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

Authors: Zhong-Yuan Li; Jing Lu; Dong-Hui Zhou; Jia Chen; Xing-Quan Zhu
Journal: BioMed Research International
 (2015)

---

## Abstract

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.


---

## Body

## 1. 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 [7–9]. 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 asPlasmodium falciparum [10, 11]. Recent studies emphasized that different clonal types ofT. gondii strains with diverse geographical distribution can cause different toxoplasmosis in animals and humans [12, 13]. In order to unveil the details ofT. gondii genetic diversity, sequence variation of the type II HSP40TgSIS1 (ToxoDB: TGME49_265310, previously named TGME49_065310) [14] amongT. gondii isolates from different hosts and geographical regions was examined in this study.


## 2. Materials and Methods

### 2.1.T. gondii Isolates and gDNA Preparation

SixteenT. 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.Table 1
Details ofToxoplasma gondii isolates used in this study.
Number
Isolate
Host
Geographical location
Genotype∗
1
CTG
Cat
United States
Reference, Type III, ToxoDB#2
2
TgCatBr64
Cat
Brazil
Reference, ToxoDB#111
3
GT1
Goat
United States
Reference, Type I, ToxoDB#10
4
RH
Human
France
Reference, Type I, ToxoDB#10
5
TgPLH
Pig
Henan, China
Type I, ToxoDB#10
6
TgToucan
Toucan
Costa Rica
Reference, ToxoDB#52
7
MAS
Human
France
Reference, ToxoDB#17
8
TgCatBr5
Cat
Brazil
Reference, ToxoDB#19
9
PTG
Sheep
United States
Reference, Type II, ToxoDB#1
10
PRU
Human
France
Type II, ToxoDB#1
11
QHO
Sheep
Qinghai, China
Type II, ToxoDB#1
12
TgC7
Cat
Guangzhou, China
ToxoDB#9
13
PYS
Pig
Panyu, China
ToxoDB#9
14
GJS
Pig
Jingyuan, Gansu, China
ToxoDB#9
15
TgCgCa1
Cougar
Canada
Reference, ToxoDB#66
16
TgWtdSc40
Deer
USA
Type 12, ToxoDB#5
∗Based on the results of Zhou et al. [25, 26] and Su et al. [20].


### 2.2. PCR Amplification and Sequencing

Three fragments (A, B, and C) (Figure1) were separated based on the HSP40 sequence ofT. 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 intoE. coli DH5α 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.Table 2
Details of primers used in this study.
Primer
Sequence (5′-3′)
F40A
ATGGGGAAGGTAATCACATT
R40A
CGACTGGAACTATCGATTTC
F40B
TGTGGGGCGAGAGCCAGAGG
R40B
TGCAGAGGTGCCTTGCGTTT
F40C
CCCTGCATCGCGTGAGCTTC
R40C
AACGAGTGGAAAGCCCCCGT
Amplicons A, B, and C were amplified from differentT. gondii strains by PCR using F40A/R40A, F40B/R40B, and F40C/R40C, respectively.Figure 1
Sequence analysis of HSP40 gene among differentToxoplasma gondii strains. Color boxes indicate 12 extrons of HSP40 gene and the gray one stands for the region of 3′-UTR. Black triangle (
▲
) and asterisk (∗) indicate the translation start condon (ATG) and the stop one (TAG), respectively. Three pairs of specific primers (F40A/R40A, F40B/R40B, and F40C/R40C) used in this study were marked with arrows. Upper or lower case, respectively, indicates different nucleotide of extron or intron/3′-UTR. Point (.) and stub (–) among different nucleotides indicate identical or no nucleotide here compared with that of CTG strain (bottom line). The number beside the sequence stands for the variable sequence position for nucleotide, and black bar indicates 500 bp in length.


### 2.3. Sequence Analysis and Phylogenetic Reconstruction

The sequences of all the examinedT. gondii strains were amplified step by step and sequenced. Alignment analysis based on the obtained sequences including the reference one (ToxoDB: 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 P
A
U
P
∗ 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.


### 2.4. Characterization ofT. gondii Isolates by PCR-RFLP

To evaluate whether HSP40 gene was suitable for genotyping ofT. 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 enzymesMscI andEarI 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].


## 2.1.T. gondii Isolates and gDNA Preparation

SixteenT. 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.Table 1
Details ofToxoplasma gondii isolates used in this study.
Number
Isolate
Host
Geographical location
Genotype∗
1
CTG
Cat
United States
Reference, Type III, ToxoDB#2
2
TgCatBr64
Cat
Brazil
Reference, ToxoDB#111
3
GT1
Goat
United States
Reference, Type I, ToxoDB#10
4
RH
Human
France
Reference, Type I, ToxoDB#10
5
TgPLH
Pig
Henan, China
Type I, ToxoDB#10
6
TgToucan
Toucan
Costa Rica
Reference, ToxoDB#52
7
MAS
Human
France
Reference, ToxoDB#17
8
TgCatBr5
Cat
Brazil
Reference, ToxoDB#19
9
PTG
Sheep
United States
Reference, Type II, ToxoDB#1
10
PRU
Human
France
Type II, ToxoDB#1
11
QHO
Sheep
Qinghai, China
Type II, ToxoDB#1
12
TgC7
Cat
Guangzhou, China
ToxoDB#9
13
PYS
Pig
Panyu, China
ToxoDB#9
14
GJS
Pig
Jingyuan, Gansu, China
ToxoDB#9
15
TgCgCa1
Cougar
Canada
Reference, ToxoDB#66
16
TgWtdSc40
Deer
USA
Type 12, ToxoDB#5
∗Based on the results of Zhou et al. [25, 26] and Su et al. [20].


## 2.2. PCR Amplification and Sequencing

Three fragments (A, B, and C) (Figure1) were separated based on the HSP40 sequence ofT. 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 intoE. coli DH5α 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.Table 2
Details of primers used in this study.
Primer
Sequence (5′-3′)
F40A
ATGGGGAAGGTAATCACATT
R40A
CGACTGGAACTATCGATTTC
F40B
TGTGGGGCGAGAGCCAGAGG
R40B
TGCAGAGGTGCCTTGCGTTT
F40C
CCCTGCATCGCGTGAGCTTC
R40C
AACGAGTGGAAAGCCCCCGT
Amplicons A, B, and C were amplified from differentT. gondii strains by PCR using F40A/R40A, F40B/R40B, and F40C/R40C, respectively.Figure 1
Sequence analysis of HSP40 gene among differentToxoplasma gondii strains. Color boxes indicate 12 extrons of HSP40 gene and the gray one stands for the region of 3′-UTR. Black triangle (
▲
) and asterisk (∗) indicate the translation start condon (ATG) and the stop one (TAG), respectively. Three pairs of specific primers (F40A/R40A, F40B/R40B, and F40C/R40C) used in this study were marked with arrows. Upper or lower case, respectively, indicates different nucleotide of extron or intron/3′-UTR. Point (.) and stub (–) among different nucleotides indicate identical or no nucleotide here compared with that of CTG strain (bottom line). The number beside the sequence stands for the variable sequence position for nucleotide, and black bar indicates 500 bp in length.


## 2.3. Sequence Analysis and Phylogenetic Reconstruction

The sequences of all the examinedT. gondii strains were amplified step by step and sequenced. Alignment analysis based on the obtained sequences including the reference one (ToxoDB: 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 P
A
U
P
∗ 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.


## 2.4. Characterization ofT. gondii Isolates by PCR-RFLP

To evaluate whether HSP40 gene was suitable for genotyping ofT. 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 enzymesMscI andEarI 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].


## 3. Results and Discussion

Our results showed that HSP40 gene of all the examined strains was between 6621 bp and 6644 bp in length spliced by amplicons A (2430 bp), B (2107–2130 bp), and C (2252 bp), and their A+T contents varied from 48.54% to 48.80% (Figure1, Table 3). The alignment of all the 17 sequences revealed nucleotide mutations at 195 positions (0.12%–1.14%) in HSP40 genomic locations and 29 positions in CDS (0.02%–0.12%) in comparison withT. gondii ME49 strain (ToxoDB: TGME49_265310), which was lower than our previous reports of GRA5 [21], ROP38 [22], ROP47 [23], eIF4A [15], and other genes ofT. gondii, such as GRA6 [24]. Moreover, 141 transitions (A
↔
G and C
↔
T) and 54 transversions (A
↔
C, A
↔
T, G
↔
T, and G
↔
C) (R = transition/transversion = 2.6) were also identified, and the distance of evolutionary divergence was 0.1%–1.0% among the examinedT. gondii strains (Table 3).Table 3
Sequence characteristics ofToxoplasma gondii HSP40 including its expressed regions and introns.
Item
DNA
cDNA
CDS
Number of intron
1
2
3
4
5
6
7
8
9
10
11
Length (bp)
6621–6644
1620
1011
471
595
320
278
501
629–651
533-534
547
287
367
473
A+T (%)
48.54–48.80
45.12–45.56
45.20–45.70
44.80–45.65
48.40–48.91
45.62–46.56
45.68–46.40
51.70–52.10
53.90–54.99
48.78–50.28
51.74–52.47
54.70–55.75
45.78–46.32
47.36–47.78
Transition
141
36
26
7
15
5
3
8
14
12
10
9
6
16
Transversion
54
5
3
2
6
0
0
4
8
10
9
5
1
4
R
∗
2.6
7.2
8.6
3.5
2.5
—
—
2
1.7
1.2
1.1
1.8
6
4
Distance (%)
0.1–1.0
0.1–0.7
0–1.1
0–1.1
0–1.4
0–0.9
0–1.1
0–1.8
0–1.9
0–2.1
0–1.8
0–2.1
0–1.1
0.2–1.5
∗
R = transition/transversion; — means no data here.Nucleotide polymorphisms analysis revealed two polymorphic restriction sitesMscI andEarI in the sequence of amplicon C (2252 bp in length), which can differentiate three classical genotypes ofT. gondii (Types I, II, and III) (Figure 2) [19, 20, 25, 26]. In brief, the PCR products ofTgToucan, MAS,TgCatBr5, andT. gondii Type I strains (GT1, RH, andTgPLH) were digested into four segments (81, 165, 811, and 1195 bp); Type II strains (PTG, PRU, and QHO), ToxoDB#9 (TgC7, PYS, and GJS),TgCgCa1, andTgWtdSc40 were composed of three parts (81, 811, and 1360 bp); and the PCR products of Type III (CTG) andTgCatBr64 were cut into five sections (81, 165, 381, 811, and 814 bp). The results suggested that all the examinedT. gondii strains could not be completely separated into their own groups by PCR-RFLP especially for ToxoDB#9 strains.Figure 2
PCR-RFLP analysis based on the PCR products of amplicon C. Lane M indicates DL2000; lanes 1–16 stand forT. gondii Type I (GT1, RH, andTgPLH);TgToucan, MAS, andTgCatBr5, Type II (PTG, PRU, and QHO); ToxoDB#9 (TgC7, PYS, and GJS),TgCgCa1, andTgWtdSc40, Type III (CTG); andTgCatBr64, respectively.Phylogenetic reconstruction was constructed based on HSP40 sequences of the 17T. gondii strains includingT. gondii ME49 isolates (ToxoDB: TGME49_265310) (Figure 3) [18]. Our results showed thatT. 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, whereasTgToucan (ToxoDB#52) was gathered into the cluster of Type I (RH, GT1, andTgPLH), suggesting that the examinedT. gondii strains could not be completely separated by MP method though three classical genotypes ofT. gondii (Types I, II, and III) were clustered into different groups.Figure 3
Phylogenetic analysis of HSP40 gene among 17Toxoplasma gondii strains using maximum parsimony (MP) method. Three clusters of the classical genotypes (Types I, II, and III) and ToxoDB#9 were denoted. The numbers along branches indicate bootstrap values (%).


## 4. Conclusion

Our data suggested that HSP40 gene is not a suitable marker forT. gondii population genetic study, though three classical genotypes ofT. gondii (Types I, II, and III) could be differentiated by polymorphic restriction endonuclease sitesMscI andEarI existing in amplicon C.



---

*Source: 209792-2015-11-03.xml*