Identification of differentially expressed genes in a Giardia lamblia WBC6 clone resistant to nitazoxanide and metronidazole
Identification of differentially expressed genes in a Giardia lamblia WBC6 clone resistant to nitazoxanide and metronidazole
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DOI：
10.1093/jac/dkn142
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发表时间：
2008-07-01
影响因子：
5.2
通讯作者：
Mueller, Norbert
中科院分区：
文献类型：
--
作者：
Mueller, Joachim;Ley, Serej;Mueller, Norbert
目的：应用基因芯片技术和实时定量聚合酶链式反应技术研究蓝氏贾第鞭毛虫WB C6株C4对甲硝唑和硝唑尼特耐药的基因表达差异。方法：在前期研究中，我们建立并鉴定了蓝氏贾第鞭毛虫WB C6克隆株。在这项研究中，使用基于微阵列的方法，我们已经确定了在C4中与其野生型WB C6相比差异表达的开放阅读框架(ORF)。利用实时定量聚合酶链式反应，我们已经验证了其中一些ORF的表达模式，重点是野生型和C4滋养体中的伴侣蛋白，如热休克蛋白。为了诱导抗原转换，两个菌株的滋养体都经历了一个内切和外排的循环。结果：与野生型相比，9115个ORF中有40个在C4中高表达，46个在C4中低表达。经过一次循环，C4对硝唑尼特和甲硝唑的抵抗力消失。抗性的形成和En-/Exystation与编码主要表面抗原的ORFs的表达变化有关，如变异的表面蛋白TSA417或AS7(抗原转移)。此外，胞质热休克蛋白HSP70B2、HSP40以及先前发现的硝唑尼特结合蛋白硝基还原酶和蛋白二硫化物异构酶PDI4的表达模式与EN-/EXSTATION后的抗性和抗性丧失相关。C4滋养体的耐热性高于野生型滋养体。结论：这些结果表明贾第鞭毛虫对硝唑尼特和甲硝唑的抗性形成与热休克蛋白等应激反应相关基因的表达变化有关。
Objectives: The characterization of differential gene expression in Giardia lamblia WB C6 strain C4 resistant to metronidazole and nitazoxanide using microarray technology and quantitative real-time PCR.Methods: In a previous study, we created and characterized the G. lamblia WB C6 clone C4 resistant to nitazoxanide and metronidazole. In this study, using a microarray-based approach, we have identified open-reading frames (ORFs) that were differentially expressed in C4 when compared with its wild-type WB C6. Using quantitative real-time PCR, we have validated the expression patterns of some of those ORFs, focusing on chaperones such as heat-shock proteins in wild-type and C4 trophozoites. In order to induce an antigenic shift, trophozoites of both strains were subjected to a cycle of en- and excystation. Expression of selected genes and resistance to nitazoxanide and metronidazole were investigated after this cycle.Results: Forty of a total of 9115 ORFs were found to be up-regulated and 46 to be down-regulated in C4 when compared with wild-type. After a cycle of en- and excystation, resistance of C4 to nitazoxanide and metronidazole was lost. Resistance formation and en-/excystation were correlated with changes in expression of ORFs encoding for major surface antigens such as the variant surface protein TSA417 or AS7 ('antigenic shift'). Moreover, expression patterns of the cytosolic heat-shock protein HSP70 B2, HSP40, and of the previously identified nitazoxanide-binding proteins nitroreductase and protein disulphide isomerase PDI4 were correlated with resistance and loss of resistance after en-/excystation. C4 trophozoites had a higher thermotolerance level than wild-type trophozoites. After en-/excystation, this tolerance was lost.Conclusions: These results suggest that resistance formation in Giardia to nitazoxanide and metronidazole is correlated with altered expression of genes involved in stress response such as heat-shock proteins.