Involvement of Mitochondrial Genes of Babesia gibsoni in ...

FULL PAPER Internal Medicine

Involvement of Mitochondrial Genes of Babesia gibsoni in Resistance to Diminazene 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 causative pathogen of canine babesiosis [34]. It is difficult to eliminate this parasite from infected dogs, although a number of drugs, including diminazene aceturate (Da), clindamycin, metronidazole, pentamidine, and atovaquone combined with azithromycin, are used for treatment of the disease [9, 16, 22, 29]. in the treatment of canine babesio-sis, possible relapses and the development of drug-resistant isolates are matters of concern.

Da, an aromatic diamidine derivative, has been used as a first-line agent for the treatment of B. gibsoni infection in dogs [22]; however, Da cannot eliminate B. gibsoni from infected dogs, and relapses often occur [5, 8, 17, 29]. in addition, a Da-resistant B. gibsoni isolate was previ-ously developed in vitro in our laboratory [8]. However, the mechanism of action of Da on B. gibsoni has not been elucidated, and the mechanism of the development of Da resistance in B. gibsoni is still not known. To treat canine babesiosis with Da effectively, these problems have to be clarified. The DA-resistant B. gibsoni isolate developed by Hwang et al. was more resistant to pentamidine, clindamy-cin and doxycycline than the wild-type [8]. Furthermore,

the transcription levels of the heat shock protein 70 (hsp70) gene in this isolate decreased during the development of the Da-resistant isolate, suggesting that the hsp70 gene is involved but does not play a major role in the development of the Da-resistant isolate. Thus, we have very little knowl-edge of the mechanism of the Da resistance of B. gibsoni. in Trypanosoma and Leishmania species, aromatic diami-dine analogues, such as DB75 and DB820, localize within the mitochondria [11, 13–15, 23, 26], and ultrastructural changes to the mitochondria and diminished mitochondrial membrane potential are observed in Leishmania exposed to those drugs [21]. moreover, in resistant parasites, the de-creased mitochondrial transmembrane potential correlates with the downregulation of the expression of numerous mitochondrial dehydrogenases and also F1F0-aTPase [21], leading to a pleiotropic decrease in cytochrome c oxidase activity [24]. in addition, the loss of P2 nucleoside trans-porter function in Trypanosoma brucei brucei has been

*CorrespondenCe to: Yamasaki, m., Laboratory of Veterinary internal medicine, Department of Veterinary clinical sciences, Graduate school of Veterinary medicine, Hokkaido University, sapporo 060–0818, Japan.

e-mail: [email protected]©2012 The Japanese society of Veterinary science

Footnote: Nucleotide sequence data of Babesia gibsoni isolates reported in this study are available in the GenBankTm, emBL and DDBJ databases under accession numbers: GenBank iD: aB685182 (complete mtDNa of wild-type 1), aB685183 (complete mtDNa of Da200-i isolate), aB685184 (complete mtDNa of Da200-ii isolate), aB685185 (COXI gene of Da1 variant), aB685186 (COXIII gene of Da1 variant), aB685187 (CYTb gene of Da1 variant), aB685188 (COXI gene of Da non-responsible Hyogo isolate), aB685189 (COXIII gene of Da non-responsible Hyogo isolate), aB685190 (CYTb gene of Da non-responsible Hyogo isolate), aB685191 (COXI gene of wild-type 2).

B. K. WicKramaseKara raJaPaKsHaGe ET AL.1140

implicated in resistance to Da [3]. Thus, there are some known mechanisms of Da resistance in Trypanosoma and Leishmania parasites. similarly, it is possible that B. gibsoni could also have multiple mechanisms of Da resistance. in the present study, accordingly, we focused on mitochondria of B. gibsoni as a target of Da among the above candidates.

in apicomplexa, mitochondrion is an attractive target for therapeutic drugs [4, 18]; however, the alteration of mito-chondrial DNa (mtDNa), such as cytochrome c oxidase subunit I (COXI), cytochrome c oxidase subunit III (COXIII) and cytochrome b (CYTb) gene, has been identified in the de-velopment of drug resistance in parasites, fungi, and cancer cells [6, 16, 19, 22]. especially in B. gibsoni, mutation of the CYTb gene has been described in atovaquone-resistant isolates [16, 22]. mutation of the CYTb gene in atovaquone resistance is also reported in Plasmodium spp. [12, 25]. in fungi, it is reported that the main mechanism conferring resistance to quinol group drugs, the most successful fun-

gicides, is target site modification, involving mutations in the CYTb gene [6]. in cancer cells, COXI gene mutations are detected in human tumors, which are chemoresistant to anticancer drugs in vitro as well as in vivo [19]: therefore, it is supposed that mtDNa in B. gibsoni might be involved in the mechanisms of Da resistance.

In the present study, we initially confirmed the stability of the characteristics of Da resistance in a Da-resistant B. gibsoni isolate. subsequently, we investigated the nucleotide and amino acid sequences and gene transcription of COXI, COXIII, and CYTb genes of the mtDNa of Da-resistant B. gibsoni variants.

maTeriaLs aND meTHODs

Isolates of Babesia gibsoni: The wild-type B. gibsoni used in the present study originated from a naturally infected dog in the city of Nagasaki, Japan in 1973. The infected dog was

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.

miTOcHONDriaL GeNe OF Da-resisTaNT B. GIBSONI 1141

not treated with any drug when B. gibsoni was isolated. since then, this wild-type has been maintained in cultures in our laboratory [31]. Da-resistant B. gibsoni isolate-i (Da200-i isolate) was previously developed from this wild-type in vitro by Hwang et al. [8]. To analyze the mtDNa using a dif-ferent Da-resistant isolate, Da-resistant B. gibsoni isolate-ii (Da200-ii isolate) was also developed from this wild-type according to the method of Hwang et al. [8]. Briefly, the wild-type B. gibsoni isolate was cultured in culture medium containing 1 ng/ml Da, and then the concentration of Da was gradually increased from 1 to 200 ng/ml after 252 days (Fig. 1). Thereafter, the Da-resistant B. gibsoni isolates were maintained in culture medium containing 200 ng/ml Da for more than six months. This Da200-ii isolate was also used to develop the Da-cessation isolate in the present study.

To observe the alteration to the sequence of mtDNa by exposure to low concentration of Da, Da1 variant was separated during the development of Da-resistant B. gibsoni isolate-ii. When the concentration of Da was increased, the parasites needed roughly 2 weeks to adapt to each concen-tration of Da. The parasites that have adapted to 1 ng/ml Da were separated as Da1 variant, which was maintained in culture medium containing 1 ng/ml Da for 7 weeks and used for DNa extraction at week 7.

additionally, the Hyogo isolate of B. gibsoni was obtained from a naturally B. gibsoni infected dog in Hyogo Prefec-ture, Japan in 2010. The dog had been treated with Da and atovaquone; however, canine babesiosis relapsed repeatedly, and it is supposed that this isolate might have resistance to

Da and atovaquone.In vitro culture of Babesia gibsoni: The wild-type B. gib-

soni, Da1 variant, and Da-resistant B. gibsoni isolates have been maintained in cultures in our laboratory according to the method of Yamasaki et al. [31]. in brief, the parasites were incubated at 38°C in a humidified atmosphere of 5% cO2, 5% O2 and 90% N2 in a culture medium consisting of RPMI-1640 (Invitrogen, Carlsbad, CA, U.S.A.), 20% dog serum and canine HK red blood cells (rBcs) that contain a high concentration of potassium [10], sufficient to yield a packed cell volume (PCV) of 5%. Every 24 hr, 60% of the culture supernatant was removed and replaced with an equal volume of fresh culture medium [32]. every 7 days, a half-volume of the erythrocyte suspension was removed and replaced with an equal volume of uninfected fresh erythrocyte suspension as a subculture. The wild-type, Da1 variant, and Da-resistant isolates were maintained in culture media without Da, with 1 ng/ml Da, and with 200 ng/ml Da, respectively.

Stability of the characteristics of DA-resistant B. gibsoni isolate in vitro: To investigate the stability of the character-istics of resistance to Da, a Da-cessation isolate was devel-oped from Da200-ii isolate. in brief, the Da200-ii isolate was cultured with 200 ng/ml Da on week 1. From week 2 to 5, part of this isolate was cultured in culture medium without Da. after week 5, this isolate was cultured in culture me-dium with 200 ng/ml Da again. in addition, the wild-type and Da200-ii isolate were maintained during the entire experimental period as controls. Thin smears were prepared

Table 1. Oligonucleotides used for analysis of the mtDNa of Babesia gibsoni

Name sequences UsecYTbF1 3′-atgttgtcctatttggttcc-5′ Amplification/SequencingcYTbr1a) 3′-atatgcaaacttcccggcta-5′ Amplification/SequencingcYTbF2 3′-tgcaggtttaattgctatgg-5′ sequencingcYTbr2a) 3′-ctgttgctccccaataactc-5′ sequencingmaF1 3′-ggtatggtgagacgacatgg-5′ Amplification/Sequencingmar1a) 3′-agtatacctatgattccagc-5′ Amplification/SequencingmaF2 3′-catccaaggcaaagaatcgt-5′ sequencingmar2a) 3′-gtaaagatgaaggaagtagg-5′ sequencingmBF1 3′-ataggttaacttacatagtcc-5′ Amplification/SequencingmBr1a) 3′-atcatacagtgccagcagcc-5′ Amplification/SequencingmcF1 3′-cagcatgggattataaaacagt-5′ Amplification/Sequencingmcr1a) 3′-gtggagacaatagagaagtcg-5′ Amplification/SequencingmDF1 3′-aggcatgcaataccgaacag-5′ Amplification/SequencingmDr1a) 3′-caccggtttacattcatcac-5′ Amplification/SequencingmeF1 3′-tcaggtttggaagcagatac-5′ Amplification/Sequencingmer1a) 3′-tgtttcgacttcgtactgac-5′ Amplification/SequencingmFF1 3′-ggwagtggwacaggwtggac-5′ Amplification/SequencingmFr1a) 3′-cataatctggtattctccttgg-5′ Amplification/SequencingmGF1 3′-ctgttgctccccaataactc-5′ Amplification/SequencingmGr1a) 3′-cttaacccaactcacgtacc-5′ Amplification/SequencingmHr1a) 3′-gctgatacaatataggatctcc-5′ Amplification/Sequencing5′F1 3′-agcttaataaaagagaattt-5′ Amplification/SequencingmiF1 3′-gttggttaaagccctgtttcc-5′ Amplification/Sequencing3′R1a) 3′-agcttaataaaagagaattt-5′ Amplification/Sequencing

a) antisense primers.

B. K. WicKramaseKara raJaPaKsHaGe ET AL.1142

every 24 hr, and the level of parasitemia was determined by counting the number of parasitized erythrocytes per 1,000 erythrocytes. The experiment was conducted three times.

DNA extraction, amplification, and sequencing: The ge-nomic DNa of B. gibsoni isolates was extracted as described previously [33]. Genomic DNa was extracted and used as a template for polymerase chain reaction (Pcr). The Pcr primers used for amplification of the mtDNA of B. gibsoni were designed based on sequences conserved among the mtDNa of Babesia bovis (GenBank iD: eU075182), Theile-ria parva (Z23263), Theileria annulata (NW_001091933), B. gibsoni NrcPD strain (aB499087), and the CYTb gene 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 according to the protocol supplied (ExTaq polymerase; Ta-kara, Tokyo, Japan). The Pcr was repeated for 35 cycles with denaturation for 1 min at 95°c, annealing for 1 min at 55°C, extension for 1 min at 72°C, and final extension for 5 min at 72°c using a VeritiTm 96 Well Thermal cycler (applied Biosystems, Tokyo, Japan).

The nucleotide sequences of the amplification products were determined according to the method of Yamasaki et al. [30]. The nucleotide sequences of the full-length mtDNa from the wild-type and Da-resistant B. gibsoni isolates (Da200-i and Da200-ii) were analyzed. The primers used for amplification were also utilized for sequencing analy-sis. some sequencing primers were designed based on the analyzed nucleotide sequence. The purified PCR products were sequenced using the BigDye Terminator v3.1 cycle 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-pensate for the possible misincorporation of nucleotide from Taq polymerase, a minimum of three amplified products were sequenced directly. The nucleotide sequence of each fragment overlapped and matched perfectly. From the Da1 variant and the Hyogo isolate, COXI, COXIII, and CYTb genes in mtDNA were amplified and sequenced. Sequence analysis and comparison of the nucleotide sequences and their deduced amino acid sequences were performed using computer software GeNeTYX-mac ver. 11.2 (Genetyx co., Tokyo, Japan).

Semi-quantitative RT-PCR: The gene transcription levels of COXI, COXIII, and CYTb genes of B. gibsoni were ob-served by semi-quantitative reverse transcription (rT)-Pcr. The infected rBcs were harvested and lysed. The liberated parasites were pelleted for rNa extraction using an rNeasy mini kit (QiaGeN, Valencia, ca, U.s.a.) according to the manufacturer’s instructions. consequently, cDNa was synthesized from total rNa using High capacity rNa-to-cDNa master mix (applied Biosystems, Tokyo, Japan) according to the manufacturer’s instructions. The expression levels of COXI, COXIII, and CYTb genes were assayed by rT-Pcr using the primers shown in Table 2. The set of prim-ers was tested in a range from 20 to 35 cycles using 800 ng cDNA. In order to confirm that product accumulation was in the linear phase of the curve, a similar protocol to that of montero et al. [20] was used. The annealing temperature and

cycles were 60°c and 35 cycles, respectively. The 18s rrNa was used as a control in order to guarantee that an equal amount of cDNa from each sample was being evaluated correctly. The samples were separated on 1.5% agarose gels and visualized under UV light by ethidium bromide staining. The absolute integrated optical density (iOD) of each band was analyzed by scanning densitometry with Lumi Vision analyzer 2.1 software (aisin seiki co., aichi, Japan). The amount of COXI, COXIII, and CYTb gene transcriptions was established as a ratio referring to 18s rrNa of B. gibsoni, which is expressed at a constant rate [9]. Data were collected in terms of the band intensity of target genes per band in-tensity of 18s rrNa. The experiment was conducted three times. Two-sample t-tests were used to compare the statisti-cal difference of the transcription of each gene. P < 0.05 was considered significant.

resULTs

Stability of the characteristic of resistance to DA: To ob-serve the stability of resistance to Da, Da-cessation isolate was developed from Da200-ii isolate. During the Da-free period, the Da-cessation isolate grew and proliferated in the same way as Da200-ii isolate (Fig. 2). interestingly, when the Da-cessation isolate was newly exposed to 200 ng/ml Da in week 6 and 7, this isolate could grow and proliferate in the same way as the Da200-ii isolate (Fig. 2). in addition, when compared to the wild-type and Da-resistant isolate of B. gibsoni, no morphological change was observed in the Da-cessation isolate throughout the experimental period. meanwhile, the levels of parasitemia of the Da200-ii isolate and Da-cessation isolate were comparatively lower than those of the wild-type throughout the experimental period (Fig. 2).

Comparison of COXI, COXIII, and CYTb genes from wild-type and DA-resistant B. gibsoni isolates: To investigate the involvement of mtDNa in Da resistance in B. gibsoni, we determined the nucleotide sequence of full-length mtDNa from the wild-type and Da-resistant B. gibsoni isolates. The lengths of mtDNa from B. gibsoni in our laboratory were 5,867 base pairs. The mtDNa of the B. gibsoni consisted of three functional genes, COXI, COXIII, and CYTb, and six discontinuous ribosomal RNA, as Hikosaka et al. previously reported (NrcPD strain) [7]. additionally, terminal inverted

Table 2. Oligonucleotides used for the COXI, COXIII, and CYTb gene expressions of Da-resistant and wild-type Babesia gibsoni

Name sequences UsecOXiF1 3′-ggaagtggtactggatggac-5′ COXImHr1a) 3′-gctgatacaatataggatctcc-5′ COXIcOXiiiF1 3′-gaatactcatcaccttgataag-5′ COXIIIcOXiiir1a) 3′-gcaattgtaaatagtattagag-5′ COXIIIcYTbF1 3′-ctcatatgttgtcctatttggttcc-5′ CYTbcYTbr2a) 3′-ctgttgctccccaataactc-5′ CYTb18srF 3′-gataaccgtgctaattgtag-5′ 18s rrNa18srra) 3′-cgaataattcaccggatcac-5′ 18s rrNa

a) antisense primers.

miTOcHONDriaL GeNe OF Da-resisTaNT B. GIBSONI 1143

repeats were found at the 3′-end and 5′-end of the mtDNA of our B. gibsoni isolate, as in the NrcPD strain.

subsequently, the nucleotide sequence and the deduced amino acid sequence of the COXI gene among the wild-type, Da200-i and Da200-ii isolates, and Da1 variant were com-pared. as a result, several nucleotide polymorphisms were found on the COXI gene. among these nucleotide polymor-phisms, nucleotide substitution at nucleotide 1006 and 1008 gave rise to a substitution at amino acid 310 on the amino acid sequence of COXI (Fig. 3). methionine at amino acid 310 on the amino acid sequence of COXI in the wild-type was substituted with isoleucine or valine in the Da200-i and DA200-II isolates, and DA1 variant. Therefore, to confirm this amino acid substitution, the COXI gene in the wild-type was cloned and sequenced again as WT2 (Fig. 3). as a result, amino acid 310 on the COXI gene in WT2 was also valine (Fig. 3). in addition, amino acid 310 on the COXI gene in the Hyogo isolate, which was exposed to Da and atovaquone, was isoleucine (Fig. 3). moreover, amino acid 179 and 425 on the COXI gene in the Hyogo isolate were isoleucine and threonine, although those in the wild-type and Da-resistant isolates were valine and isoleucine, respectively (Fig. 3).

additionally, the nucleotide sequence and the deduced amino acid sequence of COXIII and CYTb genes were compared among the wild-type, Da200-i and Da200-ii isolates, and Da1 variant. although several nucleotide poly-morphisms were detected on COXIII and CYTb genes, their amino acid sequences were identical among those isolates (Figs. 4 and 5). The COXIII and CYTb genes in the Hyogo isolate were also compared with those isolates. amino acid

102 and 158 on the COXIII gene in the Hyogo isolate were substituted with valine and arginine, respectively (Fig. 4). amino acid 121, 220, and 303 on the CYTb gene in the Hyogo isolate were substituted with isoleucine, isoleucine, and valine, respectively (Fig. 5).

Analysis of gene transcription of COXI, COXIII, and CYTb in the wild-type and DA-resistant B. gibsoni isolate: semi-quantitative rT-Pcr was employed to compare the gene transcription levels of COXI, COXIII, and CYTb genes between the wild-type and Da200-ii isolate. as a result, the band intensities of COXI, COXIII, and CYTb genes in Da200-ii isolate seemed to be slightly lower than in the wild-type. since the band intensities of 18s rrNa were comparable between the wild-type and Da200-ii isolate in each experiment, the band intensities of target genes could be compared. in the present study, product accumulation was in the linear phase of the curve during 25 to 30 cycles; therefore, the band intensities at 25 cycles were compared. subsequently, the results of densitometric analysis of bands obtained by electrophoresis of rT-Pcr products demon-strated that COXI, COXIII, and CYTb mrNa levels from the DA200-II isolate were not significantly decreased compared to the wild-type (Fig. 6).

DiscUssiON

in the present study, the stability of the Da resistance of B. gibsoni was initially examined in vitro. allowing the reversal of drug-resistant phenotypes may have value to understand the mechanisms of drug resistance [2]. although

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).

B. K. WicKramaseKara raJaPaKsHaGe ET AL.1144

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

the Da-cessation isolate was cultured without Da for 4 weeks, this isolate maintained the ability of Da resistance. in the previous study, when wild-type B. gibsoni was cul-tured in 100 ng/ml Da, the parasites were eliminated within 2 weeks [8]. These previous and present results indicated that Da resistance was stable in the Da-cessation isolate and that some genes in the Da-resistant B. gibsoni isolate might differ from those in the parasite that is sensitive to Da. since Babesia parasites multiply asexually with a reproductive time of around 10 hr under normal in vitro culture conditions [27], the genes related to Da resistance would be transferred successfully during multiplication of the parasites; however, we could not determine whether the parasites developed resistance to Da or that the Da-resistant parasites were selected from a mixed population of parasites in the present study. a similar phenomenon has also been reported in Da-resistant Trypanosoma brucei brucei [11, 26] and pentamidine-resistant Leishmania species [21] in vitro. The underlying exact genetic mechanism of Da resistance of Trypanosoma and Leishmania parasites has been well investigated, though that of Babesia parasites is unclear; therefore, determination of the genes responsible for the Da resistance of B. gibsoni is necessary.

in addition, the low parasitemia level of the Da-resistant B. gibsoni isolate compared to the wild-type suggested that the proliferation potential of the Da-resistant B. gibsoni iso-late would be lower than that of the wild-type. Because the level of parasitemia varies at different periods, the levels of parasitemia of the wild-type and the Da-resistant B. gibsoni isolate were measured concurrently in the present study. The level of parasitemia of the Da-resistant isolate was lower than that of the wild-type. a similar situation has been de-scribed for pentamidine-resistant Leishmania, which grow

slower than their parental wild-type clones in vitro [1]. The low proliferation potential of these drug-resistant parasites depends on the alteration of energy metabolism or nucleic acid synthesis. in particular, mitochondria have important roles in energy metabolism, and mtDNa is one of the targets of therapeutic drugs [4, 18]. as described above, substitu-tions in the CYTb gene on atovaquone-resistant isolates of B. gibsoni [16] and P. falciparum [12, 25] were also reported. accordingly, in the present study, we focused our attention on the mtDNa of the Da-resistant B. gibsoni isolate.

The nucleotide and amino acid sequences of COXI, COXIII, and CYTb were compared between the wild-type and the Da-resistant isolates of B. gibsoni. in the present study, to investigate if amino acid variations on mitochondri-al genes occur when exposed to only 1 ng/ml Da, the Da1 variant was separated during development of the Da200-ii isolate and was included in the present analysis. although several nucleotide variations were detected in COXI, COXIII, and CYTb genes, there were no significant amino acid substitutions in cOXi, cOXiii, and cYTb involved in Da resistance of B. gibsoni. in particular, amino acid 310 on COXI varied even among wild-type B. gibsoni; therefore, this amino acid substitution is an amino acid polymorphism on the COXI gene. These results indicated that mtDNa is not altered during the development of Da-resistant B. gib-soni isolates. meanwhile, three amino acid substitutions on CYTb related to atovaquone resistance were detected in the Hyogo isolate. since the Hyogo isolate was exposed to atovaquone in addition to Da, the three amino acid substitu-tions on CYTb were probably caused by atovaquone [16]. Furthermore, there were a few amino acid substitutions on COXI and COXIII in the Hyogo isolate of B. gibsoni. it is supposed that those amino acid substitutions might be amino

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 (*).

B. K. WicKramaseKara raJaPaKsHaGe ET AL.1146

acid polymorphisms on the COXI and COXIII genes of B. gibsoni.

Because the nucleotide and amino acid sequences of COXI, COXIII, and CYTb in Da-resistant isolates were not different from those in the wild-type, the transcription levels of these three genes were observed. as a result, the gene transcription of COXI, COXIII, and CYTb genes in the Da-resistant B. gibsoni isolate was not significantly altered. These results indicated that Da did not directly affect mtD-Na of the Da-resistant B. gibsoni. in general, COXI is one of 13 subunits of cytochrome c oxidase (complex iV), which is essential for electron transfer as a terminal oxidase in the respiratory chain [7, 28]. COXIII is highly conserved in most

of the heme-copper respiratory oxidase superfamily and is considered to be involved in the assembly and stabilization of the entire complex [7, 28]. CYTb is a subunit of complex iii, and is essential for electron transfer in the mitochondrial respiratory chain [7, 18]. accordingly, COXI, COXIII, and CYTb might also have important roles in mitochondrial res-piration, i.e., energy metabolism, in B. gibsoni. as described above, the alteration of energy metabolism might result in a change of proliferation potential. indeed, the proliferation potential of the Da-resistant B. gibsoni isolate was lower than in the wild-type in the present study; however, from the present results, it is suggested that the expression of COXI, COXIII, and CYTb in the Da-resistant B. gibsoni isolate was

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.

miTOcHONDriaL GeNe OF Da-resisTaNT B. GIBSONI 1147

not altered or suppressed by exposure to Da. The possibil-ity of specific mitochondrial changes being involved in DA resistance in african trypanosomes has also been anticipated by Teka et al. [26]. Therefore, a further study of energy metabolism, such as glycolysis pathway, Tca cycle, and electron transport system, in the Da-resistant isolate could elucidate the mechanisms of Da resistance and the mecha-nisms of action of Da against B. gibsoni.

in conclusion, the Da resistance of B. gibsoni was stable in vitro, indicating that several genes might be involved in

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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Fig. 6. analysis of gene transcription of COXI, COXIII, and CYTb in the wild-type and Da200-ii isolate. (a) Gel electrophoresis analy-sis of rT-Pcr products derived from total rNa of the wild-type (left) and Da200-ii isolate (right). The band intensities of COXI, COXIII, and CYTb were observed at 20, 25, 30, and 35 cycles. The band intensity of 18S rRNA was used as a control. The figure is representative of three experiments. (B) COXI, COXIII, and CYTb gene transcription from the wild-type (closed bar) and Da200-ii isolate (open bar) at 25 cycles of rT-Pcr. Data were collected in terms of the band intensity of target genes per band intensity of 18s rrNa (mean ± sD, n = 3).

B. K. WicKramaseKara raJaPaKsHaGe ET AL.1148

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