33.1%).

Prx are found in all organisms and their primary sequences are highly conserved. This ubiquity and high degree of conservation suggest a biological importance of this enzyme. Prx are critically involved in cellular defense, receptor signaling, protein phosphorylation transcriptional regulation, and apoptosis (Zhang et al., 1997; Jin and Jeang, 2000). TgPrx was also abundant in tachyzoites as in other eukaryotic cells of which the Prx covered 2 to 5% of total cellular proteins as a housekeeping protein (Chae et al., 1994b). And TgPrx localized in the cytoplasm of tachyzoites and maintained in intracellular stage without secretion into PV nor PVM, which suggests its role in defense mechanism against ROS produced during the metabolism within tachyzoites, although the exact function of TgPrx is still unknown.

Artemisinin, active constituent of qinghaosu as an endoperoxide which has been effective as antimalarial drug (Meshnick et al., 1996; Cumming et al., 1997), inhibited also the intracellular growth of T. gondii in vitro (Berens et al., 1998) by imposing oxidative stress within the cells. It has been proposed that artemisinin activated by the intraparasitic metals catalyzes the cleavage of this drug to ROS (Meshnick, 1998). In addition, ROS can also be cleaved from the drug in the cytoplasm of host cells, which then delivered across the PVM to reach the tachyzoites within the PVM. Resulting ROS might then damage the parasite by alkylating and poisoning one or more essential proteins. Inducible expression of Prx has been demonstrated for many mammalian cells. Inducers includes hydrogen peroxide (Kim et al., 1997), heme/heavy metals (Immenschuh et al., 1997), and others. Changes in TgPrx against artemisinin, in our results, may reduce the ROS by binding with its active thiol group. Therefore, since TgPrx is sensitive to artemisinin, it is suggested to be a good candidate for chemotherapy.