Sequence variation in Toxoplasma gondii MIC 13 gene among isolates from different hosts and geographical locations

1 College of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong Province 510642, China. 2 State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province 730046, China. 3 College of Animal Science and Technology, Yunnan Agricultural University, Kunming, Yunnan Province 650201, China.


INTRODUCTION
Toxoplasma gondii can infect almost any nucleated mammalian and avian cell types, and causes several clinical syndromes including encephalitis, chorioretinitis, systemic infections and abortion in its hosts, particularly seriously in individuals with HIV/AIDS (Montoya and Liesenfeld, 2004;Weiss and Dubey, 2009;Dubey, 2010;Zhou et al., 2011).The main measures for controlling toxoplasmosis in humans and animals are based on chemotherapy.
However, the currently used chemotherapeutic agents have some disadvantages such as inadequacy, high cost or toxicity.Obviously an effective vaccine would be ideal for the prevention and control of toxoplasmosis in humans and animals.
As sub-cellular organelles of T. gondii, micronemes, rhoptries and dense granules play key roles during the active invasion process of T. gondii into host cells (Carruthers and Sibley, 1997).Micronemal proteins (MICs) have been shown to contribute critically to host cell invasion, although their precise roles remained to be * based on genotyping results of Zhou et al. (2009Zhou et al. ( , 2010)).
fully understood (Soldati et al., 2001;Dowse and Soldati, 2004;Friedrich et al., 2010).Previous studies have also indicated that T. gondii MICs, such as MIC1, 3, 4 and 6, are potential vaccine candidate molecules against toxoplasmosis (Lourenço et al., 2006;Peng et al., 2009;Wang et al., 2009;Xiang et al., 2009).Like the T. gondii MIC1 (TgMIC1) having the microneme adhesive repeat (MAR) domain which recognizes sialic acid (Sia, a key determinant on the host cell surface for invasion by T. gondii), T. gondii MIC13 (TgMIC13), a novel Sia-binding lectin, is also involved in Sia-dependent invasion of host cells (Friedrich et al., 2010).However, it is yet to know whether there are sequence variations in TgMIC13 gene among T. gondii isolates from different hosts and geographical regions, representing different genotypes.The objective of the present study was to examine sequence variability in TgMIC13 gene among different Toxoplasma gondii isolates from different host and geographical origins.

T. gondii isolates and extraction of RNA and genomic DNA
Tachyzoites of the highly virulent RH strain of T. gondii (Type I) were prepared and collected according to the method described previously (Yan et al., 2011).Total RNA and genomic DNA (gDNA) were extracted by using Tissue RNA Kit (Biomiga) and Wizard SV Genomic DNA Purification System (Promega), respectively, according to the manufacturer's recommendations.The extracted total RNA and DNA samples were stored at -80 and -20ºC, respectively, until use.The cDNA was reverse-transcribed by using Rever Tra Ace-α Enzyme (First Strand cDNA Synthesis Kit, TOYOBO) according to the manufacturer's recommendations.All the other 17 T. gondii isolates were from different geographical locations and hosts (Table 1).These isolates have been genotyped previously (Zhou et al., 2009(Zhou et al., , 2010)).
Each amplicon (5 μL) was examined by agarose gel electrophoresis to validate amplification efficiency.The size of MIC13 amplicons was estimated using the DL5000 DNA marker (TAKARA).Positive amplicons were purified using spin columns (Wizard PCR-Preps DNA Purification System, Promega), ligated with pGEM-T Easy vector (Promega) and sequenced as reported previously (Huang et al., 2004). R=transition/transversion.

Sequence analysis and re-construction of phylogenetic relationships
The TgMIC13 gene sequences were aligned using the computer program ClustalX 1.81 (Thompson et al., 1997).Nucleotide variations in the TgMIC13 gene sequences among the examined T. gondii isolates were examined by pair-wise sequence comparison using the formula D = 1-(M/L), where M is the number of alignment positions at which the two sequences have a base in common, and L is the total number of alignment positions over which the two sequences are compared (Chilton et al., 1995).
Phylogenetic reconstruction among the examined T. gondii isolates was conducted using maximum parsimony (MP) based on the TgMIC13 gene sequences using Neospora caninum (GenBank accession No. AF421187.1)as the out-group.MP analysis was performed using PAUP* 4.0b10 (Swofford, 2002), with indels treated as missing character states.A total of 1,000 random addition searches using TBR were performed for each MP analysis.Bootstrap probability (BP) was calculated from 1,000 bootstrap replicates with 10 random additions per replicate in PAUP.Phylograms were drawn using the Tree View program version 1.65 (Page, 1996).

RESULTS AND DISCUSSION
The genomic TgMIC13 sequences were 2506-2507 bp in length for all the examined T. gondii isolates.Among them, the TgMIC13 sequences of T. gondii NT and SH isolates were 2506 bp in length, whereas those of the other isolates were 2507 bp.The TgMIC13 cDNA sequence of T. gondii RH strain was 1407 bp in length, consistent with previous report (Friedrich et al., 2010).By comparing the TgMIC13 genomic sequences and cDNA sequences, five introns were identified in the TgMIC13 gene (Table 2).A total of 21 variable nucleotide positions were identified in the TgMIC13 gene sequences, 10 of them were in the expressed regions and the other 11 were distributed among the 5 introns, with intra-specific variation of up to 0.84% (21/2507) for genomic DNA sequences and 0.71% (10/1407) for cDNA sequences.
Phylogenetic reconstruction of the examined 18 T. gondii isolates using MP analysis revealed that T. gondii isolates of the same genotypes were clustered in different clades (Figure 1), suggesting that the TgMIC13 DNA sequences were not an ideal genetic marker for intraspecies phylogenetic analysis and differential identification of T. gondii isolates of different genotypes from different hosts and geographical locations.This result was not consistent with that of previous studies using other genetic markers (e.g.PK1 and SAG2) (Su et al., 2006).

Conclusions
The present study revealed low level variability in MIC13 gene sequences among T. gondii isolates of different genotypes from different geographical regions and hosts.TgMIC13 gene sequence was not an effective genetic marker for intra-species phylogenetic and population genetic studies of T. gondii isolates of different host and geographical origins.

Figure 1 .
Figure 1.Phylogenetic relationships of Toxoplasma gondii isolates from different hosts and geographical locations inferred by maximum parsimony (MP) analyses based on the TgMIC13 gene sequences using Neospora caninum (GenBank accession number AF421187.1)as the outgroup.Numbers at nodes indicate bootstrap values (%).

Table 1 .
Details of Toxoplasma gondii isolates used in this study and the GenBank accession numbers of their MIC13 gene sequences.

Table 2 .
Characteristics of Toxoplasma gondii MIC13 (TgMIC13) gene sequences including their expressed regions and introns.