Molecular cloning, phylogenetic analysis and heat shock response of Babesia gibsoni heat shock protein 90.

The Babesia gibsoni heat shock protein 90 (BgHSP90) gene was cloned and sequenced. The length of the gene was 2,610 bp with two introns. This gene was amplified from cDNA corresponding to full length coding sequence (CDS) with an open reading frame of 2,148 bp. A phylogenetic analysis of the CDS of HSP90 gene showed that B. gibsoni was most closely related to B. bovis and Babesia sp. BQ1/Lintan and lies within a phylogenetic cluster of protozoa. Moreover, mRNA transcription profile for BgHSP90 exposed to high temperature were examined by quantitative real-time reverse transcription-polymerase chain reaction. BgHSP90 levels were elevated when the parasites were incubated at 43°C for 1 hr.

Heat shock protein 90 (HSP90) is a 90 kDa HSP and one of the molecular chaperones responsible for managing protein folding of diverse sets of proteins, including regulatory kinases and numerous other proteins [25]. Based on the function of HSP90, HSP90 of pathogens might play important roles in the pathogens’ survival and proliferation within the host. An 82 kDa protein of the HSP90 family has recently been identified in many protozoan parasites, such as Leishmania donovani, Trypanosoma cruzi, Toxoplasma gondii, Plasmodium falciparum, Eimeria tenella and E. acervulina [1, 2, 4, 11, 21]. Several studies demonstrated that this HSP90 molecule is associated with the entry of parasite into the host cells [1, 15]. In addition, experimental evidence suggested that this molecule, localized both in cytosol and nucleus, is an essential component for stage differentiation and intracellular growth inside the host cells of many protozoans [9, 10, 14, 15, 21]. Higher class eukaryotes contain two different HSP90 isoforms, which are encoded by two different genes [8]. Human HSP90-α and HSP90-β isoforms contain 86% base pair homology [6, 16]. Both isoforms are able to form homodimers and higher order structures [13]. Recently, two HSP90 isoforms were also identified in Babesia orientalis and Theileria annulata [7, 12], and one HSP90 was identified in B. bovis, Babesia sp. BQ1/Lintan and T. parva [3, 5, 17]. At the current moment, the role and function of Hsp90 of those Babesia and Theileria parasites have not been well elucidated. Babesia gibsoni is a protozoan parasite that infects dogs and causes canine babesiosis. Canine babesiosis is a worldwide disease of hemolytic anemia and thrombocytopenia. There have been no reports regarding the HSP90 of B. gibsoni. In the present study, molecular cloning of the HSP90 gene of B. gibsoni and its phylogenetic analysis in relation to other protozoan parasites, bacteria and mammals were performed. Additionally, we investigated the change in gene transcription for HSP90 of B. gibsoni after exposing to high temperatures as a first step to understand the function of this molecule.

The B. gibsoni used in the present study had been maintained in cultures for several years [24]. To prepare dog RBCs and sera for a culture, three beagle dogs were used. The dogs used had body weight of 8–12 kg and were 2–3 years old. Regarding the experimental protocols for animal care and handling, the investigators adhered to the guidelines of Hokkaido University, which basically conform to those of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. The present study was approved by the Committee for Laboratory Animals, Graduate School of Veterinary Medicine, Hokkaido University (approval number: 1022).

Genomic DNA [22] and total RNA [23] of B. gibsoni were extracted as described previously [22]. To avoid the contamination of genomic DNA, genomic DNA was digested on the column using the RNase-free DNase set (QIAGEN, Valencia, CA, U.S.A.) during total RNA extraction. cDNA was synthesized from the total RNA using High Capacity RNA-to-cDNA Master Mix (Applied Biosystems, Tokyo, Japan) according to the manufacturer’s instructions. The PCR primers used for the amplification of the partial HSP90 gene of B. gibsoni were designed based on sequences conserved among the HSP90 gene of B. bovis (AF136649) and T. parva (M57386). The primers used in the present study are listed in Table 1. These primers had a degeneracy to allow amplification of different bases. Genomic DNA and cDNA in reaction mixtures were prepared according to the manufacturer’s protocol (Ex-Taq polymerase; Takara, Tokyo, Japan), and then, it was amplified for 35 cycles (denaturation for 1 min at 95°C, annealing for 1 min at 55°C and extension for 1 min at 72°C) followed by the final extension for 5 min at 72°C in a VeritiTM 96 Well Thermal Cycler (Applied Biosystems). To determine the nucleotide sequence of 5′-end and 3′-end of BgHSP90 mRNA, the Rapid Amplification of cDNA Ends (RACE) method was performed using SMARTTM RACE cDNA Amplification Kit (Clontech Laboratory, Mountain View, CA, U.S.A.) according to the manufacturer’s instructions. The primers for the RACE method (BgHSP90-5′ and BgHSP90-3′) were designed based on the analyzed nucleotide sequence (Table 1). Each reaction product was examined by electrophoresis on a 1.5% agarose gel to confirm that it was a single product and directly utilized for the sequencing analysis. The nucleotide sequence of the amplification products was determined by an Applied Biosystems 3130 genetic analyzer (Applied Biosystems) using the ABI PRISM BigDye Terminator v 3.1 Cycle-Sequencing kit (Applied Biosystems) [22]. The primers for the amplification were also utilized for the sequencing analysis. The nucleotide sequence analyzed was confirmed as the HSP90 gene from B. gibsoni (BgHSP90) through BLAST search, because that showed the high identity with HSP90 gene from Babesia and Theileria parasites. This gene included an open reading frame of 2,148 bp, and the encoded polypeptide was comprised of 716 amino acid residues with a predicted size of 82.8 kDa, as determined using a computer-based molecular weight calculator. The accession number of BgHSP90 in DDBJ database is LC064030. The comparison of the coding sequence (CDS) of HSP90 gene was performed using GENETYX-MAC ver. 11.2 (Genetyx Co., Tokyo, Japan). The CDS of the HSP90 gene from B. gibsoni was compared with that of B. bovis, Babesia sp. BQ1/Lintan (GQ397856), T. parva, E. tenella (AF042329) and Toxoplasma gondii (AY344115), and found to have 78.9, 80.9, 74.1, 71.7 and 69.2% identity, respectively. The predicted amino acid sequence of HSP90 from B. gibsoni was also compared with that of B. bovis, Babesia sp. BQ1/Lintan, T. parva, E. tenella and T. gondii, and found to show 89.0, 89.7, 81.6, 72.0 and 70.4% identity, respectively. Among the amino acid sequences of HSP90 from Babesia parasites, amino acids from positions 1 to 9, and 231 to 267 were characteristic for each species; the remaining parts were almost the same among those Babesia parasites (Fig. 1). In the present study, BgHSP90 had higher identity with HSP90 from B. bovis and Babesia sp. BQ1/Lintan than that from other protozoan, suggesting that HSP90 gene would be well conserved among Babesia parasites.

Table 1.

Oligonucleotides for the analysis of the Babesia gibsoni HSP90 gene

Name	Sequences	Tm (˚C)d)
BgHSP90F1	5′-ggt gts ggt ttc tac tca gc-3′	64
BgHSP90F2	5′-cgt tga agg tca act cga at-3′	58
BgHSP90F3	5′-caa ggt aag tca cag gat ct-3′	58
BgHSP90F4	5′-gcc aac cgc aac aag atc gc-3′	64
BgHSP90F5	5′-agt ggg aga tgc tca aca agc-3′	64
BgHSP90F6	5′-tag ctg ctc agg aga cct ac-3′	62
BgHSP90R1a)	5′-ttg ctg aac tgc tag aac t-3′	64
BgHSP90R2a)	5′-ytt agt caa ctt cyt cca tyt t-3′	60
BgHSP90R3a)	5′-tcc cag tcg ttg cac agg ttc-3′	66
BgHSP90-5′b)	5′-cca cgc ttc aac atg tca cca gat tcg-3′	82
BgHSP90-3′c)	5′-ctt cgt gac aac agc ttc ggg agc-3′	76

a) Antisense primers. b) Primer for 5′-RACE. c) Primer for 3′-RACE. d) Melting temperature.

Fig. 1.

Alignment of the predicted amino acid sequence of Babesia gibsoni heat shock protein 90 (BgHSP90) with HSP90 sequences of Babesia sp. BQ1/Lintan and Babesia bovis. Region A (from positions 1 to 9 amino acids) and B (from 231 to 267) are characteristic sequences for each species. Identical residues are marked by asterisk (*). Additions and gaps in the sequences are indicated by dashes (−).

The BgHSP90 gene was amplified from both genomic DNA and cDNA by PCR. The amplified sequence using BgHSP90F6 and BgHSP90R3 primers from genomic DNA contained 1,038 bp, and that from cDNA contained 918 bp. Moreover, the amplified sequence using BgHSP90F2 and BgHSP90R2 primers from genomic DNA contained 1,449 bp, and that from cDNA contained 1,192 bp. The sequence of BgHSP90 gene from genomic DNA was 2,610 bp long, while that from cDNA was 2,233 bp long. These results showed that the BgHSP90 gene analyzed in the present study included two introns at positions 75 to 195 and 1,471 to 1,728 bp. Khan et al. [7] identified and characterized two novel isoforms of HSP90 from B. orientalis encoding HSP90-A (KF379584) and HSP90-B (KF379584). One intron was detected in the HSP90-A gene, although the HSP90-B gene had no intron. The role of introns in HSP90 gene of Babesia parasites is still unknown.

A phylogenetic tree was inferred using ClustalX ver. 2.1 by the neighbor-joining method [20]. In addition to B. gibsoni, CDS of the HSP90 gene from GenBank database for B. bovis, B. bovis T2Bo (XM_001611817), Babesia sp. BQ1/Lintan, B. orientalis (KF379584 [clone 14a], KF379585 [clone 14c]), T. parva, T. annulata strain Ankara (XM_947380 [TA12105], XM_948193 [TA10720], XM_948749 [TA06470]), P. falciparum 3D7 (NC_004317), P. knowlesi (XM_002259147), E. tenella, E. acervulina (AY459429), T. gondii, Cryptosporidium parvum (AF038559) and C. parvum Iowa II (XM_626924) was used in the phylogenetic analysis. To estimate the genetic distance from other species, CDS of the htpG genes from bacteria, such as Yersinia enterocolitica subsp. enterocolitica 8081 (NC_008800) and Bordetella pertussis CS (NC_017223), and mRNA of the HSP90 genes from vertebrates, such as homo sapiens (NM_001017963 [HSP90-α], NM_003299 [HSP90-β]), Sus scrofa (NM_213973 [HSP90-α], NM_214103 [HSP90-β]), Mus musculus (NM_010480 [HSP90-α], NM_011631 [HSP90-β]) and Rattus norvegicus (NM_175761 [HSP90-α], NM_001012197 [HSP90-β]) were also included in the analysis. To estimate the genetic distance from other species, the HSP70 gene of B. gibsoni (AB083510) was also included as an outgroup. A phylogenetic analysis of the HSP90 gene showed that HSP90-α and -β isoforms from mammals made separate groups and that HSP90 from protozoan made three groups located outside the paraphyletic group containing HtpG from bacteria and HSP90 from mammals (Fig. 2). This result suggested that the protozoan would have several isoforms of HSP90, as reported previously [7, 12]. HSP90s of Protozoan group 1 including B. gibsoni analyzed in the present study closely related to the group of HSP90-α from mammals (Fig. 2). Therefore, BgHSP90 might have the similar role and function to that of HSP90-α. A further detailed study might be necessary to elucidate the roles and functions of BgHSP90 in the proliferation of B. gibsoni. Additionally, T. annulata has three different HSP90s in different group, and B. bovis and B. orientalis have two different HSP90s in different group, suggesting that those parasites would have two or three HSP90 isoforms. Therefore, B. gibsoni should have two or three HSP90 isoforms.

Fig. 2.

Phylogenetic tree based on the full coding region of heat shock protein 90 (HSP90) genes. The dendrogram was constructed using the neighbor-joining method. The numbers at the nodes indicate bootstrap support from 1,000 replications. The length of lines is proportional to the bootstrap value. The scale bar represents a 70% bootstrap value. In addition to B. gibsoni, complete sequences of HSP90 gene from B. bovis (AF136649), B. bovis T2Bo (XM_001611817), Babesia sp. BQ1/Lintan (GQ397856), B. orientalis (KF379584 [clone 14a], KF379585 [clone 14c]), T. parva (M57386), T. annulata strain Ankara (XM_947380 [TA12105], XM_948193 [TA10720], XM_948749 [TA06470]), P. falciparum 3D7 (NC_004317), P. knowlesi (XM_002259147), E. tenella (AF042329), E. acervulina (AY459429), Toxoplasma gondii (AY344115), Cryptosporidium parvum (AF038559), C. parvum Iowa II (XM_626924), Yersinia enterocolitica subsp. enterocolitica 8081 (NC_008800), Bordetella pertussis CS (NC_017223), Homo sapiens (NM_001017963 [HSP90-α], NM_003299 [HSP90-β]), Sus scrofa (NM_213973 [HSP90-α], NM_214103 [HSP90-β]), Mus musculus (NM_010480 [HSP90-α], NM_011631 [HSP90-β]) and Rattus norvegicus (NM_175761 [HSP90-α], NM_001012197 [HSP90-β]) were included in the analysis. The HSP70 gene of B. gibsoni (AB083510) was also included as an outgroup.

To examine the change in the transcription of the BgHSP90 gene in the cultured B. gibsoni after shifting temperature, we performed a quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). For exposure to high temperature, the parasites cultured under normal conditions (38°C) were divided into 2 groups. One group was incubated at 38°C for 1 hr as a control. The other group was incubated at 43°C for 1 hr. Total RNA of those B. gibsoni was extracted, and cDNA was synthesized. qRT-PCR was performed using the resulting cDNA as a template and a specific primer pair, BgHSP90F5-BgHSP90R3. Reaction mixtures with 50 ng of cDNA as a template were amplified with an ABI PRISM 7300 Real-Time PCR System (Applied Biosystems) as described previously [23]. Values were expressed as raw copy numbers (per microliter of cDNA). The quantitiy of 18S rRNA was also measured by qRT-PCR as described previously [23]. To correct for differences in the amount of RNA, the calculated copy numbers of the BgHSP90 gene were adjusted according to the copy numbers of B. gibsoni 18S rRNA. Thus, values were also expressed as relative amounts. This experiment was conducted 3 times. Data on the relative amount or copy numbers of BgHSP90 gene, and copy number of 18S rRNA at 43°C were compared with those at 38°C. Data for each temperature were expressed as the mean ± SD (n=3). The statistical analysis was performed using a Student’s t-test. The difference between data was considered significant at P<0.05. Although, the copy number of 18S rRNA at 43°C was almost the same as that at 38°C (Table 2), both the copy number and the relative amount of the BgHSP90 gene significantly (P<0.05) increased when the temperature was shifted from 38°C to 43°C for 1 hr (Table 2). These results suggested that the expression of BgHSP90 was enhanced. In our previous study, the level of parasitemia at high temperature (42°C) was almost the same as that under normal conditions, and BgHSP70 was heat-inducible [23]. Based on the results of the present study and the previous report, it was suggested that the enhanced expression of BgHSP90 might be independent from the proliferation of the parasites and that BgHSP90 would also be heat-inducible. This is the first report concerning heat shock response of HSP90 from Babesia parasites. It is well known that HSP90 from mammals protects cells from heat and oxidative stress [18, 19]. However, the functions of BgHSP90 for the proliferation of the parasites are still unclear.

Table 2.

Transcription of BgHSP90 gene in Babesia gibsoni at 43°C. The real-time qRT-PCR was performed with 50 ng of cDNA. The copy number of the BgHSP90 gene and 18S rRNA was calculated in the same RT-PCR run. The relative amount of the BgHSP90 gene was also calculated by adjusting the copy number of the gene to that of 18S rRNA

Incubation temp.	38˚C	43˚C
Copy no. (/µl)
BgHSP90	5,003.1 ± 789.7	15,711.1 ± 1,093.4a)
18S rRNA	8,113.4 ± 1,893.4	8,275.9 ± 559.1
Relative amount (BgHSP90/18S rRNA)	0.62 ± 0.10	1.9 ± 1.1a)

Data are expressed as the mean ± SD (n=3). a) Significantly (P<0.05) different from the value at 38°C.