# Sequence Variation inToxoplasma gondii rop17 Gene among Strains from Different Hosts and Geographical Locations

Authors: Nian-Zhang Zhang; Ying Xu; Si-Yang Huang; Dong-Hui Zhou; Rui-Ai Wang; Xing-Quan Zhu
Journal: The Scientific World Journal
 (2014)

---

## Abstract

Genetic diversity ofT. gondii is a concern of many studies, due to the biological and epidemiological diversity of this parasite. The present study examined sequence variation in rhoptry protein 17 (ROP17) gene among T. gondii isolates from different hosts and geographical regions. The rop17 gene was amplified and sequenced from 10 T. gondii strains, and phylogenetic relationship among these T. gondii strains was reconstructed using maximum parsimony (MP), neighbor-joining (NJ), and maximum likelihood (ML) analyses. The partial rop17 gene sequences were 1375 bp in length and A+T contents varied from 49.45% to 50.11% among all examined T. gondii strains. Sequence analysis identified 33 variable nucleotide positions (2.1%), 16 of which were identified as transitions. Phylogeny reconstruction based on rop17 gene data revealed two major clusters which could readily distinguish Type I and Type II strains. Analyses of sequence variations in nucleotides and amino acids among these strains revealed high ratio of nonsynonymous to synonymous polymorphisms (>1), indicating that rop17 shows signs of positive selection. This study demonstrated the existence of slightly high sequence variability in the rop17 gene sequences among T. gondii strains from different hosts and geographical regions, suggesting that rop17 gene may represent a new genetic marker for population genetic studies of T. gondii isolates.


---

## Body

## 1. Introduction

Toxoplasma gondii can infect all warm-blooded vertebrates, including mammals and birds [1–3]. Genetic diversity ofT. gondii is widespread due to the biological and epidemiological diversity of this parasite.T. gondii isolates can be clustered into six major clades [4], and genetic diversity ofT. gondii is especially common in South America [4]. Utilizing 11 genetic markers,T. gondii isolates in North America and Europe are grouped into four major clonal lineage types (I, II, III, and 12) [5, 6] using PCR-RFLP.Rhoptry kinases are involved in mediating pathogenesis ofT. gondii[7], and they are also master regulators that manipulate the host inflammatory responses [8, 9].T. gondiirhoptry protein 17 (ROP17), a member of the ROP2 subfamily [10], was predicted to have a cellular localization on the parasitophorous vacuole membrane (PVM), which may participate in the manipulation of the host signalling pathways [9]. Previous studies have shown the existence of sequence variation in some ROP genes, such asrop7,rop9,rop13, androp38 [11–14]. However, it is yet to be known whether sequence diversity exists inrop17gene ofT. gondii. The objective of the present study was to examine sequence variation inrop17gene amongT. gondiistrains representing different genotypes and host and origins.


## 2. Materials and Methods

### 2.1.T. gondii Isolates

TenT. gondii strains collected from different hosts and locations were used for analysis in this study (Table 1). These strains have been genotyped and their genomic DNA has been prepared as described previously [15–17].Table 1
Details ofToxoplasma gondiistrains used in the present study.
Strain
Host
Geographical origin
Genotype*
GT1
Goat
United States
Reference, Type I
PTG
Sheep
United States
Reference, Type II, ToxoDB number 1
CTG
Cat
United States
Reference, Type III, ToxoDB number 2
TgCatBr5
Cat
Brazil
Reference, ToxoDB number 19
TgCatBr64
Cat
Brazil
Reference, ToxoDB number 111
TgCgCa1
Cougar
Canada
Reference, ToxoDB number 66
TgToucan (TgRsCr1)
Toucan
Costa Rica
Reference,  ToxoDB number 52
TgPLh
Pig
China
Type I,  ToxoDB number 10
QHO
Sheep
China
Type II, ToxoDB number 1
TgWtdSc40
White-tailed deer
United States
Type 12, ToxoDB number 5
*
Based on genotyping results of Zhou et al. [15, 16] and Su et al. [17].


### 2.2. Amplification ofrop17 Genes and Sequencing

Therop17 gene was amplified by PCR. Two primers were designed based on therop17 sequence ofT. gondii RH strain available in GenBank (accession number: KC997178): ROP17F, 5′-AGGACAACACTAGGTAGCGAGAACC-3′, and ROP17R, 5′-TGGCGAAGTCAAGAGACGACGCAG-3′. Each reaction was performed in a total volume of 25 μL containing 12.5 μLPremix Taq(TaKaRa, Dalian, China), ROP17F (20 pmol) 0.25 μL, ROP17R (20 pmol) 0.25 μL, template DNA (200 ng) 2 μL, and ddH2O 8 μL, and the reaction conditions were 94°C for 5 min, then 35 cycles of 30 sec at 94°C, 30 sec at 55°C, and 1 min 20 s at 72°C, and a final extension at 72°C for 10 min. All the PCR products were then cloned into pMD18-T vector (TaKaRa, China) after purification using the DNA purification kit (TIANGEN, China) and then sequenced by Songon Biotech Co., Ltd. (Shanghai, China).


### 2.3. Sequence Analysis and Reconstruction of Phylogenetic Relationships

Therop17 gene sequences of differentT. gondii strains were aligned using Multiple Sequence Alignment Program, Clustal X 1.83 [18], and the sequence differences were determined according to Chilton et al. [19] and Zhao et al. [20]. Phylogenetic reconstruction was based on therop17 gene sequences determined in the present study plus the corresponding sequences of strains TgC7, PRU, and RH available in GenBank (accession numbers: KC997176, KC997177, and KC997178) using three inference methods, namely, neighbor-joining (NJ), maximum likelihood (ML), and maximum parsimony (MP), using the sequence ofNeospora caninum (NCLIV_027930) as the outgroup. All the analyses were conducted following previous studies [20, 21]. Phylograms were drawn using the Tree View program version 1.65 [22].


## 2.1.T. gondii Isolates

TenT. gondii strains collected from different hosts and locations were used for analysis in this study (Table 1). These strains have been genotyped and their genomic DNA has been prepared as described previously [15–17].Table 1
Details ofToxoplasma gondiistrains used in the present study.
Strain
Host
Geographical origin
Genotype*
GT1
Goat
United States
Reference, Type I
PTG
Sheep
United States
Reference, Type II, ToxoDB number 1
CTG
Cat
United States
Reference, Type III, ToxoDB number 2
TgCatBr5
Cat
Brazil
Reference, ToxoDB number 19
TgCatBr64
Cat
Brazil
Reference, ToxoDB number 111
TgCgCa1
Cougar
Canada
Reference, ToxoDB number 66
TgToucan (TgRsCr1)
Toucan
Costa Rica
Reference,  ToxoDB number 52
TgPLh
Pig
China
Type I,  ToxoDB number 10
QHO
Sheep
China
Type II, ToxoDB number 1
TgWtdSc40
White-tailed deer
United States
Type 12, ToxoDB number 5
*
Based on genotyping results of Zhou et al. [15, 16] and Su et al. [17].


## 2.2. Amplification ofrop17 Genes and Sequencing

Therop17 gene was amplified by PCR. Two primers were designed based on therop17 sequence ofT. gondii RH strain available in GenBank (accession number: KC997178): ROP17F, 5′-AGGACAACACTAGGTAGCGAGAACC-3′, and ROP17R, 5′-TGGCGAAGTCAAGAGACGACGCAG-3′. Each reaction was performed in a total volume of 25 μL containing 12.5 μLPremix Taq(TaKaRa, Dalian, China), ROP17F (20 pmol) 0.25 μL, ROP17R (20 pmol) 0.25 μL, template DNA (200 ng) 2 μL, and ddH2O 8 μL, and the reaction conditions were 94°C for 5 min, then 35 cycles of 30 sec at 94°C, 30 sec at 55°C, and 1 min 20 s at 72°C, and a final extension at 72°C for 10 min. All the PCR products were then cloned into pMD18-T vector (TaKaRa, China) after purification using the DNA purification kit (TIANGEN, China) and then sequenced by Songon Biotech Co., Ltd. (Shanghai, China).


## 2.3. Sequence Analysis and Reconstruction of Phylogenetic Relationships

Therop17 gene sequences of differentT. gondii strains were aligned using Multiple Sequence Alignment Program, Clustal X 1.83 [18], and the sequence differences were determined according to Chilton et al. [19] and Zhao et al. [20]. Phylogenetic reconstruction was based on therop17 gene sequences determined in the present study plus the corresponding sequences of strains TgC7, PRU, and RH available in GenBank (accession numbers: KC997176, KC997177, and KC997178) using three inference methods, namely, neighbor-joining (NJ), maximum likelihood (ML), and maximum parsimony (MP), using the sequence ofNeospora caninum (NCLIV_027930) as the outgroup. All the analyses were conducted following previous studies [20, 21]. Phylograms were drawn using the Tree View program version 1.65 [22].


## 3. Results and Discussion

The length of therop17 genes from all examinedT. gondii isolates was 1375 bp and A+T contents varied from 49.45% to 50.11%. The alignment of 10rop17 sequences plus the corresponding sequences of the RH, PRU, and TgC7 strains available in GenBank revealed nucleotide polymorphisms at 33 positions, with an intraspecific variation of 0–2.1%. The genetic diversity inrop17 gene was higher than our previous studies for PLP1 [23], ROP7 [11], eIF4A [24], and MIC13 [25] genes and the whole genome, secretome, and kinome ofT. gondii[8]. 16 variable positions were identified as transitions and the rest variable nucleotides were classified as transversions, and no deletions were detected in the 13rop17 gene sequences.Phylogeny reconstruction using MP, NJ, and ML analyses revealed two major clusters (Figure1(a)). Topologies of all trees based on nucleotide sequences inferred by three different methods were similar, with only the small difference of bootstrap values. The classical genotypes II and III and atypical Type 12 strain were clustered in one clade. The subtree of NJ analysis further showed that genotype III (strain CTG) was separated from other strains which were supported by bootstrap analysis, and the atypical Type 12 (TgWtSc40 strain) was closely related to classical genotype II (strain PRU) (Figure 1(b)).T. gondii genotype II is one of the parental lineage of Type 12 based on the analysis of the inheritance of multilocus genotypes [6, 26]. The somewhat close relationship between Type II and Type 12 strains coincided with analyses ofUPRT andSAG1 loci [6]. All the strains belonging to genotype I in this study were clustered together, including strain TgPLh and typical strains GT1 and RH. Atypical strains TgCat1, TgToucan, TgCatBr64, and TgCatBr5 were phylogenetically clustered more closely with Type I strains. Of these, TgCatBr64 and TgCatBr5 strains which originated from cats in Brazil were grouped together. Based on the limitedT. gondii strains examined in the present study, therop17 gene sequences could distinguish the major clonal lineages, but not all, showing the weak differentiation ofT. gondii genotypes compared to analyses using GRA5, GRA6, and AK gene sequences as genetic markers [27–29]. Further validation of therop17 gene sequences as genetic marker is warranted by sampling moreT. gondiistrains from wider geographical locations and more hosts.Figure 1
Phylogenetic analysis of 13Toxoplasma gondii strains based on analysis of therop17 gene sequences. The tree was built by neighbor-joining (NJ), maximum likelihood (ML), and maximum parsimony (MP) analyses. The numbers at notes indicate bootstrap values resulting from different analyses in the order MP/NJ/ML. (a) The much higher genetic divergence inrop17 revealed two major clusters (denoted by I and II). (b) Subtree in the red box showing results of analysis using neighbor-joining (NJ).The analyses of sequence variations in nucleotides and amino acids among different strains showed high ratio of nonsynonymous to synonymous polymorphisms (>1), suggesting thatT. gondii rop17 shows signs of positive selection, although more isolates will be required to determine whetherrop17 gene is under selection at the population level. Under the immunized stresses of host cells, the positive selection occurring inrop17 gene may increase stress resistance. Ongoing positive selection is also found in several polymorphic dense granule (GRA) antigens [30, 31] and some other ROPs [8].


## 4. Conclusion

In summary, the present study demonstrated the existence of slightly high sequence variability in therop17 gene sequences amongT. gondiistrains from different hosts and regions, which may be explored as a new genetic marker for population genetic studies ofT. gondii isolates, and contributed to discovery of the new strategies for vaccination, treatment, or diagnosis.



---

*Source: 349325-2014-07-10.xml*