FULL PAPER Internal Medicine

Involvement of Mitochondrial Genes of Babesia gibsoni in Resistance to Aceturate

Bandula Kumara Wickramasekara RAJAPAKSHAGE1), Masahiro YAMASAKI1)*, Shiang-Jyi HWANG1), Noboru SASAKI1), Masahiro MURAKAMI1), Yu TAMURA1), Sue Yee LIM1), Kensuke NAKAMURA1), Hiroshi OHTA1) and Mitsuyoshi TAKIGUCHI1)

1)Laboratory of Veterinary Internal Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060–0818, Japan

(Received 8 February 2012/Accepted 24 April 2012/Published online in J-STAGE 18 May 2012)

ABSTRACT. The stability of the characteristics of the diminazene aceturate (DA)-resistant B. gibsoni isolate was initially determined in vitro. Part of the DA-resistant B. gibsoni isolate was cultured without DA for 4 weeks, and then newly exposed to 200 ng/ml DA. As a result, this isolate could proliferate the same as the DA-resistant isolate, indicating that the characteristic of DA resistance was stable in the DA- resistant isolate. Additionally, the level of parasitemia in the DA-resistant isolate was comparatively lower than in the wild-type, suggest- ing that the proliferation potential of the DA-resistant isolate would be lower than that of the wild-type. Subsequently, to investigate the involvement of mitochondrial DNA (mtDNA) in DA resistance in B. gibsoni, the nucleotide sequences and deduced amino acid sequences of mitochondrial genes such as COXI, COXIII, and CYTb genes of the DA-resistant isolate, were compared with those of the wild-type. As a result, these three genes were not altered in the DA-resistant B. gibsoni isolate. Moreover, the transcription levels of COXI, COXIII, and CYTb genes were observed by semi-quantitative RT-PCR. As a result, the gene transcription of those genes in the DA-resistant isolate was not significantly altered. These results indicated that DA did not affect mtDNA directly in DA-resistant B. gibsoni. Thus, it is suggested that mtDNA should not be deeply involved in DA resistance in B. gibsoni. KEY WORDS: Babesia gibsoni, cytochrome b, cytochrome c oxidase I, cytochrome c oxidase III, diminazene aceturate-resistant isolate. doi: 10.1292/jvms.12-0056; J. Vet. Med. Sci. 74(9): 1139–1148, 2012

Babesia gibsoni is a blood protozoan of dogs and a the transcription levels of the heat shock protein 70 (hsp70) causative pathogen of canine [34]. It is difficult gene in this isolate decreased during the development of to eliminate this parasite from infected dogs, although a the DA-resistant isolate, suggesting that the hsp70 gene is number of drugs, including diminazene aceturate (DA), involved but does not play a major role in the development , , , and of the DA-resistant isolate. Thus, we have very little knowl- combined with azithromycin, are used for treatment of the edge of the mechanism of the DA resistance of B. gibsoni. disease [9, 16, 22, 29]. In the treatment of canine babesio- In Trypanosoma and Leishmania species, aromatic diami- sis, possible relapses and the development of drug-resistant dine analogues, such as DB75 and DB820, localize within isolates are matters of concern. the mitochondria [11, 13–15, 23, 26], and ultrastructural DA, an aromatic diamidine derivative, has been used as changes to the mitochondria and diminished mitochondrial a first-line agent for the treatment of B. gibsoni membrane potential are observed in Leishmania exposed to in dogs [22]; however, DA cannot eliminate B. gibsoni those drugs [21]. Moreover, in resistant parasites, the de- from infected dogs, and relapses often occur [5, 8, 17, 29]. creased mitochondrial transmembrane potential correlates In addition, a DA-resistant B. gibsoni isolate was previ- with the downregulation of the expression of numerous ously developed in vitro in our laboratory [8]. However, mitochondrial dehydrogenases and also F1F0-ATPase [21], the mechanism of action of DA on B. gibsoni has not been leading to a pleiotropic decrease in cytochrome c oxidase elucidated, and the mechanism of the development of DA activity [24]. In addition, the loss of P2 nucleoside trans- resistance in B. gibsoni is still not known. To treat canine porter function in Trypanosoma brucei brucei has been babesiosis with DA effectively, these problems have to be clarified. The DA-resistant B. gibsoni isolate developed by Footnote: Nucleotide sequence data of Babesia gibsoni isolates Hwang et al. was more resistant to pentamidine, clindamy- reported in this study are available in the GenBankTM, EMBL cin and than the wild-type [8]. Furthermore, and DDBJ databases under accession numbers: GenBank ID: AB685182 (complete mtDNA of wild-type 1), AB685183 (complete mtDNA of DA200-I isolate), AB685184 (complete *Correspondence to: Yamasaki, M., Laboratory of Veterinary mtDNA of DA200-II isolate), AB685185 (COXI gene of DA1 Internal Medicine, Department of Veterinary Clinical Sciences, variant), AB685186 (COXIII gene of DA1 variant), AB685187 Graduate School of Veterinary Medicine, Hokkaido University, (CYTb gene of DA1 variant), AB685188 (COXI gene of DA Sapporo 060–0818, Japan. non-responsible Hyogo isolate), AB685189 (COXIII gene of e-mail: [email protected] DA non-responsible Hyogo isolate), AB685190 (CYTb gene of DA non-responsible Hyogo isolate), AB685191 (COXI gene of ©2012 The Japanese Society of Veterinary Science wild-type 2). 1140 B. K. Wickramasekara RAJAPAKSHAGE ET AL.

Fig. 1. Development of the diminazene aceturate (DA)-resistant B. gibsoni isolate II (DA200- II isolate). (A) Changes in the levels of parasitemia. (B) DA concentration in the culture medium. DA concentration was increased gradually from 1 ng/ml to 200 ng/ml, requiring 252 days. implicated in resistance to DA [3]. Thus, there are some gicides, is target site modification, involving mutations in known mechanisms of DA resistance in Trypanosoma and the CYTb gene [6]. In cancer cells, COXI gene mutations Leishmania parasites. Similarly, it is possible that B. gibsoni are detected in human tumors, which are chemoresistant to could also have multiple mechanisms of DA resistance. In anticancer drugs in vitro as well as in vivo [19]: therefore, it the present study, accordingly, we focused on mitochondria is supposed that mtDNA in B. gibsoni might be involved in of B. gibsoni as a target of DA among the above candidates. the mechanisms of DA resistance. In apicomplexa, mitochondrion is an attractive target for In the present study, we initially confirmed the stability therapeutic drugs [4, 18]; however, the alteration of mito- of the characteristics of DA resistance in a DA-resistant B. chondrial DNA (mtDNA), such as cytochrome c oxidase gibsoni isolate. Subsequently, we investigated the nucleotide subunit I (COXI), cytochrome c oxidase subunit III (COXIII) and amino acid sequences and gene transcription of COXI, and cytochrome b (CYTb) gene, has been identified in the de- COXIII, and CYTb genes of the mtDNA of DA-resistant B. velopment of drug resistance in parasites, fungi, and cancer gibsoni variants. cells [6, 16, 19, 22]. Especially in B. gibsoni, mutation of the CYTb gene has been described in atovaquone-resistant MATERIALS AND METHODS isolates [16, 22]. Mutation of the CYTb gene in atovaquone resistance is also reported in Plasmodium spp. [12, 25]. In Isolates of Babesia gibsoni: The wild-type B. gibsoni used fungi, it is reported that the main mechanism conferring in the present study originated from a naturally infected dog resistance to quinol group drugs, the most successful fun- in the city of Nagasaki, Japan in 1973. The infected dog was MITOCHONDRIAL GENE OF DA-RESISTANT B. GIBSONI 1141

Table 1. Oligonucleotides used for analysis of the mtDNA of Babesia gibsoni Name Sequences Use CYTbF1 3′-atgttgtcctatttggttcc-5′ Amplification/Sequencing CYTbR1a) 3′-atatgcaaacttcccggcta-5′ Amplification/Sequencing CYTbF2 3′-tgcaggtttaattgctatgg-5′ Sequencing CYTbR2a) 3′-ctgttgctccccaataactc-5′ Sequencing MAF1 3′-ggtatggtgagacgacatgg-5′ Amplification/Sequencing MAR1a) 3′-agtatacctatgattccagc-5′ Amplification/Sequencing MAF2 3′-catccaaggcaaagaatcgt-5′ Sequencing MAR2a) 3′-gtaaagatgaaggaagtagg-5′ Sequencing MBF1 3′-ataggttaacttacatagtcc-5′ Amplification/Sequencing MBR1a) 3′-atcatacagtgccagcagcc-5′ Amplification/Sequencing MCF1 3′-cagcatgggattataaaacagt-5′ Amplification/Sequencing MCR1a) 3′-gtggagacaatagagaagtcg-5′ Amplification/Sequencing MDF1 3′-aggcatgcaataccgaacag-5′ Amplification/Sequencing MDR1a) 3′-caccggtttacattcatcac-5′ Amplification/Sequencing MEF1 3′-tcaggtttggaagcagatac-5′ Amplification/Sequencing MER1a) 3′-tgtttcgacttcgtactgac-5′ Amplification/Sequencing MFF1 3′-ggwagtggwacaggwtggac-5′ Amplification/Sequencing MFR1a) 3′-cataatctggtattctccttgg-5′ Amplification/Sequencing MGF1 3′-ctgttgctccccaataactc-5′ Amplification/Sequencing MGR1a) 3′-cttaacccaactcacgtacc-5′ Amplification/Sequencing MHR1a) 3′-gctgatacaatataggatctcc-5′ Amplification/Sequencing 5′F1 3′-agcttaataaaagagaattt-5′ Amplification/Sequencing MIF1 3′-gttggttaaagccctgtttcc-5′ Amplification/Sequencing 3′R1a) 3′-agcttaataaaagagaattt-5′ Amplification/Sequencing a) Antisense primers. not treated with any drug when B. gibsoni was isolated. Since DA and atovaquone. then, this wild-type has been maintained in cultures in our In vitro culture of Babesia gibsoni: The wild-type B. gib- laboratory [31]. DA-resistant B. gibsoni isolate-I (DA200- soni, DA1 variant, and DA-resistant B. gibsoni isolates have I isolate) was previously developed from this wild-type in been maintained in cultures in our laboratory according to vitro by Hwang et al. [8]. To analyze the mtDNA using a dif- the method of Yamasaki et al. [31]. In brief, the parasites ferent DA-resistant isolate, DA-resistant B. gibsoni isolate-II were incubated at 38°C in a humidified atmosphere of 5% (DA200-II isolate) was also developed from this wild-type CO2, 5% O2 and 90% N2 in a culture medium consisting according to the method of Hwang et al. [8]. Briefly, the of RPMI-1640 (Invitrogen, Carlsbad, CA, U.S.A.), 20% dog wild-type B. gibsoni isolate was cultured in culture medium serum and canine HK red blood cells (RBCs) that contain containing 1 ng/ml DA, and then the concentration of DA a high concentration of potassium [10], sufficient to yield was gradually increased from 1 to 200 ng/ml after 252 days a packed cell volume (PCV) of 5%. Every 24 hr, 60% of (Fig. 1). Thereafter, the DA-resistant B. gibsoni isolates were the culture supernatant was removed and replaced with an maintained in culture medium containing 200 ng/ml DA for equal volume of fresh culture medium [32]. Every 7 days, more than six months. This DA200-II isolate was also used a half-volume of the erythrocyte suspension was removed to develop the DA-cessation isolate in the present study. and replaced with an equal volume of uninfected fresh To observe the alteration to the sequence of mtDNA by erythrocyte suspension as a subculture. The wild-type, DA1 exposure to low concentration of DA, DA1 variant was variant, and DA-resistant isolates were maintained in culture separated during the development of DA-resistant B. gibsoni media without DA, with 1 ng/ml DA, and with 200 ng/ml isolate-II. When the concentration of DA was increased, the DA, respectively. parasites needed roughly 2 weeks to adapt to each concen- Stability of the characteristics of DA-resistant B. gibsoni tration of DA. The parasites that have adapted to 1 ng/ml isolate in vitro: To investigate the stability of the character- DA were separated as DA1 variant, which was maintained istics of resistance to DA, a DA-cessation isolate was devel- in culture medium containing 1 ng/ml DA for 7 weeks and oped from DA200-II isolate. In brief, the DA200-II isolate used for DNA extraction at week 7. was cultured with 200 ng/ml DA on week 1. From week 2 to Additionally, the Hyogo isolate of B. gibsoni was obtained 5, part of this isolate was cultured in culture medium without from a naturally B. gibsoni infected dog in Hyogo Prefec- DA. After week 5, this isolate was cultured in culture me- ture, Japan in 2010. The dog had been treated with DA and dium with 200 ng/ml DA again. In addition, the wild-type atovaquone; however, canine babesiosis relapsed repeatedly, and DA200-II isolate were maintained during the entire and it is supposed that this isolate might have resistance to experimental period as controls. Thin smears were prepared 1142 B. K. Wickramasekara RAJAPAKSHAGE ET AL. every 24 hr, and the level of parasitemia was determined by Table 2. Oligonucleotides used for the COXI, COXIII, and CYTb counting the number of parasitized erythrocytes per 1,000 gene expressions of DA-resistant and wild-type Babesia gibsoni erythrocytes. The experiment was conducted three times. Name Sequences Use DNA extraction, amplification, and sequencing: The ge- COXIF1 3′-ggaagtggtactggatggac-5′ COXI nomic DNA of B. gibsoni isolates was extracted as described MHR1a) 3′-gctgatacaatataggatctcc-5′ COXI previously [33]. Genomic DNA was extracted and used as COXIIIF1 3′-gaatactcatcaccttgataag-5′ COXIII a template for polymerase chain reaction (PCR). The PCR COXIIIR1a) 3′-gcaattgtaaatagtattagag-5′ COXIII primers used for amplification of the mtDNA of B. gibsoni CYTbF1 3′-ctcatatgttgtcctatttggttcc-5′ CYTb were designed based on sequences conserved among the CYTbR2a) 3′-ctgttgctccccaataactc-5′ CYTb mtDNA of Babesia bovis (GenBank ID: EU075182), Theile- 18SrF 3′-gataaccgtgctaattgtag-5′ 18S rRNA ria parva (Z23263), Theileria annulata (NW_001091933), 18SrRa) 3′-cgaataattcaccggatcac-5′ 18S rRNA B. gibsoni NRCPD strain (AB499087), and the CYTb gene a) Antisense primers. of B. gibsoni (AB215096). The primers used for amplifica- tion of the mtDNA of B. gibsoni are listed in Table 1. The extracted genomic DNA in reaction mixtures was prepared cycles were 60°C and 35 cycles, respectively. The 18S rRNA according to the protocol supplied (ExTaq polymerase; Ta- was used as a control in order to guarantee that an equal kara, Tokyo, Japan). The PCR was repeated for 35 cycles amount of cDNA from each sample was being evaluated with denaturation for 1 min at 95°C, annealing for 1 min correctly. The samples were separated on 1.5% agarose gels at 55°C, extension for 1 min at 72°C, and final extension and visualized under UV light by staining. for 5 min at 72°C using a VeritiTM 96 Well Thermal Cycler The absolute integrated optical density (IOD) of each band (Applied Biosystems, Tokyo, Japan). was analyzed by scanning densitometry with Lumi Vision The nucleotide sequences of the amplification products Analyzer 2.1 software (Aisin Seiki Co., Aichi, Japan). The were determined according to the method of Yamasaki et amount of COXI, COXIII, and CYTb gene transcriptions was al. [30]. The nucleotide sequences of the full-length mtDNA established as a ratio referring to 18S rRNA of B. gibsoni, from the wild-type and DA-resistant B. gibsoni isolates which is expressed at a constant rate [9]. Data were collected (DA200-I and DA200-II) were analyzed. The primers used in terms of the band intensity of target genes per band in- for amplification were also utilized for sequencing analy- tensity of 18S rRNA. The experiment was conducted three sis. Some sequencing primers were designed based on the times. Two-sample t-tests were used to compare the statisti- analyzed nucleotide sequence. The purified PCR products cal difference of the transcription of each gene. P < 0.05 was were sequenced using the BigDye Terminator v3.1 Cycle considered significant. Sequencing Kit (Applied Biosystems) for 35 cycles (94°C for 1 min, 60°C for 1 min, and 72°C for 1 min). To com- RESULTS pensate for the possible misincorporation of nucleotide from Taq polymerase, a minimum of three amplified products Stability of the characteristic of resistance to DA: To ob- were sequenced directly. The nucleotide sequence of each serve the stability of resistance to DA, DA-cessation isolate fragment overlapped and matched perfectly. From the DA1 was developed from DA200-II isolate. During the DA-free variant and the Hyogo isolate, COXI, COXIII, and CYTb period, the DA-cessation isolate grew and proliferated in the genes in mtDNA were amplified and sequenced. Sequence same way as DA200-II isolate (Fig. 2). Interestingly, when analysis and comparison of the nucleotide sequences and the DA-cessation isolate was newly exposed to 200 ng/ml their deduced amino acid sequences were performed using DA in week 6 and 7, this isolate could grow and proliferate computer software GENETYX-MAC ver. 11.2 (Genetyx in the same way as the DA200-II isolate (Fig. 2). In addition, Co., Tokyo, Japan). when compared to the wild-type and DA-resistant isolate of Semi-quantitative RT-PCR: The gene transcription levels B. gibsoni, no morphological change was observed in the of COXI, COXIII, and CYTb genes of B. gibsoni were ob- DA-cessation isolate throughout the experimental period. served by semi-quantitative reverse transcription (RT)-PCR. Meanwhile, the levels of parasitemia of the DA200-II isolate The infected RBCs were harvested and lysed. The liberated and DA-cessation isolate were comparatively lower than parasites were pelleted for RNA extraction using an RNeasy those of the wild-type throughout the experimental period mini kit (QIAGEN, Valencia, CA, U.S.A.) according to (Fig. 2). the manufacturer’s instructions. Consequently, cDNA was Comparison of COXI, COXIII, and CYTb genes from wild- synthesized from total RNA using High Capacity RNA- type and DA-resistant B. gibsoni isolates: To investigate the to-cDNA Master Mix (Applied Biosystems, Tokyo, Japan) involvement of mtDNA in DA resistance in B. gibsoni, we according to the manufacturer’s instructions. The expression determined the nucleotide sequence of full-length mtDNA levels of COXI, COXIII, and CYTb genes were assayed by from the wild-type and DA-resistant B. gibsoni isolates. The RT-PCR using the primers shown in Table 2. The set of prim- lengths of mtDNA from B. gibsoni in our laboratory were ers was tested in a range from 20 to 35 cycles using 800 ng 5,867 base pairs. The mtDNA of the B. gibsoni consisted of cDNA. In order to confirm that product accumulation was three functional genes, COXI, COXIII, and CYTb, and six in the linear phase of the curve, a similar protocol to that of discontinuous ribosomal RNA, as Hikosaka et al. previously Montero et al. [20] was used. The annealing temperature and reported (NRCPD strain) [7]. Additionally, terminal inverted MITOCHONDRIAL GENE OF DA-RESISTANT B. GIBSONI 1143

Fig. 2. Development of DA-cessation B. gibsoni isolate from DA200-II isolate in vitro. To determine the stability of the resistance to DA, the DA-cessation B. gibsoni isolate (open triangles) was cultured without DA from week 2 to 5, and then with 200 ng/ml DA from week 6 to 7. As controls, the wild- type (closed squares) and DA200-II isolate (closed circles) were cultured in culture media without DA and with 200 ng/ml DA, respectively, during the entire experimental period. Data are expressed as the means ± SD (n = 3). repeats were found at the 3′-end and 5′-end of the mtDNA of 102 and 158 on the COXIII gene in the Hyogo isolate were our B. gibsoni isolate, as in the NRCPD strain. substituted with valine and arginine, respectively (Fig. 4). Subsequently, the nucleotide sequence and the deduced Amino acid 121, 220, and 303 on the CYTb gene in the amino acid sequence of the COXI gene among the wild-type, Hyogo isolate were substituted with isoleucine, isoleucine, DA200-I and DA200-II isolates, and DA1 variant were com- and valine, respectively (Fig. 5). pared. As a result, several nucleotide polymorphisms were Analysis of gene transcription of COXI, COXIII, and found on the COXI gene. Among these nucleotide polymor- CYTb in the wild-type and DA-resistant B. gibsoni isolate: phisms, nucleotide substitution at nucleotide 1006 and 1008 Semi-quantitative RT-PCR was employed to compare the gave rise to a substitution at amino acid 310 on the amino gene transcription levels of COXI, COXIII, and CYTb genes acid sequence of COXI (Fig. 3). Methionine at amino acid between the wild-type and DA200-II isolate. As a result, 310 on the amino acid sequence of COXI in the wild-type the band intensities of COXI, COXIII, and CYTb genes in was substituted with isoleucine or valine in the DA200-I and DA200-II isolate seemed to be slightly lower than in the DA200-II isolates, and DA1 variant. Therefore, to confirm wild-type. Since the band intensities of 18S rRNA were this amino acid substitution, the COXI gene in the wild-type comparable between the wild-type and DA200-II isolate in was cloned and sequenced again as WT2 (Fig. 3). As a result, each experiment, the band intensities of target genes could amino acid 310 on the COXI gene in WT2 was also valine be compared. In the present study, product accumulation (Fig. 3). In addition, amino acid 310 on the COXI gene in the was in the linear phase of the curve during 25 to 30 cycles; Hyogo isolate, which was exposed to DA and atovaquone, therefore, the band intensities at 25 cycles were compared. was isoleucine (Fig. 3). Moreover, amino acid 179 and 425 Subsequently, the results of densitometric analysis of bands on the COXI gene in the Hyogo isolate were isoleucine and obtained by electrophoresis of RT-PCR products demon- threonine, although those in the wild-type and DA-resistant strated that COXI, COXIII, and CYTb mRNA levels from the isolates were valine and isoleucine, respectively (Fig. 3). DA200-II isolate were not significantly decreased compared Additionally, the nucleotide sequence and the deduced to the wild-type (Fig. 6). amino acid sequence of COXIII and CYTb genes were compared among the wild-type, DA200-I and DA200-II DISCUSSION isolates, and DA1 variant. Although several nucleotide poly- morphisms were detected on COXIII and CYTb genes, their In the present study, the stability of the DA resistance amino acid sequences were identical among those isolates of B. gibsoni was initially examined in vitro. Allowing the (Figs. 4 and 5). The COXIII and CYTb genes in the Hyogo reversal of drug-resistant phenotypes may have value to isolate were also compared with those isolates. Amino acid understand the mechanisms of drug resistance [2]. Although 1144 B. K. Wickramasekara RAJAPAKSHAGE ET AL.

Fig. 3. alignment of the deduced amino acid sequences of cytochrome c oxidase I (COXI) gene from the wild-type (WT-1 and WT-2), DA1 variant (DA1), DA-resistant B. gibsoni isolates (DA200-I and DA200-II), and Hyogo isolate (Hyogo). Identical residues are denoted by periods (.). Amino acids conserved among the isolates are marked by asterisks (*). Arrowhead (▲) indicates the location of substitutions of the amino acid sequence among the isolates. MITOCHONDRIAL GENE OF DA-RESISTANT B. GIBSONI 1145

Fig. 4. alignment of the deduced amino acid sequences of cytochrome c oxidase III (COXIII) gene from the wild-type (WT), DA1 variant (DA1), DA-resistant B. gibsoni isolates (DA200-I and DA200-II), and Hyogo isolate (Hyogo). Identical residues are denoted by periods (.). Amino acids conserved among the isolates are marked by asterisks (*). the DA-cessation isolate was cultured without DA for 4 slower than their parental wild-type clones in vitro [1]. The weeks, this isolate maintained the ability of DA resistance. low proliferation potential of these drug-resistant parasites In the previous study, when wild-type B. gibsoni was cul- depends on the alteration of energy metabolism or nucleic tured in 100 ng/ml DA, the parasites were eliminated within acid synthesis. In particular, mitochondria have important 2 weeks [8]. These previous and present results indicated roles in energy metabolism, and mtDNA is one of the targets that DA resistance was stable in the DA-cessation isolate of therapeutic drugs [4, 18]. As described above, substitu- and that some genes in the DA-resistant B. gibsoni isolate tions in the CYTb gene on atovaquone-resistant isolates of B. might differ from those in the parasite that is sensitive to gibsoni [16] and P. falciparum [12, 25] were also reported. DA. Since Babesia parasites multiply asexually with a Accordingly, in the present study, we focused our attention reproductive time of around 10 hr under normal in vitro on the mtDNA of the DA-resistant B. gibsoni isolate. culture conditions [27], the genes related to DA resistance The nucleotide and amino acid sequences of COXI, would be transferred successfully during multiplication of COXIII, and CYTb were compared between the wild-type the parasites; however, we could not determine whether the and the DA-resistant isolates of B. gibsoni. In the present parasites developed resistance to DA or that the DA-resistant study, to investigate if amino acid variations on mitochondri- parasites were selected from a mixed population of parasites al genes occur when exposed to only 1 ng/ml DA, the DA1 in the present study. A similar phenomenon has also been variant was separated during development of the DA200-II reported in DA-resistant Trypanosoma brucei brucei [11, 26] isolate and was included in the present analysis. Although and pentamidine-resistant Leishmania species [21] in vitro. several nucleotide variations were detected in COXI, The underlying exact genetic mechanism of DA resistance COXIII, and CYTb genes, there were no significant amino of Trypanosoma and Leishmania parasites has been well acid substitutions in COXI, COXIII, and CYTb involved in investigated, though that of Babesia parasites is unclear; DA resistance of B. gibsoni. In particular, amino acid 310 therefore, determination of the genes responsible for the DA on COXI varied even among wild-type B. gibsoni; therefore, resistance of B. gibsoni is necessary. this amino acid substitution is an amino acid polymorphism In addition, the low parasitemia level of the DA-resistant on the COXI gene. These results indicated that mtDNA is B. gibsoni isolate compared to the wild-type suggested that not altered during the development of DA-resistant B. gib- the proliferation potential of the DA-resistant B. gibsoni iso- soni isolates. Meanwhile, three amino acid substitutions late would be lower than that of the wild-type. Because the on CYTb related to atovaquone resistance were detected in level of parasitemia varies at different periods, the levels of the Hyogo isolate. Since the Hyogo isolate was exposed to parasitemia of the wild-type and the DA-resistant B. gibsoni atovaquone in addition to DA, the three amino acid substitu- isolate were measured concurrently in the present study. The tions on CYTb were probably caused by atovaquone [16]. level of parasitemia of the DA-resistant isolate was lower Furthermore, there were a few amino acid substitutions on than that of the wild-type. A similar situation has been de- COXI and COXIII in the Hyogo isolate of B. gibsoni. It is scribed for pentamidine-resistant Leishmania, which grow supposed that those amino acid substitutions might be amino 1146 B. K. Wickramasekara RAJAPAKSHAGE ET AL.

Fig. 5. alignment of the deduced amino acid sequences of cytochrome b (CYTb) gene from the wild-type (WT), DA1 variant (DA1), DA-resistant B. gibsoni isolates (DA200-I and DA200-II), and Hyogo isolate (Hyogo). Identical residues are denoted by periods (.). Amino acids conserved among the isolates are marked by asterisks (*), and arrowheads (▲) represent the positions of reported substitutions related to atovaquone resistance. acid polymorphisms on the COXI and COXIII genes of B. of the heme-copper respiratory oxidase superfamily and is gibsoni. considered to be involved in the assembly and stabilization Because the nucleotide and amino acid sequences of of the entire complex [7, 28]. CYTb is a subunit of complex COXI, COXIII, and CYTb in DA-resistant isolates were III, and is essential for electron transfer in the mitochondrial not different from those in the wild-type, the transcription respiratory chain [7, 18]. Accordingly, COXI, COXIII, and levels of these three genes were observed. As a result, the CYTb might also have important roles in mitochondrial res- gene transcription of COXI, COXIII, and CYTb genes in the piration, i.e., energy metabolism, in B. gibsoni. As described DA-resistant B. gibsoni isolate was not significantly altered. above, the alteration of energy metabolism might result in These results indicated that DA did not directly affect mtD- a change of proliferation potential. Indeed, the proliferation NA of the DA-resistant B. gibsoni. In general, COXI is one potential of the DA-resistant B. gibsoni isolate was lower of 13 subunits of cytochrome c oxidase (complex IV), which than in the wild-type in the present study; however, from the is essential for electron transfer as a terminal oxidase in the present results, it is suggested that the expression of COXI, respiratory chain [7, 28]. COXIII is highly conserved in most COXIII, and CYTb in the DA-resistant B. gibsoni isolate was MITOCHONDRIAL GENE OF DA-RESISTANT B. GIBSONI 1147

the mechanism of DA resistance; however, there were no significant substitutions in mtDNA, i.e., COXI, COXIII, and CYTb genes, in DA-resistant B. gibsoni isolate compared with the wild-type. Additionally, the gene transcription of COXI, COXIII, and CYTb genes was not altered in the DA- resistant isolate, suggesting that DA did not affect mtDNA directly.

ACKNOWLEDGMENTS. This work was supported in part by a grant from the Science Research Fund of the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Ms. Aiko Ohnuma at the Research Center for Zoonosis Control, Hokkaido University, Japan, for the sequencing and Ms. Yukiko Kamihara at Kamihara Animal Hospital in Hyogo Prefecture, Japan, for providing the Hyogo isolate of B. gibsoni.

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