Sequence Variation in HSP40 Gene among 16 Toxoplasma gondii Isolates from Different Hosts and Geographical Locations

Toxoplasma gondii with worldwide distribution has received substantial medical and scientific attentions as it causes serious clinical and veterinary problems especially for pregnant women and immunocompromised patients. Heat shock protein 40 (HSP40) plays a variety of essential roles in the pathogenesis of this protozoan parasite. In order to detail the genetic diversity of HSP40 gene, 16 T. gondii strains from different hosts and geographical locations were used in this study. Our results showed that HSP40 sequence of the examined strains was between 6621 bp and 6644 bp in length, and their A+T content was from 48.54% to 48.80%. Furthermore, sequence analysis presented 195 nucleotide mutation positions (0.12%–1.14%) including 29 positions in CDS (0.02%–0.12%) compared with T. gondii ME49 strain (ToxoDB: TGME49_265310). Phylogenetic assay revealed that T. gondii strains representing three classical genotypes (Types I, II, and III) were completely separated into different clusters by maximum parsimony (MP) method, but Type II and ToxoDB#9 strains were grouped into the same cluster. These results suggested that HSP40 gene is not a suitable marker for T. gondii population genetic research, though three classical genotypes of T. gondii could be differentiated by restriction enzymes MscI and EarI existing in amplicon C.


Introduction
Toxoplasma gondii infects almost all the warm-blooded animals including about one-third population of humans [1,2] and can cause serious clinical diseases, especially in pregnant women and immunocompromised individuals such as tumor sufferers and AIDS patients [3,4]. T. gondii can also cause abortion and congenital toxoplasmosis in livestock, leading to considerable economic losses [5,6].
Heat shock proteins (HSPs) involved in antigen presentation and cross-presentation play important roles in activation of immune-related cells such as macrophages, lymphocytes, and DCs [7][8][9]. As the important member of HSPs, HSP40 associated with DNA replication, protein folding, assembling and degradation, translocation across membranes, signal transduction, and endocytosis participates in the pathogenesis of apicomplexan parasites such as Plasmodium falciparum [10,11]. Recent studies emphasized that different clonal types of T. gondii strains with diverse geographical distribution can cause different toxoplasmosis in animals and humans [12,13]. In order to unveil the details of T. gondii genetic diversity, sequence variation of the type II HSP40TgSIS1 (ToxoDB: TGME49 265310, previously named TGME49 065310) [14] among T. gondii isolates from different hosts and geographical regions was examined in this study.

T. gondii Isolates and gDNA Preparation.
Sixteen T. gondii isolates harvested from different hosts and geographical locations were used in the present study (Table 1). Genomic DNA was extracted as normal and stored at −20 ∘ C till used.

PCR Amplification and Sequencing.
Three fragments (A, B, and C) ( Figure 1) were separated based on the       HSP40 sequence of T. gondii ME49 isolate (ToxoDB: TGME49 265310), and the amplifications were performed by PCR using three pairs of specific primers, respectively ( Table 2). Thermal cycling conditions were according to the following protocol: initial denaturation at 94 ∘ C for 10 min followed by 35 cycles composing of 94 ∘ C for 1 min, 54.7 ∘ C (amplicon A), 66.8 ∘ C (amplicon B) or 64.0 ∘ C (amplicon C) for 45 s respectively, and 72 ∘ C for 2 min, and the additional extension step was carried out at 72 ∘ C for 10 min. Negative control without gDNA was included in each amplification. PCR amplifications were confirmed by agarose gel electrophoresis as previously described [15]. All the PCR products were purified using spin columns (Promega, Madison, USA), ligated with pMD18-T vector (TaKaRa, Dalian, China), and transformed into E. coli DH5 competent cells (Promega) according to the manufacturers' instructions. The positive colonies confirmed by PCR were sequenced by Shanghai Sangon Biological Engineering Biotechnology Company. All the experiments were run in triplicate.

Sequence Analysis and Phylogenetic Reconstruction.
The sequences of all the examined T. gondii strains were amplified step by step and sequenced. Alignment analysis based on the obtained sequences including the reference one (Tox-oDB: TGME49 265310) was carried out with Clustal X 1.83 [16], and the number of sequence variation compared with ME49 strain was calculated as previously described [17]. Phylogenetic reconstructions were performed by maximum parsimony (MP) method using PAUP * 4.0b10 [18], and 100 random addition searches using tree-bisection reconnection (TBR) were carried out for each MP assay. Bootstrap probability (BP) was calculated from 1,000 bootstrap replicates with 10 random additions per replicate in PAUP.

Characterization of T. gondii Isolates by PCR-RFLP.
To evaluate whether HSP40 gene was suitable for genotyping of T. gondii isolates, PCR-RFLP method was also used in this study as previously described [19,20]. All the PCR products of amplicon C were digested with two restriction enzymes MscI and EarI by incubating at 37 ∘ C for 4 h according to the manufacturer's instructions (NEB, Beijing, China). And the restriction fragments were separated by electrophoresis as previously described [21].
Phylogenetic reconstruction was constructed based on HSP40 sequences of the 17 T. gondii strains including T. gondii ME49 isolates (ToxoDB: TGME49 265310) (Figure 3) [18]. Our results showed that T. gondii strains belonging to Type II (PRU, QHO, ME49, and PTG), Type 12 (TgWtdSc40), or ToxoDB#9 (PYS, TgC7 and GJS) were grouped into the same cluster, whereas TgToucan (ToxoDB#52) was gathered into the cluster of Type I (RH, GT1, and TgPLH), suggesting that the examined T. gondii strains could not be completely separated by MP method though three classical genotypes of T. gondii (Types I, II, and III) were clustered into different groups.

Conclusion
Our data suggested that HSP40 gene is not a suitable marker for T. gondii population genetic study, though three classical genotypes of T. gondii (Types I, II, and III) could be differentiated by polymorphic restriction endonuclease sites MscI and EarI existing in amplicon C.