MIC16 gene represents a potential novel genetic marker for population genetic studies of Toxoplasma gondii

The zoonotic agent Toxoplasma gondii is distributed world-wide, and can infect a broad range of hosts including humans. Microneme protein 16 of T. gondii (TgMIC16) is responsible for binding to aldolase, and is associated with rhomboid cleavage and presence of trafficking signals during invasion. However, little is known of the TgMIC16 sequence diversity among T. gondii isolates from different hosts and geographical locations. In this study, we examined sequence variation in MIC16 gene among T. gondii isolates from different hosts and geographical regions. The entire genomic region of the MIC16 gene was amplified and sequenced, and phylogenetic relationship was reconstructed using Bayesian inference (BI) and maximum parsimony (MP) based on the MIC16 gene sequences. The results of sequence alignments showed two lengths of the sequence of MIC16 gene among all the examined 12 T. gondii strains: 4391 bp for strains TgCatBr5 and MAS, and 4394 bp for strains RH, TgPLH, GT1, PRU, QHO, PTG, PYS, GJS, CTG and TgToucan. Their A+T content ranged from 50.30 to 50.59 %. A total of 107 variable nucleotide positions (0.1–0.9 %) were identified, including 29 variations in 10 exons and 78 variations in 9 introns. Phylogenetic analysis of MIC16 sequences showed that typical genotypes (Type I, II and III) were able to be grouped into their respective genotypes. Moreover, the three major clonal lineages (Type I, II and III) can be differentiated by PCR-RFLP using restriction enzyme Pst I. Phylogenetic analysis and PCR-RFLP of the MIC16 locus among T. gondii isolates from different hosts and geographical regions allowed the differentiation of three major clonal lineages (Type I, II and III) into their respective genotypes, suggesting that MIC16 gene may provide a novel potential genetic marker for population genetic studies of T. gondii isolates.


Background
Toxoplasma gondii is an important zoonotic pathogen which can infect all warm-blooded animals and humans. It is estimated that approximately one-third of human population in the world has been infected with T. gondii [1][2][3]. Toxoplasmosis causes several clinical syndromes such as chorioretinitis, encephalitis and systemic infections, particularly seriously in pregnant hosts and immuno-compromised individuals such as those with HIV/AIDS [4][5][6]. Also, toxoplasmosis can lead to considerable economic losses in livestock industry [7][8][9].
T. gondii isolates from different geographical locations and hosts have been grouped into 15 haplogroups that collectively define six major clades by PCR-restriction fragment length polymorphism (PCR-RFLP) [10,11]. Despite that genotyping T. gondii strains using polymorphisms has been focused on 12 markers including SAG1, SAG3, BTUB, GRA6, c29-2, L358, PK1, 5′-SAG2, 3′-SAG2, alt. SAG2, C22-8 and Apico, additional gene (s) may contribute to some distinct T. gondii genotypes [12]. The full knowledge of genetic diversity in T. gondii is a key to better understand pathogenicity and epidemiological patterns, and thus to explore a new way for vaccination, treatment and diagnosis of toxoplasmosis.
Microneme proteins (MICs) of T. gondii play key roles in T. gondii survival and the invasion process, and thus affecting host cell signaling, as well as participating in the binding to host cell receptors [13][14][15]. Among those MICs, the microneme protein 16 of T. gondii (TgMIC16) is responsible for binding to aldolase, is associated with rhomboid cleavage, and thus involved in the trafficking signals during invasion [16]. Although the major functions of TgMIC16 were uncovered, little is known of the sequence variation in MIC16 gene among T. gondii isolates.
Therefore, the objectives of the present study were to examine sequence variation in MIC16 gene among 12 T. gondii strains from different hosts and geographical locations, as well as to assess whether MIC16 gene can provide a potential marker for population genetic studies of T. gondii by phylogenetic analysis and PCR-RFLP.

Methods
Genomic DNA samples of T. gondii A total of 12 genomic DNA samples of T. gondii were used in this study (Table 1). These T. gondii DNA samples had been genotyped in our previous studies [17][18][19]. The DNA samples of the reference strains GT1, MAS, TgCatBr5, PTG, CTG and TgToucan were kindly provided by Associate Professor Chunlei Su at the Department of Microbiology, the University of Tennessee, Knoxville, USA.

PCR amplification
The entire genomic sequence of the MIC16 gene was amplified by PCR using a pair of specific primers (forward primer: 5′-ATGGTTGTTTCCTGTCTCTGT AC-3′; reverse primer: 5′-TTAGAGGTAGTTGTCC CGTGTCC-3′) designed based on the MIC16 gene sequence of T. gondii GT1 strain (TGGT1_289630, http://www.toxodb.org/toxo/). The amplification reaction was carried out in a volume of 25 μl containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 3 mM MgCl2, 250 μM each of dNTP, 0.2 μM of each primer, 100-200 ng of template DNA, and 0.25 U La Taq polymerase (TaKaRa). Amplification of DNA samples from individual isolates was carried out in a thermocycler (Bio-Rad, Hercules, CA, USA) under the following conditions: the initial denaturation at 95°C for 5 min, followed by 34 cycles consisting of 95°C for 1 min (denaturation), 64°C for 45 s (annealing for the two primers), 72°C for 4 min 30 s (extension for the two primers), and a final extension step was at 72°C for 10 min. Each (5 μl) amplification was carried out by electrophoresis on a 1 % (w/v) agarose gel with ML5000 DNA ladder marker (TaKaRa), stained with GoldenView™ and photographed using a gel documentation system (UVPGelDoc-It™ Imaging System, Cambridge, UK) [20].

Sequencing of the MIC16 amplicons
To ensure the accuracy of MIC16 sequences from different T. gondii strains, the PCR products of MIC16 gene were purified according to manufacturer's recommendations (Wizard™ PCR-Preps DNA Purification System, Promega, USA), and then ligated with the pGEM-Teasy (Promega, USA). Thereafter, positive plasmids were transformed into the JM109 competent cells (Promega, USA). Following PCR screening, the positive colonies were sequenced by GenScript (Nanjing) Co., Ltd.

Sequence analysis and phylogenetic reconstruction
The obtained MIC16 gene sequences from different T. gondii strains were aligned using Multiple Sequence Alignment Program, Clustal X 2.11 [21], and sequence variation was determined among the examined T. gondii strains. The phylogenetic reconstructions based on the MIC16 sequences from different T. gondii strains were performed by Bayesian inference (BI) and maximum parsimony (MP). BI analyses were conducted with four independent Markov chains run for 10,000,000 metropolis coupled MCMC generations, sampling a tree every 10,000 generations in MrBayes 3.1.1 [22]. The first 250 trees were omitted as burn-in and the remaining trees were used to calculate Bayesian posterior probabilities (PP). MP analysis was performed using PAUP* 4.0b10 [23]. Bootstrap support for MP tree was calculated from 1000 bootstrap replicates with 10 random additions per replicate [24].

PCR-RFLP
PCR-RFLP was used to further assess whether MIC16 gene sequence is a potential marker for genotyping T. gondii isolates. The amplified MIC16 fragments were digested with restriction enzyme Pst I for 8 h at 37°C in the appropriate buffer, and then inactivated at 80°C for 20 min according to the manufacturer's instructions (NEB). The restriction fragments were analyzed by electrophoresis on 1 % agarose gel stained with Golden-View™ and photographed using a gel documentation system (UVP GelDoc-It™ Imaging System, Cambridge, UK).

Sequence analysis
All  Figure S1). Sequences analysis of polymorphisms in the three references genotypes (RH/GTI, PRU/QHO/PTG, CTG) revealed the existence of polymorphic restriction sites. Using PCR-RFLP method, digestion of the amplification products with Pst I allowed the differentiation between genotype I, II, and III (Fig. 1).

Phylogenetic analysis of T. gondii strains based on MIC16 sequences
By phylogenetic reconstruction based on MIC16 sequence data of all 12 strains, we have obtained the phylogenetic tree (Fig. 2). Phylogenetic analysis revealed three major clusters, which are corresponding to classical genotypes (I, II, III) respectively, and Type III are clustered more closely with type I than with other strains. Atypical strains TgToucan were phylogenetically linked to Type III, and thus the both strains GYS and PYS were phylogenetically linked to Type II.

Discussion
In the present study, we determined the entire genome region of the MIC16 locus for 12 T. gondii strains among different host and geographical origins, and determined  [25][26][27]. The analyses of MIC16 gene in both nucleotides and amino acids among 12 T. gondii strains showed high ratio of non-synonymous to synonymous polymorphisms, emphasized that MIC16 was undergoing positive selection together with some other loci, such as GRA5, GRA6 and ROP17 [24,28,29].
With the purpose of analyzing the evolutionary relationship among the 12 examined T. gondii isolates, the phylogenetic tree was established (Fig. 1) based on the MIC16 sequence using BI and MP methods. Phylogenetic analysis showed that T. gondii strains corresponding to classical genotypes (I, II, III) were grouped into their respective clusters separately, which were consistent with the results from other genetic markers including GRA6 and GRA5 genes [24,28].
Moreover, the three major clonal lineages (Type I, II and III) can be differentiated by digestion of MIC16 PCR products using endonuclease Pst I (Fig. 2), which further suggested that the MIC16 locus may be a potential new genetic marker for PCR-RFLP genotyping of T. gondii strains. However, some atypical strains including TgCatBr5, TgToucan, PYS, GJS and MAS can not be distinguished from the three major lineage types by PCR-RFLP (not shown), suggesting that PCR-RFLP genotyping with a single locus is not enough for the identification of non-clonal genotypes.

Conclusions
The present study examined sequence variation in MIC16 and demonstrated the existence of sequence variability in MIC16 gene among the examined T. gondii strains from different hosts and geographical localities. Phylogenetic analysis and PCR-RFLP genotyping of the examined T. gondii strains suggest that MIC16 gene may provide a potential marker for population genetic studies of T. gondii isolates, and further research sampling more isolates from different hosts and geographical separations need to be done to verify this possibility.

Availability of data and materials
The data sets supporting the results of this article are included within the article and its additional file. GenBank accession numbers for these T. gondii MIC16 sequences are KX147274-KX147285. Study Accession URL of deposition of our phylogenetic data in TreeBase is http://purl.org/phylo/treebase/phylows/study/ TB2:S19062.
Authors' contributions XQZ and JC conceived and designed the study and revised the manuscript. WGL, XPX and SLL performed the experiments and analyzed the data. QMX helped in the study design and manuscript revision. WGL and XPX wrote the manuscript. All authors read and approved the final manuscript.

Competing interests
The authors declare that they have no competing interests.

Consent for publication
Not applicable.
Ethics approval and consent to participate Not applicable.   Table 1 for isolate information