Hwang Shiang-Jyi, Yamasaki Masahiro, Nakamura Kensuke, Sasaki Noboru, Murakami Masahiro, Kumara Bandula, Rajapakshage Wickramasekara, Ohta Hiroshi, Maede Yoshimitsu, Takiguchi Mitsuyoshi
Laboratory of Veterinary Internal Medicine, Department of Veterinary Clinical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Jpn J Vet Res. 2010 Nov;58(3-4):155-64.
In our previous report, we developed a diminazene aceturate (DA)-resistant Babesia gibsoni strain that was maintained in culture with 200 ng/ml DA. While developing this strain, we also obtained DA-resistant B. gibsoni variants, which were maintained in culture with DA from 1 to 175 ng/ml for more than 8 weeks. Because heat shock protein 70 (Hsp70) seems to play important roles in adaptation to a stress environment in protozoan parasites, in the present study, we examined the copy number of B. gibsoni Hsp70 (BgHsp70) transcripts of those DA-resistant variants using quantitative real-time reverse transcription-polymerase chain reaction. We found that when wild-type B. gibsoni was exposed to 1 ng/ml DA, the level of BgHsp70 transcripts was decreased at day 14. The copy number of BgHsp70 transcripts in the DA-resistant variant cultured with 1 ng/ml DA was significantly lower than in wild-type B. gibsoni, while those in DA-resistant variants increased with escalating doses of DA from 1 to 75 ng/ml, although they were lower than in wild-type B. gibsoni. However, those in DA-resistant variants cultured with >125 ng/ml DA were almost the same as wild-type B. gibsoni. These results indicated that the transcript levels of the BgHsp70 gene might be reduced when the parasites are exposed to a low concentration of DA, and then might recover to the normal level after achieving resistance against DA. We expect that further study of the function of BgHsp70 will elucidate the mechanism of drug resistance against DA in B. gibsoni.
在我们之前的报告中，我们培育出了一种对乙酰马啉（DA）具有抗性的吉氏巴贝斯虫菌株，该菌株在含200 ng/ml DA的培养基中维持培养。在培育该菌株的过程中，我们还获得了对DA具有抗性的吉氏巴贝斯虫变体，这些变体在含1至175 ng/ml DA的培养基中维持培养超过8周。由于热休克蛋白70（Hsp70）似乎在原生动物寄生虫适应应激环境中发挥重要作用，因此在本研究中，我们使用定量实时逆转录 - 聚合酶链反应检测了那些对DA具有抗性的变体的吉氏巴贝斯虫Hsp70（BgHsp70）转录本的拷贝数。我们发现，当野生型吉氏巴贝斯虫暴露于1 ng/ml DA时，第14天BgHsp70转录本水平下降。在含1 ng/ml DA培养的DA抗性变体中，BgHsp70转录本的拷贝数显著低于野生型吉氏巴贝斯虫，而在含1至75 ng/ml DA递增剂量培养的DA抗性变体中，其转录本拷贝数增加，尽管低于野生型吉氏巴贝斯虫。然而，在含>125 ng/ml DA培养的DA抗性变体中，其转录本拷贝数与野生型吉氏巴贝斯虫几乎相同。这些结果表明，当寄生虫暴露于低浓度DA时，BgHsp70基因的转录水平可能会降低，然后在获得对DA的抗性后可能恢复到正常水平。我们期望对BgHsp70功能的进一步研究将阐明吉氏巴贝斯虫对DA的耐药机制。
